Download DEFINING THE ROLE OF THE SHP2 PROTEIN TYROSINE

DEFINING THE ROLE OF THE SHP2 PROTEIN TYROSINE
PHOSPHATASE IN FcɛRI SIGNALING IN MAST CELLS
by
Victor Allon McPherson
A thesis submitted to the Department of Biochemistry
In conformity with the requirements for
the degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
(August, 2008)
Copyright ©Victor Allon McPherson, 2008
i
Abstract
Mast cells are granulocytes that are a key component of the innate and adaptive immune
system, and contribute to allergic disorders. Mast cell activation following clustering of
the high affinity IgE receptor (FcεRI) by multivalent antigens requires reversible tyrosine
phosphorylation of myriad signaling proteins. Activated mast cells rapidly release
granule contents (eg. histamine and serine proteases) that cause vascular permeability,
and in a more delayed manner they also synthesize and secrete eicosanoids and numerous
cytokines (eg. IL-6 and TNFα) that recruit activated leukocytes. FcεRI signaling is
initiated by Lyn, a Src Family Kinase (SFK), that phosphorylates immunoreceptor
tyrosine-based activation motifs (ITAMs) found on the FcεRI β and γ chains. This allows
recruitment of Fyn SFK and Syk kinase that bind ITAMs and phosphorylate numerous
downstream targets. Src Homology 2 domain-containing Phosphatase 2 (SHP2, encoded
by ptpn11/shp2) is known to be recruited to several phosphorylated proteins following
FcεRI aggregation in mast cells, however attempts to define the role of SHP2 have been
hampered by its essential role during embryonic development and hematopoiesis in mice.
Recently, conditional SHP2 knock-out mice (shp2fl/fl) have been created allowing for
shp2 inactivation in a tissue-specific manner by Cre recombinase. Here we describe the
use of transgenic mice expressing a modified estrogen receptor-Cre Recombinase
(TgCreER*) on a shp2fl/fl genetic background, that allows for maturation of bone marrowderived mast cells (BMMCs) prior to shp2-inactivation using 4-hydroxytamoxifen (4OHTM). SHP2-depleted BMMCs display reduced phosphorylation of the FcεRI β chain, but
exhibit extended phosphorylation of Syk kinase. Additionally, SHP2-deficient cells
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display a defect in the activation of both Erk mitogen-activated protein kinase and Akt,
which correlates with an observed defect in the production of TNFα and Leukotriene C4.
Finally, we show that SHP2-deficient BMMCs display elevated FcεRI-evoked
phosphorylation of Csk-Binding Protein (Cbp or PAG) on residue Y317, which recruits
C-terminal Src kinase (Csk) that phosphorylates SFKs on an inhibitory tyrosine. This
hyperphosphorylation of Cbp correlates with elevated phosphorylation of the C-terminal
inhibitory tyrosine on Fyn kinase. This study provides new insights into the role of SHP2
as a positive effector of FcεRI signaling and cytokine production in mast cells.
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Co-Authorship
Stephanie Everingham1, Gen-Sheng Feng2 and Andrew WB Craig1.
1
Department of Biochemistry, Queen’s University, Kingston, ON; 2Burnham Institute, La Jolla,
California
SE performed mouse genotyping and ELISAs
GSF provided the conditional SHP2 Knockout mouse model
AWBC helped in the experimental design and analyses
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Acknowledgements
I would like to thank Dr. Andrew Craig for all of his help and support throughout the
completion of this study. Without his insight and guidance, I would never have been able
to enjoy the success that I have found during this time. I would also like to thank my
fellow labmates, Stephanie Everingham, Julie Smith, Jinghui Hu and Dr. Alka
Mukhopadhyay, and my former labmates Rob Karisch, Andrew Sage and Vanessa
DiPalma for their knowledge and aid and for making the last two years immensely
enjoyable. Finally, I would like to thank CIHR for their funding in support of this
research.
v
Table of Contents
Abstract............................................................................................................................................ii
Co-Authorship ................................................................................................................................iv
Acknowledgements.......................................................................................................................... v
Table of Contents............................................................................................................................vi
List of Figures ...............................................................................................................................viii
List of Abbreviations ......................................................................................................................ix
Chapter 1 Introduction ..................................................................................................................... 1
1.1 Mast Cells Origins and Development .................................................................................... 1
1.2 Mast Cell Involvement in Immunity...................................................................................... 4
1.3 The FcɛRI Receptor ............................................................................................................... 6
1.4 Degranulation Pathways ...................................................................................................... 10
1.5 FcɛRI Mediated Transcription Factor Activation and Associated Chemokine Production . 12
1.6 Bioactivity of Mast Cell-Derived Inflammatory Mediators................................................. 14
1.7 Negative Feedback of FcɛRI Signaling................................................................................ 16
1.8 Kit Receptor Activation Potentiates FcεRI Signaling.......................................................... 20
1.9 Mast Cell Dysregulation and Disease .................................................................................. 21
1.10 The Non-Receptor Protein Tyrosine Phosphatase, SHP2 .................................................. 22
1.11 SHP1 is a Negative Regulator of Mast Cell Degranulation and Inflammation ................. 25
1.12 SHP2 Recruitment Sites Within the FcεRI Pathway ......................................................... 27
1.13 Currently Identified Roles for SHP2 in Signal Regulation................................................ 28
1.14 SHP2 Trapping Assays ...................................................................................................... 30
1.15 Conditional SHP2 Knock-out Mouse Models and Mast Cells........................................... 31
1.16 Study Rationale, Hypothesis and Objectives ..................................................................... 33
Chapter 2 Materials and Methods .................................................................................................. 35
2.1 Antibodies ............................................................................................................................ 35
2.2 Transgenic Mouse Lines ...................................................................................................... 36
2.3 BMMC Cultures................................................................................................................... 36
2.4 BMMC SHP2 Inactivation and FcεRI Stimulations ............................................................ 37
2.5 Cytokine ELISAs ................................................................................................................. 38
2.6 Degranulation Assays .......................................................................................................... 38
2.7 Immunoprecipitation and Immunoblots Analysis................................................................ 39
2.8 Densitometry and Statistical Analysis ................................................................................. 40
vi
Chapter 3 Results ........................................................................................................................... 41
3.1 A Transgenic Mouse Model Allowing for Temporal Inactivation of shp2 in BMMCs....... 41
3.2 SHP2 is Not Required for Degranulation ............................................................................ 45
3.3 SHP2 Modulates FcεRI-Proximal Signaling ....................................................................... 48
3.4 SHP2 Promotes Akt Activation ........................................................................................... 51
3.5 SHP2 Promotes Activation of Erk MAPK........................................................................... 53
3.6 SHP2 promotes Leukotriene C4 & TNFα Release ............................................................... 56
3.7 Evidence that Cbp is a SHP2 Substrate that Limits Fyn Activity in BMMCs..................... 58
Chapter 4 Discussion ..................................................................................................................... 61
References...................................................................................................................................... 71
vii
List of Figures
Figure 1. Schematic of the signaling events downstream of FcεRI. ................................................ 7
Figure 2. FcεRI Structure................................................................................................................. 9
Figure 3. Negative Regulation of SFKs by Csk............................................................................. 18
Figure 4. SHP2 Structure and Autoinhibition of Phosphatase Activity......................................... 23
Figure 5. A Transgenic Mouse Model Allowing for Temporal Inactivation of shp2 in BMMCs. 42
Figure 6. Characterization of shp2 inactivation model. ................................................................. 44
Figure 7. SHP2 is Not Required for Degranulation. ...................................................................... 47
Figure 8. SHP2 Modulates FcεRI-Proximal Signaling. ................................................................. 49
Figure 9. SHP2 Promotes Akt Activation...................................................................................... 52
Figure 10. SHP2 Promotes Activation of Erk MAPK. .................................................................. 54
Figure 11. SHP2 promotes Leukotriene C4 & TNFα Release........................................................ 57
Figure 12. Evidence that Cbp is a SHP2 Substrate that Limits Fyn Activity in BMMCs. ............ 59
Figure 13. Proposed functions for SHP2 in Regulating the FcεRI Pathway.................................. 63
viii
List of Abbreviations
4OH-TM
4-hydroxytamoxifen
BBB
Blood Brain Barrier
BMMC
Bone Marrow Derived Mast Cell
c-Kit
c-Kit Receptor
Cbp
Csk Binding Protein
CD34
Cluster of Differentiation 34
CMV
Cytomegalovirus
CSF
Colony Stimulating Factor
Csk
C-Terminal Src Kinase
DAG
Diacylglycerol
DNP
2,4-Dinitrophenyl
Dok1
Docking Protein 1
Dok2
Docking Protein 2
EGFR
Epidermal Growth Factor Receptor
ELISA
Enzyme-Linked ImmunoSorbent Assay
ER
Endoplasmic Reticulum
Erk
Extracellular Signal-Related Kinase
Fer
Fes-Related Protein
FcεRI
High Affinity IgE Receptor
FGF4
Fibroblast Growth Factor 4
fl
Floxed
Fps/Fes
Fujinami Poultry Sarcoma/Feline Sarcoma
Oncogene
Gab2
Grb2-Associated Binder 2
GAP
GTPase Activating Protein
GEF
Guanine Nucleotide Exchange Factor
Grb2
Growth Factor Receptor-Bound Protein 2
HSA
Human Serum Albumin
HuMC
Human Mast Cell
ix
IB
Immunoblot
IgE
Immunoglobulin E
IKKα
ΙκB Kinase α
IL
Interleukin
IP
Immunoprecipitate
IP3
Inositol 1,4,5-Trisphosphate
ITAM
Immunoreceptor Tyrosine-Based Activation Motif
ITIM
Immunoreceptor Tyrosine-Based Inhibitory Motif
JMML
Juvenile Myelomonocytic Leukemia
JNK
Jun-Amino Terminal Kinase
LAT
Linker for activation of T cells
LPS
Lipopolysaccharide
LT
Leukotriene
MAdCAM
Mucosal Addressin Cell Adhesion Molecule
MAFA
Mast Cell Function-Associated Antigen
MAPK
Mitogen Activated Protein Kinase
MBP
Myelin Basic Protein
MEF
Mouse Embryonic Fibroblast
MHC
Major Histocompatibility Complex
MIP
Macrophage Inflammatory Protein
mMCP
Mouse Mast Cell Protease
MS
Multiple Sclerosis
NFAT
Nuclear Factor of Activated T-Cells
NFκB
Nuclear Factor κ B
NTAL
Non-T Cell Activation Linker
RhoGAP
Rho GTPase Activating Protein
PAMP
Pathogen Associated Molecular Pattern
PDK-1
Phosphatidylinositol(3,4,5)P3-Dependent Protein
Kinase-1
Pecam-1
Platelet-Endothelial Cell Adhesion Molecule 1
PG
Prostaglandin
PI(4,5)P2
Phosphatidylinositol 4,5-Bisphosphate
x
PI3K
Phosphoinositide-3 kinase
PIP3
Phosphatidylinositol (3,4,5)-trisphosphate
PKC
Protein Kinase C
PLA2
Phospholipase A2
PLCγ
Phospholipase C γ
PKB
Protein Kinase B
PTP
Protein Tyrosine Phosphatase
RasGAP
Ras GTPase Activating Protein
RasGEF
Ras Guanine Nucleotide Exchange Factor
RBL
Rat Basophilic Leukemia
SCF
Stem Cell Factor
SEM
Standard Error of the Mean
SFK
Src Family Kinase
SH2
Src Homology 2 Domain
SHIP
SH2 Domain–Containing 5′-Ionositol Phosphatase
SHP1
Src Homology 2 domain-Containing Phosphatase 1
SHP2
Src Homology 2 domain-Containing Phosphatase 2
SLP-76
SH2 domain–containing leukocyte protein of 76
kDa
SOC
Store Open Channel
SOS
Son-of-Sevenless
STAT
Signal Transducer and Activator of Transcription
Th2
T Helper 2
TLR
Toll-Like Receptor
TNFα
Tumour Necosis Factor α
TNP
Trinitrophenylated
TS
Trophoblast Stem
VCAM-1
Vascular Cell Adhesion Molecule-1
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Chapter 1
Introduction
1.1 Mast Cells Origins and Development
Mast Cells were first described by Paul Ehrlich in the late 1800s1. They were
initially termed “Mastzellen” (meaning “well fed cells”) due to their abundance of
cytoplasmic granules; Ehrlich also noted that tissues affected by chronic inflammation
exhibited a striking increase in the presence of these cells1. Mast cells are granulocytes
of the myeloid lineage2 that are key components of the innate and adaptive immune
system3. They are involved in the inflammatory response through the combined activities
of a variety of cell surface receptors, including the Toll Like Receptor (TLR) family and
the high affinity IgE receptor, FcεRI3. The activities of the receptors expressed by mast
cells allows them to be involved in the immune responses to bacterial, viral and parasitic
infections4.
Mast cells derive from multipotent, CD34+/c-kit+ hematopoietic progenitors that
are present in the bone marrow (BM). Following their development in the BM, immature
mast cells subsequently enter the blood stream and then migrate to mucosal or connective
tissues throughout the body5. This migration is dependent upon integrin recognition of
extracellular matrix proteins, including fibrinogen and fibronectin, and adhesion proteins
expressed on the surface of intestinal epithelial cells and lung stromal cells, including
MAdCAM-1 and VCAM-16. The integrins that are involved in this tissue homing process
include α4β1, α4β7 and αIIbβ36.
1
Upon arrival at peripheral tissues, the committed mast cell progenitors develop
into mature mucosal or connective tissue mast cells (with specialized granule contents,
etc.) in response to factors released by the local tissue5. The two varieties of mast cells
are differentiated by their tissue of residence and can be identified by their differential
histological staining properties and the proteases they contain within their secretory
granules6. Mucosal mast cells are present in mucosal epithelial tissues of the intestine and
lungs, while connective tissue mast cells are present in the peritoneal cavity and skin6.
Mucosal mast cells do not stain with the histological stain safranin and predominantly
express mouse mast cell protease (mMCP) 1 and mMCP-2. Connective tissue mast cells
stain positively with safranin as a result of heparin being contained within their granules,
while they express mMCP-3, mMCP-4, mMCP-5 and mMCP-67,8.
Stem Cell Factor (SCF) is the ligand for the Kit receptor and is the primary mast
cell growth factor in vivo5. However, many other cytokines and growth factors have been
shown to promote development and determine the exact phenotype of the resulting mast
cells in vitro5. The Kit receptor is expressed on the surface of mast cells through all
stages of development9. SCF is constitutively expressed in both membrane bound and
soluble forms by stromal cells, including fibroblasts and epithelial cells9. SCF interaction
with the Kit receptor promotes receptor oligomerization, activation of receptor tyrosine
kinase activity and autophosphorylation at many tyrosine residues that serve to recruit Src
homology 2 (SH2) domain containing proteins5. Kit receptor activation results in the
initiation of multiple signal cascades, including Ras-Mitogen Activated Protein Kinase
(MAPK) activation, which acts to promote cell growth and proliferation5. SCF may be
2
used in vitro to drive the growth and development of hematopoietic stem cells into
murine bone marrow-derived mast cells (BMMCs) or human mast cells in their
respective systems9.
Several other cytokines also participate in the regulation of mast cell proliferation
and development under in vitro conditions5. The first of these cytokines to be identified
as being able to drive in vitro mast cell growth and development was Interleukin-3 (IL3)10,11. IL-3 is able to independently promote the differentiation of murine hematopoeitic
stem cells into mucosal type BMMCs and is sufficient to support the growth and survival
of the resulting culture10,11. IL-3 at concentrations of approximately 1ng/ml can also
enhance the SCF-induced proliferation of the BMMC culture5. However, at higher
concentrations (above 5ng/ml), IL-3 can stimulate the development of granulocyte and
macrophage colonies as well; therefore, lower IL-3 concentrations are desirable in cell
culture in order to ensure a homogeneous culture of BMMCs. In contrast, IL-3 has been
shown to be dispensable for in vivo mast cell development and proliferation, as IL-3
knockout mice exhibit no reduction in the number of mast cells in their tissues12.
Furthermore, human mast cells do not express the IL-3 receptor and thus do not respond
to this cytokine13.
A number of other cytokines can augment the in vitro affects of SCF and IL-3.
IL-4 can act in concert with IL-3 to promote the development of murine mast cells that
exhibit the connective tissue mast cell phenotype14. IL-6 also promotes murine mast cell
development15, and has been shown to have mast cell growth promoting effects16 and
anti-apoptotic effects in cultures of human cord blood derived mononuclear cells17. IL-9
3
promotes the viability of SCF driven BMMC cultures, while also promoting a phenotypic
change associated with the later stages of mucosal mast cell development18. This
promotion of maturation occurs through the initiation of the expression of the mMCP-1
and mMCP-2 serine proteases18. IL-9 mediated expression of these two proteases, as well
as mMCP-4, is inhibited by both IL-3 and IL-4; therefore, despite promoting BMMC
proliferation, these two ILs actually serve to inhibit the final steps of mast cell
development in vitro18.
1.2 Mast Cell Involvement in Immunity
Mast cells are present at locations adjacent to openings to the outside
environment, are associated with nerves and blood vessels4, and often accumulate at sites
of infection19. Mast cells can act as antigen presenting cells by binding and processing
exogenously derived peptides, and presenting small portions of these peptides on the
major histocompatibility complex 1 (MHC1) and MHC2 receptors20,21. These receptors
allow the presentation of exogenous molecules to B and T lymphocytes, which can serve
to promote both cellular and humoral immune responses22.
Mast cells also express the TLRs, which are Pattern Recognition Receptors
(PRRs) that are expressed on the cell surface and are involved in the recognition of
Pathogen Associated Molecular Patterns (PAMPs). PAMPs are highly conserved
sequences23 present in exogenously derived molecules which originate from either
bacteria or viruses24. Specifically, it has been shown that BMMCs express TLR2, TLR4,
TLR6 and TLR825. Through TLR-mediated recognition of exogenous particles, mast cells
are involved in the immune response to bacterial, viral and parasitic infections and
4
function to regulate the local tissue immune response to infection by releasing a
combination of pre-formed and newly synthesized inflammatory mediators4. In BMMCs,
the subset of mediators that are released in response to TLR activation include many that
are regulated by the transcription factor NfκB, including tumour necrosis factor α
(TNFα), IL-1β, IL-3 and IL-625. These mediators are involved in coordinating the actions
of other nearby cells and recruiting other types of immune cells to the area, including
neutrophils4. Leukocyte recruitment plays a critical role in the resolution of the bacterial
or viral infection.
TLR4 has been shown to promote mast cell mediated stimulation of the immune
response in the event of a bacterial infection4. TLR4 expressed on the surface of BMMCs
allows for mast cell activation in response to stimulation with the major components of
bacterial cells walls – the gram negative bacterial component lipopolysaccharide (LPS),
and the gram positive derived peptidoglycan24. The study indicates that TLR4 is of
primary importance in the generation of immune mediators in response to bacterial
infection25 and that the main downstream effect of TLR stimulation in mast cells is the
release of TNFα, which acts to recruit neutrophils to the area4.
Mast cells are also involved in the immune response that occurs following viral
infection4. Mast cell activation in reaction to viral products can be mediated by TLR3,
TLR7 and TLR84, and serves to initiate the secretion of IL-1β, IL-6, macrophage
inflammatory protein MIP)-1α and MIP-1β26. These mediators increase vascular
permeability and are chemotactic factors involved in recruitment of T cells and natural
killer cells, which are directly involved in the resolution of viral infection4.
5
Additionally, mast cells play key protective roles in the immune response elicited
against parasitic infection, including nematodes27, the protozoa Giardia lamblia28, and
gastrointestinal helminth29. This response is dependent on IL-6 for Giardia lamblia28 and
on secreted mast cell proteases, including MCP-129 and MCP-927 for nematodes and
gastrointestinal helminth. Finally, mast cells also have a protective role in sepsis30 and in
response upon exposure to bee and snake venoms31.
1.3 The FcɛRI Receptor
The FcεRI is the high affinity IgE receptor expressed on mast cells and the closely
related blood-borne basophils32. The receptor plays a key role in activating these cells and
initiating the inflammatory response in response to antigen exposure3. Mast cells store
granules containing inflammatory mediators that are exocytosed upon cell stimulation;
this release of pre-formed inflammatory mediators is termed degranulation3. In parallel to
degranulation, mast cells also secrete eicosanoids, chemokines and cytokines in response
to the initiation FcεRI signaling (Figure 1)3,33.
The FcεRI is one mechanism to allow the mast cell to participate in the humoral
immune response; following initial systemic exposure to an antigen, a subset of B cells
produce IgE antibodies that specifically recognize that antigen. These IgE antibodies bind
the FcεRI and sensitize the mast cell to the antigen, allowing the cell to trigger the
inflammatory response upon subsequent exposure. FcεRI activation is initiated by
receptor aggregation, which occurs as a result of several IgE molecules binding to a
single multivalent antigen molecule3. Binding of several FcεRI bound IgE molecules to
the same multivalent antigen molecule causes a clustering of multiple FcεRI receptors,
6
Cbp
Figure 1. Schematic of the signaling events downstream of FcεRI.
Schematic Diagram of the FcεRI signaling cascade. IgE binds the α subunit of the FcεRI;
multivalent antigen mediated receptor clustering leads to Lyn activation, phosphorylation
of β and γ chain ITAMs and activation of downstream effectors, including Syk and Fyn
kinases. Lyn also phosphorylates Cbp, which recruits Csk. Csk phosphorylates inhibitory
C-terminal tyrosine sites on SFKs, including Lyn and Fyn, leading to a negative feedback
loop initiated attenuation of FcεRI signaling. Major downstream events associated with
receptor signaling include PLCγ mediated degranulation and MAPK dependent cytokine
and eicosanoid synthesis and secretion. Adapted from Ref. 34.
7
and initiates various signaling cascades.
The FcεRI receptor is composed of an IgE binding α chain, a tetramembrane
spanning β chain, and a dimeric γ chain (Figure 2)35. The β chain is necessary for
amplification of the FcεRI signal36. The Src Family Kinase (SFK) Lyn is lipid modified
and localized to lipid rafts in the cell membrane37, and is involved in recruiting FcεRI
receptors to these rafts while also mediating low levels of receptor phosphorylation38.
Upon antigen initiated FcεRI aggregation, Lyn trans-phosphorylates both itself and the
immunoreceptor tyrosine-based activation motifs (ITAMs) present on the β and γ chains
of the receptor3. These phosphorylated tyrosine residues (Y219, Y225 and Y229 on the β
chain, and Y47 and Y58 on the γ chain) act as recruitment sites for signaling molecules
involved in the downstream signaling cascade, including Syk and the SFK Fyn35.
Recent studies have shown that the β chain serves to recruit both positive and
negative regulators of FcεRI receptor signaling3. Tyrosine residues Y219 and Y229 of the
β chain have been shown to play a role in the recruitment of SH2 domain–containing 5′inositol phosphatase (SHIP-1) and the p85 subunit of Phosphoinositide-3 kinase (PI3K)39.
PI3K acts by phosphorylating PI(4,5)P2 to generate PIP33. PIP3 serves to recruit
molecules that contain pleckstrin homology (PH) domains to the plasma membrane3.
Phospholipase Cγ (PLCγ) is among the molecules recruited through this process and is
essential for proper degranulation response3. In order to regulate this recruitment process,
β chain-recruited SHIP-1 dephosphorylates PIP3, generating PI(3,4)P23. Thus, SHIP-1
decreases the level of PIP3 in the membrane surrounding the FcεRI receptor, and serves
to downregulate the recruitment of proteins such as PLCγ to the membrane3; this reduced
8
Figure 2. FcεRI Structure.
The FcεRI is composed of 3 subunits. The first is the IgE binding α chain. The second is the
tetramembrane spanning β subunit binds Lyn and, upon antigen-mediated receptor clustering,
becomes phosphorylated on ITAM residues. β chain ITAMs recruit positive and negative
signaling proteins, including SHIP. The third is the γ subunit, which is a disulphide linked
homodimer and contains additional ITAMs; these ITAMs recruit Syk and SHP1. Adapted from
Ref. 3.
9
recruitment decreases FcεRI signaling39. Recently, SHIP-2 has also been shown to have a
role in regulating FcεRI signaling40.
The γ chain has also been shown to recruit both positive and negative signaling
molecules3. Syk kinase promotes downstream signaling, and has been reported to require
Lyn activity for activation41, while also being downstream of Fyn42. The γ chain ITAM
sites have been shown to recruit Syk and promote downstream signaling through the
phosphorylation of effector molecules43, while γ chain recruitment of the dual SH2
domain containing phosphatase 1 (SHP1) can serve to reduce signaling via
dephosphorylation of proteins within the signaling cascade44.
1.4 Degranulation Pathways
The activation of the FcεRI results in mast cell activation and exocytosis of preformed inflammatory mediators that are stored in cytoplasmic granules3. A 2005 paper
written by Nishida et al has shown evidence that the degranulation response can be
broken down into two separate, concurrently acting pathways that cooperatively interact
to allow for proper degranulation45. The first of these pathways is calcium dependent and
signals through a Syk/Linker for activation of T cells (LAT)/PLCγ pathway, which
results in calcium mobilization and allows for granule fusion with the cell membrane3,46.
The second cascade is termed the calcium independent pathway, and involves the
Fyn/Grb2-associated binder 2 (Gab2)/RhoA mediated formation of microtubules, which
is crucial for the translocation of the cytoplasmic granules from the cytoplasm to the
plasma membrane45.
10
The calcium-dependent pathway requires Lyn45. Lyn activation leads to the
formation of a complex composed of the adaptor proteins LAT and SH2 domain–
containing leukocyte protein of 76 kDa (SLP-76), as well as PLCγ45. The complexing of
these proteins leads to PLCγ activation via phosphorylation; active PLCγ then catalyses
the hydrolysis of plasma membrane located PIP2 into Diacylglycerol (DAG) and IP347.
Increased IP3 leads to a rise in intracellular Ca2+ 47 that occurs in two phases46. First, IP3
binds receptors in the endoplasmic reticulum48, which leads to the release of Ca2+ stores
located in the endoplasmic reticulum (ER), followed by an influx of external Ca2+ into
the cell through Store Open Channels (SOCs) in response to the depletion of ER Ca2+
stores46. This Ca2+ influx (termed Capacitive Calcium Entry) from the surroundings is
required for degranulation, as it plays a role in promoting the disassembly of a cortical
actin ring46. Disassembly of this actin ring is necessary for granule fusion with the cell
membrane and release of the granule contents into the surrounding tissue46.
As mentioned, Fyn is recruited to the FcεRI receptor upon cell stimulation by
multivalent antigens41. Activated Fyn acts upstream of the phosphorylation of the adapter
protein Gab2 – most likely via activation of Syk42. Phosphorylated of Gab2 is known to
recruit the p85 subunit of PI3K, which catalyses the phosphorylation of
phosphatidylinositides present in the plasma membrane at the 3’ position47. The
phosphorylated phosphatidylinositides provide binding sites for molecules that contain
PH domains, which, as previously mentioned, includes PLCγ47. It has been shown that
PI3K activation is required for the degranulation response, and contributes to the final
stages of calcium mobilization associated with degranulation47. A proposed model for the
11
mechanism of involvement of PI3K in calcium mobilization hypothesizes that the
generation of 3’ phosphorylated phosphatidylinositides causes a sustained localization of
PLCγ to the area surrounding the FcεRI47.
The 2005 Nishida et al paper provides the first description of the second, Ca2+
independent signaling pathway involved in the degranulation process45. This branch of
the pathway acts through a Fyn/Gab2/RhoA signaling path and was shown to be linked to
formation of microtubules near the cell periphery that allow for the translocation of the
cytoplasmic granules to the plasma membrane45. A 2006 paper by Sulimenko et al shows
that FcεRI stimulation leads to the formation of complexes containing both Fyn and Syk
PTKs along with several other proteins, including γ-tubulin, which is essential to
microtubule formation49. Sulimenko’s results provide additional support for the role of
Fyn and Syk as key regulators of the early stages of microtubule formation in the
degranulation process49.
1.5 FcɛRI Mediated Transcription Factor Activation and Associated
Chemokine Production
In addition to the immediate degranulation-associated release of inflammatory
mediators contained in the exocytosed cytoplasmic granules, FcεRI stimulation activates
MAPKs, including extracellular-signal related kinase (Erk), p38 MAP kinase (p38) and
Jun-amino terminal kinase (JNK), as well as Protein Kinase B (PKB)/Akt (hereafter
referred to as Akt)48. The result of the activation of these molecules is to initiate
transcription pathways that result in the de novo synthesis of a variety of cytokines,
chemokines and eicosanoids33. A recent review summarized the current knowledge
12
regarding the pathways that lead to MAPK and Akt activation and subsequently to the
activation of transcription factors and the synthesis of these inflammatory mediators48.
The list of mediators shown to be produced downstream of the FcεRI include the
cytokines: TNFα, granulocyte-macrophage colony stimulating factor (CSF), IL-1-IL-6,
IL-9-14, IL-16 and IL-1848; the chemokines include: IL-8, mast cell protease 1 (MCP-1),
MCP-4, MIP-1, and MIP-348; while the eicosanoids include Prostaglandin D2 (PGD2)50
and Leukotriene C4 (LTC4)51.
As has been discussed, the combined recruitment of the p85 regulatory subunit of
PI3K to the β chain of the FcεRI and to Gab2 results in the formation of PIP3 and the
recruitment of PH domain containing signaling molecules. These molecules include Akt,
which gets activated upon recruitment to the plasma membrane by another kinase that is
recruited to the signaling complex through the interaction between PIP3 and its PH
domain, phosphatidylinositol(3,4,5)P3-dependent protein kinase-1 (PDK1)52,48. Akt
activity results in the activation of a number of transcription factors, including NFκB,
NFAT and AP-148.
The combined kinase activity of Lyn and Syk result in the phosphorylation of the
transmembrane adapter protein, LAT48. Phosphorylated LAT serves to recruit a variety of
signaling molecules, including Grb2 and Shc, which recruit son of sevenless (Sos). Sos is
a Ras Guanine nucleotide exchange factor (RasGEF), which when activated serves to
stimulate Ras by loading GTP. Ras activity turns on the Ras/Raf/Mek/Erk cascade48. Erk
serves to phosphorylate and initiate phospholipase A2 (PLA2) activity, as well as activate
the transcription factors Elk and c-Myc48. The consequences of PLA2 activity involve the
13
release of arachidonic acid from lipids located in the plasma membrane; arachidonic acid
liberation results in the generation of eicosanoids48.
Another downstream target of Syk kinase is the Rac GTPase (Rac)/RhoGEF Vav1. When activated by Syk-mediated tyrosyl phosphorylation, Vav-1 activates Rac48. Rac
serves to initiate two separate signaling pathways that proceed to stimulate p38 and JNK
activities48. In a similar manner to Akt and Erk, these MAPKs activate transcription
factors; Elk and ATF2 are activated by p38; cJun and ATF2 are activated by JNK48.
As has also been described above, FcεRI-mediated PLCγ activation initiates
calcium mobilization47; the increase in the concentration of intracellular Ca2+ results in
the activation of Protein Kinase C (PKC)48. PKC can directly activate p38 and JNK and
thus represents a mechanism for the activation of these MAPKs that is independent of
Vav-1/Rac pathway48.
1.6 Bioactivity of Mast Cell-Derived Inflammatory Mediators
There are a wide variety of pro-inflammatory mediators that are released upon
engagement of the FcεRI present on mast cells48. Histamine is one of the main bioactive
molecules released within seconds of mast cell activation, while LTC4, TNFα and IL-6
are synthesized in the hours following receptor activation48.
Histamine is a key component of the granule contents of mast cells and is
involved in the FcεRI-initiated inflammatory response3. This bioactive amine is released
upon mast cell degranulation and elicits its potent vasodilatory activity through binding to
histamine receptors53, which promotes relaxation of the smooth muscles of the local
vasculature54. Histamine-mediated vasodilation leads to an increase in the blood flow to
14
the local tissue54. Through increasing the local blood flow, histamine increases the
number of leukocytes in the local vasculature, thus resulting in an increase in the local
chemoattractant-mediated extravasation and recruitment of leukocytes to the location of
the inflammation-initiating antigen55. Histamine also increases the permeability of the
vascular endotheilia to plasma; therefore, the net result of the functions of histamine is an
increase in the number of leukocytes in the area of infection55, along with tissue swelling
as a result of an increase in the local extracellular fluid54. There are four currently
identified histamine receptors (H1-H4); H1 antagonists are the commonly known
antihistamines and are used to treat allergies53.
Leukotrienes, including LTC4, are arachidonic acid derivatives that are
synthesized by activated mast cells. Leukotrienes are both chemoattractants for immune
cells56 and act on receptors located on the cell surface of smooth muscle to cause
transient vasoconstriction57. Leukotrienes initiate vasoconstriction which leads to the
leakage of plasma from postcapillary venules, causing tissue swelling57. Pharmacological
leukotriene inhibitors are currently used in the treatment of asthma58.
TNFα acts as a chemotactic agent that results in neutrophil recruitment to the site
of its generation4. Neutrophils are specialized granulocytes that are often the first
leukocytes that arrive at a site of injury or infection54. Neutrophils have large cytoplasmic
granules that contain bactericidal molecules and lysosomal enzymes54. These cells
phagocytose pathogens and subsequently die; neutrophil cell death is associated with the
release of the bactericidal compounds and chemotactic agents that act to recruit additional
neutrophils to the area54. As mentioned earlier, mast cell TLR4-mediated production of
15
TNFα and subsequent neutrophil recruitment plays a crucial role in the resolution of
bacterial infections4. Thus, for IgE/Antigen-triggered mast cell responses, TNFα
production is also likely linked to recruitment of neutrophils to aid in clearing the source
of antigen that served to initiate the IgE-mediated immune response.
IL-6 is a cytokine that has been shown to play a key role in initiating the final
stages of B cell differentiation, which is associated with antibody production; thus, IL-6
production is associated with initiation of the humoral immune response following
infection59. IL-6 also positively regulates tissue leukocyte recruitment through the
stimulation of chemokine production by endothelial cells60. Lastly, IL-6 plays an
important role in inflammation through the activation of C-reactive protein, serum
amyloid A protein and fibrinogen61.
1.7 Negative Feedback of FcɛRI Signaling
There are negative feedback systems in place that serve to limit the degranulation
response in mast cells. The classical example of FcεRI inhibitory signaling involves the
antigen initiated co-clustering of the FcεRI with FcγRII. FcγRII is an inhibitory IgG
receptor that is expressed in a wide variety of immune cells and has been shown to play
an important role in suppressing immune receptor function. FcγRII contains
immunoreceptor tyrosine-based inhibitory motifs (ITIM), which serve to recruit negative
regulators of immune receptor signaling62. Co-aggregation of FcεRI and FcγRII via IgE
and IgG molecules that recognize the same antigen results in Lyn-mediated
phosphorylation of the ITIMs present on FcγRIIB, which leads to the recruitment of
16
SHP1, SHP2 and SHIP1, which serve to inhibit FcεRI signaling63. IgG mediated FcγRIIB
suppression of FcεRI signaling has been shown to inhibit in vivo IgE initiated
anaphylaxis through antigen clearance, preventing antigen binding to IgE molecules and
co-clustering of FcγRIIB and FcεRI64,65,66.
Another important negative feedback loop involves the inhibitory C-terminal
phosphorylation of SFKs via C-terminal Src kinase (Csk)67,68,69,70. Recruitment of Csk
occurs through its SH2 domain-dependent association with a tyrosine residue present on
Csk binding protein (Cbp) that is phosphorylated by Lyn upon FcεRI receptor activation
(Y317 in humans, Y314 in mice)68. Upon recruitment, Csk phosphorylates the C-terminal
tyrosine residues present in the SFKs involved in the signaling cascade, including both
Lyn71 and Fyn70. Phosphorylation on this site reduces kinase activity through mediating
an autoinhibitory intramolecular association between this tyrosine reside and the kinase’s
SH2 domain (Figure 3)72. Thus, Lyn is involved in a negative feedback loop which
results in its own inactivation, along with the inactivation of Fyn71,73. This inactivation is
crucial to the cessation of FcεRI signaling events, as can be seen by the
hyperdegranulation response displayed by Lyn deficient mast cells73. This phenotype is
attributed primarily to the lack of negative feedback provided by this Lyn dependent
pathway73.
An additional negative feedback system involves the platelet-endothelial cell
adhesion
molecule
1
(Pecam-1).
Pecam-1
knock-out
mast
cells
show
a
hyperdegranulation phenotype as a result of unregulated FcεRI mediated signaling
events74,75. A 2002 paper by Dr. Craig and Dr. Greer published data indicating that Fer
17
CSK
PTPs
(Eg. CD45)
Figure 3. Negative Regulation of SFKs by Csk.
Csk-mediated phosphorylation of the C-terminal tyrosine residue of SFKs mediates an
autoinhibitory association with the SH2 Domain, reducing catalytic activity. This Cterminal phosphorylation is antagonized by PTPs, including CD45. Adapted from Ref.
72.
18
and Fps/Fes (Fps/Fer) tyrosine kinases are phosphorylated in response to FcεRI receptor
activation76. A further article published by Udell et al in 2006 shows that the
phosphorylation of Fps/Fer as well as Y685 of Pecam-1 is Lyn-dependent75. Y685 is
necessary for the recruitment of both SHP1 and SHP2, which are initially inactive but are
proposed to become activated upon Fps/Fer phosphorylation of Pecam-1’s C-terminal
ITIM, Y66275. There is evidence that the dual SH2 domains present in SHP1 and SHP2
bind both Y662 and Y685, which allows their phosphatase domains to act on substrates75.
The action of these protein tyrosine phosphatases (PTPs) is proposed to down-regulate
FcεRI mediated signaling events via the dephosphorylation of proteins involved in the
signaling cascade, although the exact substrates involved are currently unknown76.
Pecam-1 could also serve a role in transcriptional regulation in response to FcεRI
signaling; this regulation could occur through Fps/Fer mediated phosphorylation of
Pecam-1 Y70075. The phosphorylated Y700 residue may then serve as a recruitment site
for either Signal Transducer and Activator of Transcription-3 (Stat-3) or Stat5; Stat5 has
been shown to be recruited to this residue77 and both of these molecules have been shown
to be activated downstream of Fer/Fps in mature monocytes78. This Stat3/5 protein
activation could then initiate transcription of target genes78.
Non-T cell activation linker (NTAL) is a transmembrane adapter protein that is
phosphorylated transiently downstream of FcεRI in HuMCs and BMMCs79 and has been
shown to play a significant negative role in FcεRI signaling80,81. NTAL is closely related
to LAT, with the major difference between the two adapter molecules being that in
contrast to LAT, NTAL cannot directly bind PLCγ and additionally, it contains an
19
inhibitory sequence in the N-terminus82. NTAL can be directly phosphorylated by two of
the major FcεRI effector kinases, Lyn and Syk83 and has a predicted SHP1 recruitment
site84. Studies have investigated the affects of NTAL deficiency in BMMCs and have
showed that these cells display: hyperdegranulation; hyperphosphorylation of LAT;
elevated activities of PI3K, SHP2 and Erk; elevated activation of PLCγ1 and PLCγ2
along with a corresponding increase in calcium mobilization; and increased production of
cytokines, including IL-2, IL-3, IL-4, IL-6, MIP-1α, TNFα80,81. Conversely, a study in
which NTAL was overexpressed in a Rat Basophilic Leukemia (RBL) cell line displayed
decreased phosphorylation of the β and γ FcεRI subunits, Syk and LAT. In addition, the
study showed that NTAL appears to compete with LAT for binding of Grb2 within the
FcεRI pathway and that NTAL recruitment of Grb2 reduces formation of active Grb2PI3K complexes85. Finally, a recent study indicates a positive role for NTAL in FcεRI
evoked signaling through the promotion of Akt activation by downregulating SHIP-1
recruitment to LAT86.
1.8 Kit Receptor Activation Potentiates FcεRI Signaling
Since mast cells respond to multiple signals simultaneously in vivo, increasing
evidence has emerged for cross-talk between key signaling pathways. Kit receptor
activation has been shown to cross-talk with the FcεRI signaling cascade, acting to
enhance the FcεRI mediated release of histamine and LTC487 through synergistic
activation of PLCγ, the MAPKs, and Akt88. Furthermore, NTAL phosphorylation is
20
potentiated by concurrent Kit and FcεRI activation, indicating that NTAL may play a role
in the receptor crosstalk mechanism79.
1.9 Mast Cell Dysregulation and Disease
Mast cells have been implicated in the development of many immune disorders,
including allergy, asthma and Multiple Sclerosis (MS). In allergic hypersensitivity
reactions, dysregulation of the levels of T helper (Th) 2 cells produced in response to
allergen stimulation results in the hypersecretion of IgE by B cells89. This elevated level
of circulating IgE causes an increased rate of mast cell activation upon subsequent
exposure to the original antigen, which results in an exaggerated mast cell FcεRImediated inflammatory response89.
Mast cells release PGD2, LTC4 and histamine; these mediators produce many of
the symptoms seen in asthma, including mucous secretion, edema of mucosal tissues, and
bronchoconstriction90. Mast cells serve to promote inflammation through the release of
pro-inflammatory mediators, including IL-4, IL-5 and IL-1390,91. These mediators can
promote both eosinophil mediated inflammation and IgE synthesis, while other mediators
secreted by mast cells can promote fibroblast activity91. Furthermore, asthmatics show
indications of chronic activation of these pathways as well as abnormal accumulation of
mast cells in airway smooth muscle tissue90.
Finally, mast cells have been shown to play a role in the pathogenesis of MS22. A
pair of studies have shown high levels of histamine92 and tryptase93 in the cerebral spinal
fluid (CSF) of patients with MS, while other studies have shown that mast cells
accumulate in MS plaques94. Mast cell degranulation has been shown to be associated
21
with increased permeability of the blood brain barrier (BBB) that occurs in the early
stages of MS95, and mast cells degranulate in response to myelin basic protein (MBP)96.
In vitro studies have determined that some of the proteases released by mast cells in
response to this FcεRI receptor activation can directly lead to local demyelination, which
is the primary cause of the symptoms associated with MS96.
1.10 The Non-Receptor Protein Tyrosine Phosphatase, SHP2
My project focuses on the role of the non-receptor PTP SHP2 in FcεRI mediated
mast cell activation. SHP2 is encoded by the ptpn11/shp2 gene and is ubiquitously
expressed97. In addition, SHP2 is known to be required for hematopoiesis and embryonic
development as well as the intracellular response to cytokines and growth factors98,99.
Over the last number of years, SHP2 has been identified as a proto-oncogene that is
implicated in many cancer types100; activating mutations have been found in both colon
and lung cancers, leukemias, melanomas, and neuroblastomas101.
SHP2 is composed of three domains: an N-terminal domain composed of tandem
SH2 domains, a central PTP domain, and a C-terminal tail containing two sites of
tyrosine phosphorylation98. In resting cells, SHP2 activity is restricted via the N-terminal
SH2 domain binding to the catalytic cleft of the phosphatase domain, resulting in
autoinhibition of catalytic activity (Figure 4)98. In order for SHP2 to become activated,
this interaction must be disrupted97. There are two models of SHP2 activation; in the first
model, the tandem SH2 domains present in SHP2 bind to another protein containing two
phosphorylated tyrosine residues – this binding prevents the autoinhibitory conformation,
allowing substrate access to the phosphatase domain’s catalytic cleft97. The second model
22
A
Inactive
Active
B
Point Mutations Lead to
Constitutive SHP2 PTP Activity
Normal Individual
(Inactive)
Noonan Syndrome:
Partial Activation
JMML:
Consitutive Activation
Figure 4. SHP2 Structure and Autoinhibition of Phosphatase Activity.
SHP2 is composed of tandem N-terminal SH2 domains, a phosphatase domain and a pair
of C-terminal tyrosine residues. A. The inactive SHP2 conformation occurs as a result of
an association between the N-terminal SH2 domain and the phosphatase domain catalytic
cleft; the association prevents substrate access and represses phosphatase activity. SHP2
is proposed to become activated by the SH2 domains binding to either phosphorylated
binding partners or to the N-terminal tyrosine residues of SHP2 upon phosphorylation.
SH2 domain binding to phosphorylated tyrosine residues disrupts the interaction between
the N-terminal SH2 domain and the phosphatase domain, thus allowing the phosphatase
domain to access and dephosphorylate substrates. B. Activating mutations associated
with pathological conditions cause a level of constitutive SHP2 activation as a result of
disrupting the association between the N-terminal SH2 domain and the PTP domain.
Moderately activating mutations are associated with the development of Noonan
Syndrome, while severe activating mutations result in oncogensis. Adapted from Ref. 97.
23
proposes that the dual SH2 domains bind intramolecularly to the C-terminal tyrosine
residues once they become phosphorylated – once again, this is proposed to dissociate the
N-terminal SH2 domain from the catalytic cleft of the phosphatase domain97. A number
of disease associated point mutations have been identified that result in varying degrees
of reduced autoinhibition and thus a level of SHP2 activation (Figure 4)98. For instance,
the D61Y and E76K mutations are known to stimulate phosphatase activity by reducing
the affinity of the N-terminal SH2 domain to bind to the PTP domain, thus relieving
autoinhibition98,97.
A 2004 paper published by Loh et al showed that activating mutations in SHP2
were also frequently found in Juvenile Myelomonocytic Leukemia (JMML)102.
Furthermore, SHP2 is a known upstream activator of Ras and Erk, and is therefore able to
promote cellular growth and proliferation101,103. Mutations that result in either increased
Ras or SHP2 activation have been shown to be mutually exclusive in cancers, indicating
a redundancy of function in growth signal promotion102. Unregulated SHP2 activity has
been shown to result in hypersensitivity to growth factors, further supporting the role of
SHP2 as a growth promoter103. Finally, there have been many papers published that show
a link between the upregulation of SHP2 activity and the generation of Human Adult
Leukemia104. Also, the proliferative ability of these leukemic cells was shown to be
proportional to the level of SHP2 protein activity104. Therefore, appropriate SHP2
regulation is critical for the prevention of oncogenesis. Finally, SHP2 activating
mutations have been identified in roughly fifty percent of patients with Noonan
Syndrome, which is a disease characterized by cardiac defects, facial dysmorphia, and
24
short stature; these mutations typically result in a lower level of SHP2 activation than do
the oncogenic variants105,98.
A 2006 study investigated the role of SHP2 in embryogenesis106. The group
showed that shp2 null mouse embryos die peri-implantation106. Although the expected
number of shp2 null blastocysts were generated when shp2+/- mice were bred, very few
shp2-/- embryos were found post-implantation106. Additionally, the implanted embryos
were observed to be highly necrotic and lacked appropriate tissue differentiation106. The
study concluded that SHP2 plays a role in Trophoblast Stem (TS) cells in promoting
Src/Ras/Erk activation downstream of the fibroblast growth factor-4 (FGF4)106. In the
absence of SHP2, Erk signaling is defective, which causes the stabilization of the proapoptotic protein Bim and subsequent apoptosis of the TS cells106. TS cells are essential
for forming cells of the trophoblast lineage in the developing fetus; thus, shp2
inactivation confers embryonic lethality106.
1.11 SHP1 is a Negative Regulator of Mast Cell Degranulation and
Inflammation
SHP2 is closely related to another non-receptor PTP, SHP1 (PTPN6), that shares
a conserved domain organization107. Like SHP2, SHP1 is also autoinhibited via an
intramolecular association between N-terminal SH2 domain and PTP domain108. SHP1
plays a role in regulating hematopoiesis109, IL-3 signaling110,111, and is a key negative
regulator of the immune response. However, while SHP2 expression is ubiquitous, SHP1
is expressed primarily in hematopoietic cells112,113,114,115. Another key difference between
25
SHP1 and SHP2 is that while many studies have shown a positive role for SHP2 in
signaling pathways, SHP1 is primarily a negative regulator of cell signaling116.
The difference in SHP1 and SHP2’s roles in signaling pathways are the result of
differential substrate specificity. This substrate specificity has been shown to be reliant
upon intrinsic differences in their PTP domains as opposed to a difference in SH2 domain
mediated localization and association with phosphorylated binding partners117,118.
There have been studies that have investigated the sites of SHP1 recruitment
within the FcεRI pathway, and others that have identified regulatory roles for SHP1
within the pathway. SHP1 has been shown to bind to the β119 and γ chain of the FcεRI44,
Pecam-175, and there is also a predicted binding site contained within NTAL84. In
addition, overexpression of wild type SHP1 in a RBL cell line was shown to have no
affect on degranulation, but decreased the phosphorylation of Syk and of the β and γ
chains of the FcεRI120. Conversely, overexpression of a dominant negative trapping
mutant version of SHP1 produced the opposite affects on the phosphorylation status of
these proteins120. Conversely, the same study found that SHP1 overexpression increases
JNK activation and TNFα production120. Finally, another study identified a negative
regulatory role for SHP1 in the FcεRI evoked production of the cytokines IL-6 and IL-13
in BMMCs derived from SHP1-/+ mice and hyperinflammatory responses in vivo121.
Overall, SHP1 appears to play a predominantly negative role in regulating FcεRI
signaling, although the molecular mechanism is not well defined.
26
1.12 SHP2 Recruitment Sites Within the FcεRI Pathway
SHP2 has been shown to be recruited to a variety of signaling proteins within the
FcεRI signaling cascade, including the FcεRI β chain39, Pecam-175, and Gab2111, while
NTAL has been shown to modulate SHP2 actvity80.
In one study, recombinant β chain peptides were shown to be able to isolate SHP2
from whole cell lysates, indicating that the β chain of the FcεRI may play a role in
recruiting SHP2 upon receptor activation39.
The ITIMs Y662 and Y685 on Pecam-1 have been shown to play roles in the
recruitment of both SHP1 and SHP275. Pecam-1 knock-out mast cells display a
hyperdegranulation phenotype, which implies that Pecam-1 recruitment of SHP1/2 could
promote the downregulation of FcεRI signaling through dephosphorylation of pathwayassociated proteins74.
SHP2 has been shown to bind Gab2 in a wide variety of signaling contexts. A
2006 study showed that SHP2 binds Gab2 and plays a role in promoting signaling
downstream of the Kit receptor in BMMCs122, while another study indicated that SHP2
associates with Gab2 downstream of the IL-3 receptor, which is also present on
BMMCs111.
The 2004 NTAL study by Volná et al provided data which indicates that NTAL
inhibits SHP2 activity. In NTAL-deficient BMMCs, SHP2 immunoprecipitates had
increased catalytic activity when compared to those isolated from WT cells, as measured
by in-gel phosphatase assays. The mechanistic basis for this observation has not been
established.
27
1.13 Currently Identified Roles for SHP2 in Signal Regulation
SHP2 is a known positive regulator of both SFK activity, as well as Akt and Erk
activation. In addition, several known SHP2 substrates are signaling molecules involved
in the FcεRI pathway, including Syk kinase123, the SFK regulatory protein Cbp124, the
Akt activating adapter protein Gab2111, and p190-B Rho GTPase activating protein
(RhoGAP)125. Additionally, SHP2 plays a role in promoting Erk signaling downstream of
the Epidermal Growth Factor Receptor (EGFR)126. Understanding the roles of SHP2 in
signaling regulation in other pathways may provide insight into possible roles for SHP2
in the FcεRI signaling cascade.
Shp2 has been shown to be able to decrease the FcεRI evoked phosphorylation of
Syk kinase under in vitro conditions123. The study implicates SHP2 in mediating
inhibition of FcεRI signaling upon treatment of RBL cells with mast cell functionassociated antigen (MAFA), which is a glycoprotein that suppresses FcεRI signaling in a
dose dependent manner123. This SHP2 mediated inhibition is proposed to be a result of
MAFA induced co-clustering of SHP2 and Syk, with subsequent dephosphorylation of
Syk kinase123.
A 2004 paper by Zhang et al shows that SHP2 promotes SFK activity by
dephosphorylating the tyrosine residue present on Cbp that recruits Csk124. This
dephosphorylation prevents the inhibitory Csk kinase activity on SFKs proximal to the
FcεRI receptor124. This study shows that SHP2 promotes activation of SFKs and
subsequent Erk signaling downstream of EGFR activation in Mouse Embryonic
Fibroblasts (MEFs)124. Another study also implicates SHP2 in promoting SFK activity
28
and the integrin-mediated activation of NFAT and subsequent myofibril formation and
skeletal muscle growth127.
Additionally, SHP2 has been shown to both bind and dephosphorylate the Gab2
adapter protein111. In the EGFR pathway, SHP2 has been shown to regulate the
phosphorylation of the Y472 on the closely related Gab1128,129. In a similar manner, Gab2
phosphorylation causes the recruitment of the p85 regulatory subunit of PI3K130; tyrosine
residue Y452 (in human Gab2; Y441 in mouse Gab2) on Gab2 is homologous to the
PI3K recruitment site on Gab1. In the context of the EGFR pathway, reduced SHP2
activity results in hyperphosphorylation of Y472 on Gab1, which causes an increase in
Akt activation128,129. However, in contrast to this result in the EGFR pathway, it was also
reported that in some signaling pathways, including the Platelet Derived Growth Factor
Receptor (PDGFR) signaling cascade, SHP2 null cells display the opposite affect and
show reduced Akt activation128,129.
Furthermore, it has been shown that SHP2 dephosphorylates p190-B RhoGAP125,
which has been shown to be phosphorylated downstream of the FcεRI34.
Dephosphorylation of
p190-B RhoGAP inhibits RhoGAP activity and results in
activation of RhoA125. This study shows that SHP2 plays a positive role in myogenesis
through dephosphorylation of p190-B RhoGAP125.
As shown in many studies of the oncogenic effects of SHP2 activation, SHP2
promotes the activation of the Erk MAPK pathway. In the EGFR pathway, the
recruitment of RasGAP to the signaling complex acts in a negative feedback role to
inactivate Ras and shut off Erk signaling. SHP2 has been shown to promote Erk
29
activation through dephosphorylation of RasGAP recruitment sites on both the EGFR131
and on Gab1132. Dephosphorylation of these sites reduces the recruitment of an inhibitor
of Erk signaling, therefore resulting in signaling propagation. The major effect of Erk
activation in many cell types is the promotion of cell survival and proliferation; however,
antigen-mediated FcεRI aggregation and activation does not induce mast cell survival
responses133. In addition to its proliferative effects, Erk is involved in regulation of gene
transcription134.
1.14 SHP2 Trapping Assays
Phosphatase substrate trapping mutants are phosphatases that are mutated in their
phophatase domain. Substrate trapping constructs are typically composed of either the
full length phosphatase or of the phosphatase domain in addition to a subset of the
proteins’ regulatory domains. The constructs can be used to generate recombinant
proteins for in vitro use, or they can be transfected into cell lines to analyze their in vivo
associations and affects135. The mutations that confer trapping activity are designed in
such a way as to prevent the successful completion of the phosphatase’s catalytic activity,
and cause the irreversible association between the phosphatase and substrate135.
Importantly, the substrate specificity of the phosphatase is unaltered. There are a variety
of phosphatase substrate trapping variants, including a variety of SHP2 variants135.
Among the SHP2 variants hat have been described are the dual mutation containing
variants, the CS/DA136 and the DA/QA125.
The CS/DA trapping mutant contains two amino acid substitutions that generate
trapping action136. The first involves a cysteine to serine mutation137,138,139. In the
30
functional protein, this cysteine residue acts as a nucleophile, attacking the phosphorus
atom in the phosphate group attached to the substrate137. This attack is a central step in
the process of dephosphorylation, and the replacement of the cysteine with a serine
residue removes the negative charge at this position, preventing nucleophilic attack on
the phosphate137. The prevention of the attack results in the creation of a catalytically
dead phosphatase domain137,138,139. The DA mutation within the Phosphatase domain is an
aspartate to alanine mutation that is located within a WPD loop that is involved in
holding the substrate in place within the catalytic cleft140,141. The alanine residue changes
the conformation of the loop such that it permanently inhibits release of the substrate135.
The DA/QA trapping mutant has been shown to have a higher affinity for
substrate binding than the CS/DA variant and also contains a pair of trapping
mutations142. The DA component consists of the same aspartate to alanine mutation in the
WPD loop that is contained within the CS/DA mutant135. The QA mutation is distinct and
involves the substitution of a glutamine residue for an alanine residue142. This glutamine
residue is involved in phosphatase catalysis by stabilizing a water molecule that attacks a
Cys-PO3 intermediate structure during enzyme activity142. Mutation of this residue to
alanine prevents this activity and promotes the stabilization of the substrate in the
catalytic cleft142.
1.15 Conditional SHP2 Knock-out Mouse Models and Mast Cells
Mice that are SHP2 deficient die in utero because SHP2 is required for TS cell
survival and embryo implantation99. Alternative approaches have been developed to
allow for the study of genes that cause embryonic lethality when inactivated. The LoxP
31
system involves site directed excision of a section of DNA that is flanked by two
identical LoxP sites143. These sites are composed of 34 base pair (bp) sequences
containing a central 8 bp sequence flanked on each side by a 13 bp palindromic
sequence143. LoxP sites are recognized by Cre recombinase, which was originally
obtained from bacteriophage P1143. Upon recognition, Cre subsequently excises the DNA
located between the two LoxP sites (“floxed” or fl allele)143. A transgenic mouse line has
been established by Dr. Feng and coworkers (The Burnham Institute) that contains LoxP
sites on either side of exon 4 located within the coding segment of SHP2144. These
modifications are designed to introduce a premature stop codon into the resulting mRNA
sequence, causing destabilization of the mRNA and thus preventing the production of
functional SHP2 protein products144.
Several studies have reported the utilization of shp2 floxed mice in order to study
the affects of tissue specific shp2 inactivation. This is accomplished by breeding the mice
containing a floxed shp2 allele (shp2fl) with transgenic mouse lines that express Cre
recombinase in a tissue specific manner. This approach has been validated in several
studies. Through the utilization of a neuronal SHP2 knock-out (KO) model, SHP2 was
shown to play a role in energy balance and metabolism144 and neuronal SHP2 KO is
associated with the onset of diabesity, which is a condition that is the combination of
diabetes and obesity145. Another study using a liver specific SHP2 KO found that SHP2
inactivation in the liver causes a reduction in liver regeneration following partial
hepatectomy; this reduced regenerative ability is associated with a defect in Erk
activation146.
32
One study described the use of a mouse line147 that contains an ubiquitously
expressed form of the Cre recombinase that is linked to a modified estrogen receptor
(ER*, CreER*)148 in order to inactivate SHP2 in cell culture in BMMCs122. This CreER*
construct represents a ligand activated form of the Cre recombinase; in the absence of
ER* ligand, the CreER* fusion protein binds to Heat Shock Protein 90 (Hsp90) and is
sequestered in the cytoplasm149. The modified ER* binds the compound 4hydroxytamoxifen (4OH-TM); upon binding 4OH-TM, Hsp90 dissociates from ER*,
allowing the translocation of Cre to the nucleus147. This nuclear localization initiates
Cre’s function of site-directed recombination between any LoxP sites present in the cell
genome147. The study that utilized this experimental approach to inactivate shp2 in
BMMCs showed that SHP2 plays a role in promoting Kit receptor signaling and
proliferation in mast cells147. However, no results were reported for the role of SHP2 in
the FcεRI signaling cascade; that topic is the focus of this thesis.
1.16 Study Rationale, Hypothesis and Objectives
The rationale for defining the role of SHP2 in FcεRI signaling and cytokine
production in mast cells is that several reports have documented SHP2 recruitment to
downstream effectors of FcεRI in mast cells39,75,111; thus, SHP2 is likely regulating FcεRI
signaling, but to date no function has been ascribed to SHP2. Since SHP2 is a PTP and it
is recruited to signaling structures within the FcεRI signaling cascade, we hypothesize
that following recruitment to the cascade, SHP2 regulates FcεRI-triggered mast cell
responses via dephosphorylation of specific signaling proteins within the pathway.
33
Our first objective for this study was to define the differences between the FcεRItriggered signaling and cellular responses in WT and in SHP2-deficient BMMCs. Our
second objective was to then identify the molecular mechanism for the observed defects
in the FcεRI-evoked signaling and cellular responses.
34
Chapter 2
Materials and Methods
2.1 Antibodies
Antibodies kindly provided by researchers from other institutions include: Rabbit
anti-phospho-CBP serum (Y317; as described150, kindly provided by Dr. Schraven and
Dr. Lindquist - Institute of Immunology, Otto-von-Guericke University, Magdeburg,
Germany); anti-SHIP-1 (rabbit serum; Provided by Jerald Krystal; University of British
Columbia); anti-Syk and anti-Lyn (rabbit serums; Joan Brugge; Harvard University);
anti-FcεRI β subunit (mouse mAb; Juan Rivera; National Institutes of Health).
Antibodies obtained from Santa Cruz Biotechnology Inc. include: anti-SHP2
(rabbit pAb; sc-280), anti-phospho-tyrosine pY99 (pY; mouse mAb; sc-7020); anti-Akt
1/2 (rabbit pAb; sc-8312); anti-Erk 1 (rabbit pAb; sc-94); anti-phospho-ERK (Y204;
mouse mAb; sc-7383); anti-Fyn (mouse mAb; sc-434); anti-PLCγ1 (rabbit pAb; sc-81);
anti-PLCγ2 (rabbit pAb; sc-407). In addition, our group procured the following
antibodies from Cell Signaling Technology: anti-phospho-Src Y416 (Y416; rabbit pAb;
2101); anti-phospho-Src Y527 (Y527; rabbit pAb; 2105); anti-phospho-Gab2 (Y452;
rabbit pAb; 3882); anti-phospho-AKT (S473; rabbit mAb; 4058); anti-p38 (rabbit pAb;
9212);
anti-phospho-p38
(T180/Y182;
rabbit
pAb;
9211);
anti-phospho-JNK
(T183/Y185; mouse mAb; 9255); anti-phospho-PLCγ1 (Y783; rabbit pAb; 2821); antiphospho-PLCγ2 (Y1217; rabbit pAb; 3871); anti-phospho-Syk (Y352; rabbit pAb; 2701);
35
anti-phospho-SHIP1 (Y1020; rabbit pAb; 3941); anti-phospho-IKKα/IKKβ (α:S180,
β:S181; rabbit pAb; 2681); anti-IKKα (rabbit pAb; 2682). Finally, we obtained antiGab2 (rabbit pAb; 06-967) from Upstate Biotechnology, anti-phospho-Lyn (Y507; rabbit
mAb; clone EP504Y) from Epitomics, and anti-α tubulin from Sigma-Aldrich (mouse
mAb; T 6074).
2.2 Transgenic Mouse Lines
The exon 4, LoxP-targeted shp2fl transgenic mouse line was kindly provided by
Dr. Gen-Sheng Feng (The Burnham Institute) and was previously described144. The
TgCreER* transgenic mouse line was previously described147 and was procured from The
Jackson Laboratory. The shp2fl/fl and the TgCreER* transgenic mouse lines were interbred
at Queen’s Animal Care Services to generate a dual transgenic, shp2fl/fl TgCreER* mouse
line. All mouse studies were approved by the Queen’s University Animal Care
Committee.
2.3 BMMC Cultures
BMMC cultures were generated in a manner similar to what has been previously
described75. Femurs were isolated from shp2fl/fl; TgCreER* or shp2fl/fl mice, flushed with
BMMC growth media (Iscove’s modified Dulbecco’s medium, 10% (v/v) fetal bovine
serum, 1% (v/v) antimicrobial-antimycotic solution (Invitrogen), 1 mM sodium pyruvate
(Invitrogen), 1% (v/v) nonessential amino acids (Invitrogen), 5% (v/v) conditioned
medium from X63-IL-3 cells, 5% (v/v) conditioned medium from HEK293-SCF cells
(prepared in the Craig lab by C. Udell), and 50 μM α-monothioylglycolate (Sigma)) and
36
bone marrow cells were cultured for 3/4 weeks to generate BMMC cultures.
Homogeneity of BMMC cultures and cell maturity was confirmed by sensitizing the
BMMCs with 20% volume anti-DNP IgE conditioned media (SPE7) and detected using
anti-IgE-fluorescein isothiocyanate (FITC; Southern Biotechnology Associates, Inc.) and
anti-Kit-phycoerythrin (PE; Caltag Labs), or with isotype controls: rat anti-IgG1-FITC
(Caltag Labs) and rat anti-IgG2b-phycoerythrin (Caltag Labs) to stain the cells.
Subsequently, the cells were analyzed by flow cytometry (by use of an EPICS Altra HSS;
operated by the Queen’s University Cancer Resarch Institute) in order to ensure that the
BMMC cultures were composed of >90% FcεRI+ve/Kit+ve cells.
2.4 BMMC SHP2 Inactivation and FcεRI Stimulations
BMMCs were subjected to treatment with 4-hydroxy-tamoxifen (4OH-TM) in
order to activate the CreER* protein and then inactivate the shp2 allele in the shp2fl/fl;
TgCreER* cells. Concurrently, an additional 10% (v/v) IL-3 and SCF conditioned media
was added to the culture medium. Cells were then cultured for a further 3 days in order to
allow for SHP2 protein downregulation prior to use in experimental procedures.
In order to stimulate the FcεRI, the BMMCs (approx. 107 cells/timepoint) were
then starved and sensitized with 20% (v/v) IgE conditioned media (SPE7) for 6 hours,
washed in Tyrodes buffer (10mM HEPES, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM
CaCl, 1mM MgCl, 5.6 mM glucose, 0.1% bovine serum albumin) and stimulated (11.5x106 cells/mL) with either vehicle control or 100ng/mL DNP-HSA (40 mol DNP/mol
HSA; Sigma) in Tyrodes at 37°C for the times indicated in the figures.
37
Soluble cell lysates (SCLs) were then generated from the stimulated BMMCs as
follows. Cells were rinsed in ice-cold phosphate buffered saline (PBS; 137 mM NaCl, 10
mM phosphate, 2.7 mM KCl, and a pH of 7.4) containing sodium orthovanadate, and
then lysed in kinase lysis buffer (KLB; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 10 μg of aprotinin/ml,
10 μg of leupeptin/ml, 1 mM vanadate, 100 μM phenylmethylsulfonyl fluoride). The
lysates were then centrifuged at 12000 rpm and the soluble material was separated from
the insoluble pellet to generate the SCL.
2.5 Cytokine ELISAs
BMMCs were 4OH-TM treated for 3 days in order to generate the appropriate
SHP2 protein expression levels, as described above. Subsequently, BMMCs (5x106 cells
per sample; 2x106 cells/mL) were starved and sensitized with anti-TNP IgE (1 μg/ml; BD
Biosciences) for 6 hours and then stimulated (2x106 cells/mL) with either vehicle control
or 10ng/mL DNP-HSA for either 2 (LTC4) or 6 hours (TNFα, IL-6) at 37°C. Cells were
then pelleted and supernatants were collected. Cell supernatants were analyzed for TNFα,
IL-6 or LTC4 concentration via the use of commercial ELISA kits (IL-6: BD Bioscience;
TNFα: eBioscience; LTC4: Cayman Chemical), according to manufacturer's instructions.
2.6 Degranulation Assays
BMMCs were 4OH-TM treated for 3 days in order to generate the appropriate
SHP2 protein expression levels, as described above. The BMMCs (105 cells/sample)
were then starved and sensitized with anti-TNP IgE (1 μg/ml; BD Biosciences) for 6
38
hours, washed with warm Tyrodes buffer and then stimulated for 1 hour (106 cells/mL)
in Tyrodes buffer supplemented with vehicle control, 10ng/mL DNP-HSA, or 1μM
calcium ionophore at 37°C. Cells were pelleted, supernatants were collected and cell
pellets were lysed in Tyrodes buffer supplemented with 0.5% Triton X100 to generate
whole cell lysates. All samples (supernatants and pellet samples) were analyzed for the
concentration of a marker of degranulation, β-hexosaminidase, via a colorimetric assay
described previously151.
2.7 Immunoprecipitation and Immunoblots Analysis
SCLs were generated as described above, were subjected to Immunoprecipitations
(IPs) using the indicated antibodies and GammaBind Sepharose (GE Healthcare)
overnight at 4°C. The IPs were then centrifuged at 12000 rpm and washed 3 times with
KLB, followed by the addition of SDS sample buffer.
IPs or SCL samples (SCL with SDS sample buffer) were resolved by SDS-PAGE
and then electrophoretically transferred to Polyvinylidene Difluoride (PVDF) Immobilon
P (Millipore) membranes using a Trans-Blot SD semidry transfer apparatus (BIO-RAD).
Immunoblot analysis was then conducted by incubation of the PVDF membranes in
antibodies diluted in either 5% (w/w) non-fat milk power or bovine serum albumin in
Tris-buffered saline with tween 20 (TBST) overnight at 4°C. The membranes were then
washed in TBST and subsequently probed with the appropriate horseradish peroxidase
(HRP) conjugated donkey anti-rabbit IgG (GE Healthcare) or sheep anti-mouse Ig (GE
39
Healthcare). The membranes were washed again in TBST and then visualized with
chemiluminescence reagent (Applied Biological Materials Inc.) and x-ray film (Fujifilm).
2.8 Densitometry and Statistical Analysis
Densitometry was carried out on autorads with non-saturated signals, which were
scanned and analyzed using Corel Photo-Paint. Relative levels of phosphorylationspecific signal over total loading control signal were calculated using Excel.
Statistical significance was defined as p ≤ 0.05 using student’s T-test.
40
Chapter 3
Results
3.1 A Transgenic Mouse Model Allowing for Temporal Inactivation of shp2
in BMMCs
In order to study the function of SHP2 in mast cell FcεRI signalling, we obtained
a LoxP-targeted shp2 (shp2fl) mouse line from Dr. Gen-Sheng Feng (The Burham
Institute, in La Jolla, California) which has been described previously144. These mice
were interbred with a transgenic mouse line that ubiquitously expresses a CMV/β actin
promoter-driven Cre recombinase-modified estrogen receptor fusion (CreER*) protein in
all tissues of the transgenic animals147. These two strains were interbred to yield mice
homozygous for the shp2fl allele (shp2fl/fl), and either hemizygous or negative for the
CreER* transgene (Figure 5A; shp2fl/fl; TgCreER and shp2fl/fl). The genotypes of the mice
were verified via PCR analysis as previously described144,147.
Femurs were isolated from littermate shp2fl/fl; TgCreER and the shp2fl/fl mice and
the bone marrow was flushed into BMMC culture medium containing IL-3 and SCF.
Bone marrow cells were cultured for 3 weeks in order to generate homogenous BMMC
cultures of the corresponding genotype (Figure 5B). In order to inactivate the shp2fl
alleles in the shp2fl/fl, TgCreER BMMC cultures, and to provide an appropriate control
experimental group via the use of the shp2fl/fl cultures, 100nM 4OH-TM was
administered to a subset of the BMMCs from cultures of both genotypes for 3 days.
41
A
shp2fl/fl
TgCreER*
X
Exon 4
5’
3’
Cre Mediates Exon 4 Excision
5’
3’
CMV/β Actin Promoter Drives Cre
ER*
Expression
B
“WT”
shp2
shp2fl/fl
shp2fl/fl;TgCreE
BMMC Cultures
(IL3/SCF; >3
Weeks); SHP2
Protein Expression
Intact
4OH-TM
Administered
(100nM, 3
Days)
fl
Allele Intact
SHP2 Protein Level s
Normal
“KO”
BMMCs
Starved and
Sensitized 6Hrs
With Anti-DNP
IgE prior to
experiments
CreER* Mediated
shp2fl Exon4 Deletion
Reduced SHP2 Protein
Levels
Figure 5. A Transgenic Mouse Model Allowing for Temporal Inactivation of shp2 in
BMMCs.
A. We obtained a LoxP-targeted shp2 (shp2fl) mouse line from Dr. Gen-Sheng Feng.
These mice contain a modified shp2 allele which contains LoxP repeat sequences on
either side of exon 4; Cre-mediated excision results in the introduction of a premature
stop codon. These mice were then interbred with transgenic TgCreER* mice which have a
CMV/β actin promoter-driven Cre recombinase, modified estrogen receptor fusion
(CreER*) protein. B. Experimental flow chart. shp2fl/fl; TgCreER* and shp2fl/fl mice are
used to generate BMMC cultures. BMMCs are cultured for 3/4 weeks in the presence of
IL3 and SCF and are then subjected to treatment with 100nM 4OH-TM in order to
activate the CreER* construct, which inactivates the shp2fl allele in the shp2fl/fl; TgCreER*
cells. Cells are cultured for a further 3 days in order to allow for SHP2 protein depletion,
starved and sensitized with IgE and then used for experimental procedures.
42
4OH-TM administration to the BMMC culture results in the release of CreER* fusion
proteins from the cytoplasmic HSP90 chaperone proteins, which allows for nuclear
accumulation of the CreER* protein and subsequent excision of exon 4 within the shp2fl
alleles, which we observed by PCR (data not shown). At the end of the 3 day 4OH-TM
treatment, we observed an approximately 80% decrease in the SHP2 within soluble cell
lysates of the CreER* expressing BMMCs (Figure 6A). For simplicity, shp2fl/fl; TgCreER*
cells that have been 4OH-TM treated will be hereafter referred to as “SHP2 KO” cells
and 4OH-TM treated shp2fl/fl cells will be referred to as “SHP2 WT” cells.
SHP2 plays a key role in regulating myelopoiesis99 and has also been implicated
in regulating the IL-3111 and SCF122 pathways – the two main growth factor pathways that
are used to promote the growth and development of BMMC cultures in vitro. Thus, it was
important to ensure that the presence of the CreER* protein did not affect BMMC culture
maturation through “leaky” CreER* activation and subsequent partial or complete shp2fl
inactivation at any stage in BMMC development; or due to unexpected effects associated
with expression of the transgene. When BMMCs are mature, they express both the FcεRI
and the Kit receptor5,9 and the expression of both of these receptors is used in order to
verify the maturity of BMMC cultures75. Thus, 3 weeks after the initial generation of the
cultures, we measured the expression levels of the FcεRI and Kit receptors via flow
cytometry in both shp2fl/fl; TgCreER* and shp2fl/fl BMMC cultures (Figure 6B). Both
genotypes displayed similar maturity (≥ 90% FcεRI+ Kit+), with similar levels of
43
A
WT
KO
SCL
IB: SHP2
70
SCL
IB: Erk
43
1
2
1.0
0.18
Relative SHP2
B
SHP2fl/fl BMMCs
- 4OH-TM
Kit Expression
Kit Expression
+ 4OH-TM; SHP2 WT
FcεRI Expression
FcεRI Expression
SHP2fl/ fl TgCr eER* BMMCs
- 4OH-TM
Kit Expression
Kit Expression
+ 4OH-TM; SHP2 KO
FcεRI Expression
FcεRI Expression
Figure 6. Characterization of shp2 inactivation model.
SHP2fl/fl and SHP2fl/fl; TgCreER* BMMCs were treated with 100nM 4OH-TM, then cultured for 3
fl/fl
CreER*
days to generate SHP2 WT and KO BMMCs. A. SCLs were generated from shp2 ; Tg
and shp2fl/fl BMMCs treated with 100nM 4OH-TM and subjected to IB with anti-SHP2
and anti-Erk antibodies. SHP2 protein levels in WT and KO BMMC cultures are shown;
approximately 18% of SHP2 protein remains in the KO cells compared to SHP2 WT
cells. B. shp2fl/fl; TgCreER* and shp2fl/fl BMMCs both express comparable levels of FcεRI
and Kit receptor before and after 4OH-TM treatment as detected via flow cytometry.
44
expression of both receptors. This shows that the CreER* protein did not affect the
growth and development of the BMMC cultures (Figure 6B). It is also important to note
that the results were consistent when using either shp2fl/fl or shp2wt/wt; TgCreER* BMMCs
as a control in other experiments.
Furthermore, in order to ascertain the affects of both 4OH-TM treatment of the
cultures, and the potential effects SHP2 downregulation on the surface expression of
these receptors, we also measured the expression levels of the FcεRI and Kit receptors
using cytometry following 3 days of 100nM 4OH-TM treatment. 4OH-TM treatment did
not alter the surface expression of either of these two receptors in either WT or KO
BMMCs (Figure 6B). Thus, SHP2 downregulation does not alter the surface expression
of either the FcεRI or the Kit receptor. This is an important control experiment in order to
ensure that the signalling pathways downstream of the FcεRI are not affected due to the
expression level of the receptor itself, in SHP2 WT and KO BMMCs.
3.2 SHP2 is Not Required for Degranulation
One of the major affects of FcεRI engagement is degranulation, which involved
the coordinated activities of two parallel pathways. The calcium dependent pathway
involves a Syk/LAT/PLCγ signalling cascade47, which leads to calcium mobilization,
while the calcium independent pathway involves the activities of Fyn/Gab2/RhoA and is
responsible for granule translocation to the cell membrane45. SHP2 has previously been
shown to act on Syk123 and p190-B RhoGAP125, giving it potential regulatory roles in
45
both pathways. Thus, these potential roles for SHP2 action in regulating degranulaion led
us to analyze the affects of SHP2 KO on degranulation.
We first analyzed the activation profiles of PLCγ1 and PLCγ2. Following IgE
sensitization and DNP-triggered activation of the FcεRI, soluble cell lysates were
generated from SHP2 WT and KO cells stimulated for 0, 3, 9 and 27 minutes. IB analysis
was performed using anti-phospho-PLCγ1 and anti-phospho-PLCγ2 as well as control
anti-PLCγ1 (Y783) and anti-PLCγ2 (Y1217) antibodies; the phosphorylation of these
tyrosine residues correlates with activation152,153. PLCγ1 and PLCγ2 activation appeared
to be unaffected by SHP2 depletion (Figure 7A; compare lanes 1-4 with lanes 5-8).
In order investigate SHP2’s role in regulating the degranulation response, we
generated SHP2 WT and KO BMMCs as described above, starved and sensitized the
BMMCs with anti-TNP IgE for 6 hours, and then the stimulated cells with control
tyrodes buffer, tyrodes buffer containing 10ng DNP-HSA (antigen) or tyrodes buffer with
1μM calcium ionophore for 1 hour. Cells were pelleted and both the pellets and the
supernatants were analyzed for β-hexosaminidase content (as previously described151). βhexosaminidase release was shown to be similar between SHP2 KO and SHP2 WT
BMMCs in both control and antigen stimulated samples (Figure 7B). In addition, βhexosaminidase release upon administration of calcium ionophore (A23187), was similar
between SHP2 KO and SHP2 WT BMMCs. Calcium ionophore serves as a positive
control by stimulating FcεRI independent degranulation; this result indicated that the
granules contents within the SHP2 WT and KO BMMCs were unaffected by SHP2
46
WT
A
0
3
KO
9
27
0
3
9
27
IgE/Ag (min)
SCL
IB: pPLCγ1
130
SCL
IB: PLCγ1
130
1
2
3
4
5
6
7
8
SCL
IB: pPLCγ2
130
SCL
IB: PLCγ2
130
1
2
3
4
5
6
7
8
% Beta Hexosaminidase Release
B
100
90
80
70
60
50
40
30
20
10
0
WT
KO
Control
Antigen
Ionophore
Figure 7. SHP2 is Not Required for Degranulation.
A. SHP2 WT and KO BMMCs were starved and sensitized with 20% volume SPE7 IgE
conditioned media for 6 hours and stimulated with either tyrodes or 100ng/mL DNP-HSA
in tyrodes for the times indicated. SHP2 WT and SHP2 KO SCLs were generated, then
were resolved by SDS-PAGE and subjected to IB analysis was performed using antiphospho-PLCγ1 and anti-phospho-PLCγ2 as well as control anti-PLCγ1 and anti-PLCγ2
antibodies. Molecular markers are indicated on the left side of the IBs. B. SHP2 WT and
SHP2 KO BMMCs were starved and sensitized with anti-TNP IgE for 6 hours and
stimulated with either vehicle control, 10ng/mL DNP-HSA or 1μM calcium ionophore at
37°c for 1 hour. Cells were pelleted, supernatants were collected and cell pellets were
lysed in tyrodes supplemented with 0.5% Triton X100 to generate whole cell lysates.
Samples were analyzed assayed for β-hexosaminidase content to determine % βhexosaminidase release. Results are the mean of 3 independent experiments assayed in
triplicate, +/- SEM.
47
depletion (Figure 7B). SCL IB analysis of the SHP2 WT and KO BMMCs verified that
SHP2 protein levels were effectively depleted by 4OH-TM administration (data not
shown). Thus, SHP2 is not required for the FcεRI-evoked signaling events which lead to
degranulation.
3.3 SHP2 Modulates FcεRI-Proximal Signaling
SHP2 recruitment to a number of proteins within the FcεRI signaling cascade has
been
previously
described,
suggesting
that
SHP2
becomes
activated
and
dephosphorylates proteins within FcεRI complexes or downstream effectors75,39. To
identify the exact role of SHP2 in regulating FcεRI evoked signaling, we performed a
biochemical characterization of FcεRI-proximal signaling. SCLs generated from SHP2
WT and KO BMMCs that had been IgE/DNP stimulated for 0, 3, 9 and 27 minutes were
subjected to IP with an anti FcεRI-β chain antibody. The IPs were analyzed on duplicate
membranes by IB with anti-pY99 and anti-β chain antibodies. Upon analysis of the
results from 4 independent experiments, we discovered that the β chain in SHP2 KO
BMMCs was hypophosphorylated at all assayed timepoints (Figure 8A; compare lanes 14 with lanes 5-8; p=0.023 for 9 minute timepoint - lane 3 vs lane 7). If the FcεRI β chain
was a SHP2 substrate, the opposite result would have been predicted.
IgE/antigen initiated FcεRI clustering leads to Lyn activation and subsequent
phosphorylation of the receptor chains of the FcεRI3. Next, we performed immunoblot
analysis of the activation loop tyrosine of Lyn (Y396) using an anti-Src Y416 antibody
(which cross-reacts with the corresponding tyrosine residue in Lyn, Y396) and a control
48
WT
A
0
3
KO
9
27
0
3
9
27
IgE/Ag (min)
34
IP: β chain
IB: pY99
34
IP: β chain
IB: Beta
1
0.1
2
3
2.2
1.0
4
0.4
5
0.1
6
1.3
7
8
0.6*
0.4
Fold Increase; n=4
B
SCL
IB: pY416-Src/
55
pY396-Lyn
55
SCL
IB: Lyn
1
1.7
2
1.0
3
4
5
0.8
0.8
1.5
6
7
8
1.3
1.2
0.7
Fold Increase; n=2
C
SCL
IB:pY 507-Lyn
55
SCL
IB: Lyn
55
1
2
3
4
5
0.7
1.0
0.6
0.6
0.5
6
0.8
7
8
0.6
0.6
Fold Increase; n=3
D
70
SCL
IB: pSyk
70
SCL
IB: Syk
1
2
0.5
1.0
3
2.0
4
5
6
7
8
0.4
0.4
0.9
1.9
0.9
Fold Increase; n=3
Figure 8. SHP2 Modulates FcεRI-Proximal Signaling.
A. β chain was IPed from SHP2 WT and KO SCLs, fractionated by SDS PAGE and
analyzed by IB. Membranes IBed with anti-pY99 and anti-β chain antibodies. Asterisk
indicates a statisticaly significant difference from WT (lane 3 vs lane 7 p ≤ 0.05). B.
SCLs were IBed with an anti-Src Y416 antibody (which cross-reacts with the
corresponding tyrosine residue in Lyn, Y396) and a control Lyn antibody. C. SCLs were
IBed with an anti-phospho-Lyn Y507 antibody and an anti-Lyn control antibody D. SCLs
were IBed with anti-phospho Syk (Y352) and anti-Syk antibodies. Relative fold increase
in phosphorylation signal was determined by densitometry for the number of experiments
indicated in the figures. Molecular markers are indicated on the left side of the IBs.
49
Lyn antibody. Upon analysis of the results, we found there does not appear to be
significant change in the phosphorylation of this activation loop tyrosine residue in Lyn
in SHP2 KO BMMCs (Figure 8B; compare lanes 1-4 with lanes 5-8). Next, SCL IBs
analysis of Lyn C-terminal phosphorylation was accomplished through the use of an antiphospho-Lyn Y507 antibody and an anti-Lyn control antibody. Lyn FcεRI mediated Cterminal phosphorylation was similar in SHP2 KO BMMCs when compared with WT
cells (Figure 8C; compare lanes 1-4 with lanes 5-8). Taken together, these results imply
that Lyn activity is not dependent on SHP2.
Next, we utilized anti-phospho Syk (Y352) and anti-Syk antibodies on SCL timecourses in order to analyze the phosphorylation profile of Syk kinase; phosphorylation of
Syk corresponds to activation. Phosphorylation of Syk was found to be similar in SHP2
WT and KO cells at the early timepoints when the immunoblots were analyzed by
densitometry (Figure 8D). However, Syk phosphorylation was shown to be elevated at
later times (compare lane 4 with lane 8). A 2-fold increase in the phosphorylation signal
(0.9 vs. 0.4) was observed at the 27 minute timepoint. This result is consistent with the
previously identified role of SHP2 in dephosphorylating and antagonizing Syk activity123.
Overall, the receptor proximal signaling events suggest SHP2 is a positive
regulator of FcεRI phosphorylation. SHP1 has been previously shown to bind the FcεRI
β119 and γ chain44, and SHP1 overexpression reduces β chain phosphorylation. SHP2
depletion could potentially result both in increased β chain recruitment of SHP1 and in
the phosphorylated ITAMs becoming easier to access and dephosphorylate due to a
50
decreased β chain binding of SHP2; easier substrate access could result in increased
SHP1 dephosphorylation of the β chain, resulting in the observed hypophosphorylation.
Finally, the extended activation kinetics of Syk is consistent with the predicted results of
the depletion of a phosphatase that is recruited and acts as a negative regulator of Syk.
Thus, it is plausible that SHP2 is a negative regulator of Syk kinase in the FcεRI
pathway.
3.4 SHP2 Promotes Akt Activation
We next went on to characterize the phosphorylation profile of another SHP2
binding partner and substrate, Gab2111. Immunoblot analysis of SCLs was conducted via
the use of an anti-phospho-Gab2 Y452 antibody and an anti-Gab2 control antibody. Upon
analysis, it was found that Gab2 is hyperphosphorylated on Y452 (on human Gab2; Y441
in mouse Gab2) in SHP2 KO cells (Figure 9A; compare lanes 1-5 with lanes 6-10). Y452
is a proposed PI3K recruitment site and the corresponding tyrosine residue on Gab1,
Y472, is dephosphorylated by SHP2128,129. Studies have implicated SHP2’s function in
regulating the phosphorylation of Y472 on Gab1 as being a means of downregulating the
recruitment of PI3K, and thus downregulation of the plasma membrane concentration of
PIP3128,129. PIP3 generation downstream of the EGFR results in the recruitment and
activation of Akt; these studies have correlated SHP2’s role in the dephophorylation of
Gab2 with the activation status of Akt128,129. Thus, it may be predicted that the
hyperphosphorylation of Gab2 that we saw in SHP2 deficient BMMCs could result in
elevated PI3K recruitment and hyperactivation of Akt.
51
WT
A
0 1
KO
3 9 27 0
1
3 9 27
IgE/Ag (min)
SCL
IB: pGab2
95
70
95
SCL
IB: Gab2
70
1
2
3
4
5
6
7
8
9
10
n=4
WT
B
0
3
KO
9
27
0
3
9
27
IgE/Ag (min)
55
SCL
IB: pAkt
55
SCL
IB: Akt
1
2
3
4
5
6
7
8
0.1
1.0
0.5
0.5
0.0
0.3*
0.2
0.1
Fold Increase; n=3
C
SCL
IB: pIKK
95
SCL
IB: IKK
95
1
2
3
4
5
6
0.1
1.0
1.4
2.2
0.1
0.9
7
8
0.6
0.7
Fold Increase; n=3
Figure 9. SHP2 Promotes Akt Activation.
A. SCLs were IBed with an anti-phospho-Gab2 Y452 antibody and an anti-Gab2 control
antibody. B. SCLs were IBed with an anti-phospho Akt antibody and an anti-Akt
antibody. Asterisk indicates a statisticaly significant difference from WT (lane 2 vs lane 6
p ≤ 0.05). C. SCLs were IBed with an anti-phospho IKKα/β antibody and a control antiIKKα antibody. Relative fold increase in phosphorylation signal was determined by
densitometry for the number of experiments indicated in the figures. Molecular markers
are indicated on the left side of the IBs.
52
Immunoblot analysis was then conducted using anti-phospho Akt and anti-Akt
antibodies to probe SCLs (Figure 9B). Interestingly, in contrast to the prediction
generated by the Gab2 phosphorylation data, Akt phosphorylation was found to be
reduced at all timepoints in SHP2 KO cells (compare lanes 1-4 with 5-8; p=0.0003 for 3
minute timepoint – lane 2 vs lane 6). Akt is a major transcriptional regulator and one of
its roles is in NFκB activation48. NFκB activation is dependent on the activation of IκB
kinase (IKK), which is one of substrates of activated Akt154. Analysis of the
phosphorylation profile of IKK, which was accomplished via the use of an anti-phospho
IKKα/β antibody and a control anti-IKKα antibody, showed hypophosphorylation of
IKK in SHP2 KO BMMCs when compared with SHP2 WT cells (Figure 9C; compare
lanes 1-4 with lanes 5-8). Hypophosphorylation of IKK corresponds with the hypoactivation of Akt, which serves to further support a role for SHP2 in promoting FcεRIevoked Akt activation of the Akt/ NFκB signaling pathway.
3.5 SHP2 Promotes Activation of Erk MAPK
We next went on to characterize the phosphorylation profiles of the Erk, p38 and
JNK MAPKs, which represent the other predominant transcriptional regulators within the
FcεRI signaling cascade (Figure 10)48. SHP2 has been shown to positively regulate Erk
activation downstream of the EGFR through antagonizing the recruitment of RasGAP
and Csk to the plasma membrane124,126. Thus, SHP2 may regulate FcεRI-mediated Erk
activation via a mechanism that has been previously identified in another cell system. By
53
WT
A
0
3
KO
9
27
0
3
9
27
43
IgE/Ag (min)
SCL
IB: pErk
SCL
IB: Erk
43
1
2
3
0.1
1.0* 1.2
4
5
6
1.0
0.1
7
8
0.5* 0.5
0.5
Fold Increase; n=3
B
43
SCL
IB: pp38
43
SCL
IB: p38
1
2
3
4
5
6
7
8
0.1
1.0
0.8
0.3
0.1
1.5
1.1
0.4
Fold Increase; n=3
C
55
SCL
IB: pJnk1/2
43
SCL
IB: Akt
55
Jnk2:
Jnk1:
1
2
3
4
5
6
7
8
0.5
0.0
1.0
1.0
1.9
1.7
0.7
0.3
0.5
0.1
1.0
0.5
1.2
0.8
0.3
0.1
Fold Increase; n=3
Figure 10. SHP2 Promotes Activation of Erk MAPK.
A. SCLs were IBed with an anti-phospho-Erk and an anti-Erk 1 control antibody.
Asterisk indicates a statisticaly significant difference from WT (lane 2 vs lane 6 p ≤
0.05). B. SCLs were IBed with an anti-phospho-p38 antibody and an anti-p38 antibody.
C. SCLs were IBed with an anti-phospho-JNK and a control anti-Akt 1/2 antibody.
Relative fold increase in phosphorylation signal was determined by densitometry for the
number of experiments indicated in the figures. Molecular markers are indicated on the
left side of the IBs.
54
immunoblotting SCLs with an anti-phospho-Erk and an anti-Erk 1 control antibody, we
found that IgE/DNP stimulated Erk phosphorylation is significantly attenuated in SHP2
KO BMMCs at all assayed timepoints, compared to WT (Figure 10A; compare lanes 1-4
with lanes 5-8; p=0.037 for lane 2 vs lane 6).
Next, SCL immunoblot analysis was conducted with anti-phospho-p38 and anti
p38 antibodies, which showed no significant differences in p38 phosphorylation in SHP2
KO BMMCs compared to WT (Figure 10B). Finally, immunoblot analysis of FcεRIevoked JNK 1/2 phosphorylation was conducted on SCLs through the use of an antiphospho-JNK and a control anti-Akt 1/2 antibody (Figure 10C). JNK 1/2 were found to
be moderately hypophosphorylated in SHP2 KO cells, with densitometric analysis of the
immunoblots indicating that JNK1 phosphorylation was reduced at the 3 and 9 minute
timepoints (compare lanes 2-3 with lanes 6-7), while JNK2 phosphorylation was reduced
at the 9 and 27 minute timepoints (compare lanes 3-4 with lanes 7-8).
Overall, the FcεRI-evoked MAPK phosphorylation profiles in SHP2 WT and KO
BMMCs are consistent with a role for endogenous SHP2 promoting Erk and JNK
activation while not affecting p38 activation. The MAPKs as a group serve to activate
transcription factors that serve to produce inflammatory mediators, while Erk also
activates eicosanoid synthesis through stimulating PLA2 activity48. Thus, it may be
expected that the net affect of these results of SHP2 KO on MAPK activity would be a
reduction in the FcεRI mediated synthesis and secretion of cytokines, chemokines and
eicosanoids.
55
3.6 SHP2 promotes Leukotriene C4 & TNFα Release
In the FcεRI signaling cascade, both Erk and Akt act on transcription factors that
initiate the transcription and production of the chemokine TNFα48. Therefore, our data
which indicated that these proteins are hypoactivated in SHP2 KO BMMCs implied that
SHP2 KO BMMCs may exhibit defects in the production of TNFα. In order to
investigate this possibility, we generated SHP2 WT and KO BMMCs as described above,
starved and sensitized the BMMCs with anti-TNP IgE for 6 hours, and then stimulated
the cells with 10ng/mL DNP-HSA for 6 hours in cell culture medium. BMMCs were then
centrifuged and the supernatants were collected and analyzed for TNFα concentrations
using a commercial ELISA kit. The FcεRI-evoked production of TNFα was found to be
significantly reduced in SHP2 KO cells when compared with the TNFα production by
WT BMMCs (Figure 11A; p=0.037).
Erk’s role in promoting the FcεRI-evoked eicosanoid production indicates that the
hypoactivation of Erk in SHP2 KO BMMCs could also result in these cells being
impaired in their ability to produce LTs48. LTC4 is one of the LTs that is produced by
mast cells following FcεRI activation51; therefore, we proceeded to measure the
production of LTC4 in SHP2 WT and KO BMMCs. We inactivated SHP2 as described
above, starved and sensitized the BMMCs with anti-TNP IgE for 6 hours and then
stimulated cells with 10ng/mL DNP-HSA for 2 hours in cell culture medium. Cell
supernatants were then analyzed for LTC4 concentrations using a commercial ELISA kit.
SHP2 KO cells were observed to secrete less LTC4 when compared with SHP2 WT
56
A
*
B
C
WT
KO
WT
KO
WT
KO
70
SCL
IB: SHP2
43
SCL
IB: Erk
1
2
3
4
5
6
Figure 11. SHP2 promotes Leukotriene C4 & TNFα Release.
A. SHP2 WT and KO cells were starved and sensitized with anti-TNP IgE for 6 hours
and stimulated with either vehicle control or 10ng/mL DNP-HSA for 6 hours. Cells were
pelleted and supernatants were collected. Supernatants were analyzed for TNFα
concentration via ELISA (results are presented as an average of 6 experiments conducted
with 5 independent sets of WT and KO cultures +/- SEM, supernatants assayed in
triplicate). Asterisk indicates a statisticaly significant difference from WT (p ≤ 0.05;
p=0.037). B. SHP2 WT and SHP2 KO BMMCs stimulated with either vehicle control or
10ng/mL DNP-HSA for 2 hours and supernatants were collected. Supernatants were
analyzed for LTC4 concentration via ELISA (results are presented as an average of
experiments conducted with 4 independent WT and KO cultures, assayed in triplicate, +/SEM). C. SHP2 WT and KO SCLs were generated and IBed with anti-SHP2 and anti-Erk
antibodies. Molecular markers are indicated on the left side of the IBs.
57
BMMCs (Figure 11B). SCL IB analysis of the SHP2 WT and KO BMMCs verified that
SHP2 protein levels were effectively depleted by 4OH-TM administration (Figure 11C).
The TNFα and LTC4 production data provided further evidence of a role for SHP2 in
potentiating Erk and Akt activation, and production of inflammatory cytokines and
eicosanoids in BMMCs following IgE/Antigen stimulation. This affect results in
decreased production of inflammatory mediators in SHP2 KO BMMCs.
3.7 Evidence that Cbp is a SHP2 Substrate that Limits Fyn Activity in
BMMCs
Cbp is phosphorylated by Lyn and has been shown to play an important role in
regulating FcεRI activation in BMMCs70. Phosphorylation of Cbp on Y317 results in the
recruitment of Csk to the membrane compartment, where it serves to phosphorylate the
C-terminal tyrosine residues of SFKs, which results in attenuation of SFK activity150.
Furthermore, Cbp is a known substrate of SHP2124 and elevated levels of Csk recruitment
has been shown to result in reduced FcεRI β chain phosphorylation in RBL cells67.
RNAi-mediated Csk knockdown was found to rescue the reduced Erk MAPK signaling in
EGF-treated SHP2 KO MEFs124. Therefore, a role for SHP2 in the regulation of Csk
recruitment downstream of FcεRI engagement could provide a molecular mechanism by
which SHP2 could promote FcεRI signaling and cytokine production in BMMCs.
Thus, we next surveyed the phosphorylation profile of Y317 of Cbp in SHP2 WT
and KO BMMCs (Figure 12A). SCL IB analysis using an anti-phospho-Y317 CBP
58
WT
A
0
3
9
KO
27
0
3
9
27
95
IgE/Ag (min)
SCL
IB: pY317-Cbp
70
SCL
IB: α-Tubulin
55
1
2
3
4
5
6
7
8
0.9
1.0
0.6
0.7
1.9
1.8
1.1
0.6
Fold Increase; n=2
B
SCL
IB: pY527-Src/
pY531-Fyn
55
SCL
IB: Fyn
55
1
2
3
4
5
6
7
0.5
1.0
1.0
2.0
1.4
2.0
1.2
8
1.8
Fold Increase; n=3
Figure 12. Evidence that Cbp is a SHP2 Substrate that Limits Fyn Activity in
BMMCs.
A. SCLs were IBed with an anti-phospho-Y317 CBP antibody and a control α-tubulin
antibody. B. SCLs were IBed with an anti-phospho-Src Y527 antibody (which crossreacts with phosphorylated Y531 of Fyn) and an anti-Fyn control antibody. Relative fold
increase in phosphorylation signal was determined by densitometry for the number of
experiments indicated in the figures. Molecular markers are indicated on the left side of
the IBs.
59
antibody and a control α-tubulin antibody served to indicate that Cbp is
hyperphosphorylated on this tyrosine residue in SHP2 KO BMMCs. This
hyperphosphorylation occurred both in resting cells and at the 0, 3 and 9 minute
timepoints following FcεRI engagement (compare lanes 1-3 with lanes 5-7; similar
results were obtained when pY317-Cbp densitometry was normalized to densitometry
values obtained via immunoblot analysis of the SCLs with an Akt 1/2 antibody; data not
shown).
As mentioned above, Cbp recruitment of Csk to the plasma membrane results in
the phosphorylation of SFKs on their C-terminal tyrosine residues. Therefore, we next
looked at the FcεRI-evoked phosphorylation profiles of the C-terminal tyrosine residues
of Fyn. SCL IB analysis of the Fyn inhibitory site was performed using an anti-phosphoSrc Y527 antibody (which cross-reacts with phosphorylated Y531 of Fyn) and an antiFyn control antibody (Figure 12B). The phosphorylation of Fyn Y531 was found to be
markedly increased in both resting BMMCs and at the 0, 3 and 9 minute timepoints postFcεRI stimulation (compare lanes 1-3 with lanes 5-7).
The Cbp and Fyn phosphorylation results provided further evidence to support a
role for SHP2 in positively regulating SFK activity and FcεRI signaling to Erk MAPK
and Akt. These results imply that this affect is elicited through SHP2 mediated downregulation of the recruitment of Csk to Cbp and thus promotion of SFK activity (Figure
13).
60
Chapter 4
Discussion
This study provides novel insight into the role of SHP2 in regulating FcεRI
signalling and cytokine production in BMMCs. Due to SHP2’s role in embryonic
development106, it was necessary to employ a conditional SHP2 KO model in order to
study the function of SHP2 in FcεRI signal regulation. Additionally, SHP2’s role in
regulating the major BMMC growth pathways downstream of both the IL-3 receptor111
and the Kit receptor122, necessitated that we temporally inactivate SHP2 in only the
subset of BMMCs that were being used for experiments (Figure 5B). In order to ensure
that there was no effect on cell viability during the 3 days of 4OH-TM administration,
while cytosolic SHP2 was being depleted, we used trypan blue to determine the
proportion of dead BMMCs at the end of 4OH-TM treatment. The results of this analysis
showed that there was no significant difference in the proportion of dead cells between
SHP2 WT and KO BMMCs (data not shown). Finally, one study implicates SHP2 in the
inhibition of Bcr-Abl proteosome-mediated degradation via an interaction with HSP90,
indicating that SHP2 depletion can alter the stability of cytosolic proteins155. However,
we found no significant differences in the stability of the signaling proteins within the
FcεRI pathway, nor in the surface expression of the Kit receptor or the FcεRI (Figure
6B).
A previous study used a comparable conditional SHP2 KO approach in order to
study the role of SHP2 in the Kit receptor pathway in BMMCs122. The group found that
61
an approximate 75% reduction in SHP2 protein resulted in a 50% reduction in Kit-driven
proliferation and also caused decreases in the SCF/Kit-mediated activation of the
Rac/JNK pathway122. In contrast, Akt and Erk activation were unaffected. Interestingly,
Ras activation appeared to be decreased. Ras can also activate Rac156,157, which provides
a potential mechanism for the reduction in Rac/JNK activation122. As an alternative
explanation for the Rac/JNK hypoactivation, the authors of the study predict that SHP2
may antagonize the activity of a RacGAP, thereby promoting Rac activation in the WT
Kit pathway122. It is interesting to note that our study also found a reduction in FcεRImediated JNK activation (Figure 10C) – as there are many signaling proteins common to
both pathways in mast cells, it is possible that there could be similar underlying
mechanisms for this result.
SHP2 has previously been shown to regulate Cbp recruitment of Csk in the EGFR
pathway, resulting in the promotion of Src activity124. Now, we have shown that SHP2
promotes Fyn kinase activity downstream of FcεRI activation through antagonizing the
Lyn-dependent phosphorylation of Cbp70 and subsequent recruitment of Csk (Figure 13).
Our results indicate that SHP2 KO BMMCs exhibit hyperphosphorylation of Y317 on
Cbp, which is the site that recruits Csk (Figure 12A)150. This effect on Cbp
phosphorylation correlates with our observations that our SHP2 KO BMMCs inhibited
hyperphosphorylation of Fyn Y531 (Figure 12B), which results in reduced kinase activity
when compared to WT BMMCs72.
62
Antigen
Clustered FcεRI
Syk
Fyn
Cbp
SHP2
SHP2
Lyn
Csk
Cell Membrane
Akt
Cytosol
RasGAP?
RasGAP?
SHP2
Eicosanoid Production;
eg. LTC 4
Gab2
PI3K
Erk
PLA 2
JNK
p38
Nuclear Membrane
Gene Transcription;
eg. TNFα , IL-6
Figure 13. Proposed functions for SHP2 in Regulating the FcεRI Pathway.
Our data suggests several roles for SHP2 in regulating the FcεRI pathway. SHP2
antagonizes Lyn-mediated phosphorylation of Cbp on Y317 and thus promotes Fyn
activity and FcεRI signaling. SHP2 also serves to dephosphorylate Gab2 and Syk kinase.
SHP2 depletion results in hyperphosphorylation of Cbp and attenuated Fyn activity,
along with elevated phosphorylation of Gab2 and extended phosphorylation of Syk. The
net result of these effects results in decreased activation of Erk and Akt with a
corresponding decrease in cytokine gene transcription and eicosanoid production.
63
Previous studies have implicated the Cbp/Csk negative feedback loop in
regulating both Lyn71 and Fyn activity70,69 in the FcεRI signaling cascade. However, it is
plausible to suggest that Fyn is the kinase that is predominantly regulated in this fashion.
Several studies have reported that reduced Lyn activity in Lyn KO BMMCs and in
BMMCs that were derived from epilepsy-resistant ASK mice correlated with an increase
in Fyn kinase activity70,69. In contrast, there is limited data that indicates that Lyn is
regulated by this negative feedback loop; one study found that Hck KO mast cells
displayed increased Lyn activity, which is proposed to be a result of reduced Cbp
phosphorylation by Hck and subsequently reduced Csk-mediated negative feedback to
Lyn71. However, this study did not see a significant difference in C-terminal
phosphorylation of Lyn in Hck KO cells, and Cbp phosphorylation was increased in Hck
KO cells - presumably as a result of the increased Lyn activity71. In support of their
proposed model of Hck Cbp-mediated Lyn kinase activity regulation, the study showed
data that indicated that Hck constitutively associated with Cbp, before and after FcεRI
engagement71. This result correlated with their observation that Lyn activity was
increased constitutively in Hck KO BMMCs71. Taken together, these studies provide
more substantial evidence for Fyn regulation than for Lyn. This idea is consistent with
our results, which suggest that while the Fyn C-terminal tyrosine is hyperphosphorylated
(Figure 12B), Lyn C-terminal phosphorylation is not significantly affected by SHP2 KO
(Figure 8C).
Thus, our model for SHP2 regulation of FcεRI signaling suggests that Fyn
activity is preferentially regulated by Cbp, and is reduced in SHP2 KO BMMCs.
64
Therefore, it is important to evaluate the similarities in the signaling data and cell
phenotypes between our SHP2 KO BMMCs and the data generated in Fyn KO BMMC
studies. Similar to our findings in SHP2 KO BMMCs, Fyn KO cells displayed deficient
cytokine (TNFα and IL-6) and leukotriene production (LTC4)158. In Fyn KO BMMCs,
this decrease was correlated with decreased activation of Akt/IKK and JNK. This effect is
consistent with our results (Akt/IKK: Figure 9B/C; JNK: Figure 10C)158. One interesting
difference is that our study found no significant differences in p38 activation, while the
Fyn KO studies showed a decrease in p38 activation158.
However, there are also several dissimilarities between our results and the
consequences of Fyn KO. For instance, while SHP2 KO did not significantly affect
degranulation (Figure 7B), Fyn KO results in an approximately 78% reduction in this
cellular response. The degranulation defect seen in Fyn KO cells could have been the
result of Gab2 hypophosphorylation that was observed in Fyn KO BMMCs. However, in
our study, we found hyperphosphorylation of Y452 on Gab2 (Figure 9A), which is
consistent with the notion that Gab2 is a SHP2 substrate; thus, SHP2 depletion should
result in elevated Gab2 phosphorylation. Thus, although Fyn-mediated Gab2
phosphorylation may be occurring at a reduced rate, SHP2 depletion may offset this
effect by resulting in a decreased rate of dephosphorylation, which may allow Gab2 to
play its role in the promotion of degranulation. Additionally, Syk activation is prolonged
in SHP2 KO cells (Figure 8D); Syk is another signaling molecule that is important in the
promotion of degranulation. Thus, the extended Syk activation kinetics seen in SHP2 KO
BMMCs could provide an additional means to offset any potential reduction in
65
degranulation associated with a reduction in Fyn kinase activity. Thus, there are multiple
explanations for the striking difference in the degranulation phenotypes that occur as a
result of Fyn KO and SHP2 KO.
As mentioned above, we observed extended Syk phosphorylation in our study
(Figure 8D). However, two studies have provided conflicting evidence and have shown
Syk activation to be either reduced42 or unaffected in Fyn KO BMMCs41. This difference
in the effects of Fyn KO and SHP2 KO may be explained by the fact that Syk is a
substrate of SHP2123; thus, in the absence of competing effects, SHP2 depletion would be
expected to result in hyperphosphorylation of Syk. It is possible that if Syk activation
truly is partially dependent on Fyn activity, then reduced Fyn activity could be reducing
the rate of phosphorylation of Syk, while SHP2 depletion may be resulting in a reduced
rate of dephosphorylation. These conflicting effects may be effectively cancelled out at
the early timepoints post FcεRI activation, resulting in WT Syk activation kinetics. At the
later timepoints, the kinases have been shut off by the inhibitory branches of the signaling
cascade and SHP2 depletion results in prolonged Syk activation, as there is a dramatic
reduction in the rate of SHP2-mediated Syk dephosphorylation.
β chain phosphorylation is unaffected in Fyn KO BMMCs, while Lyn KO
BMMCs display only low levels of FcεRI-evoked β chain phosphorylation41. As our
study found a significant reduction in β chain phosphorylation (Figure 8A), our proposed
model of SHP2 promoting Fyn activity without affecting Lyn kinase activity fails to
explain the effects of SHP2 KO on β chain phosphorylation. One possibility is that
66
although we do not currently have data to support Cbp regulation of Lyn activity, Cbp
may still be regulating C-terminal phosphorylation of Lyn activity. Decreased Lyn
activity in SHP2 KO BMMCs resulting from increased Csk recruitment could have
resulted in the β chain hypophosphorylation (Figure 8C). An alternative explanation is
that SHP2 protein depletion results in increased access to β and γ chain binding sites for
SHP1. SHP1 overexpression leads to decreased β chain phosphorylation, possibly as a
result of SHP1 directly dephosphorylating the receptor120; it is plausible that increased
SHP1 receptor-localization in SHP2 KO BMMCs could result in a similar effect.
In our study, one of the most striking downstream consequences of SHP2 KO was
a reduction in the IgE/Antigen-mediated activation of Erk MAPK (Figure 10A).
However, in Fyn KO BMMCs, FcεRI-mediated activation of Erk is unaffected158.
Therefore, although SHP2 has been shown to promote Erk signaling by antagonizing Csk
recruitment to Cbp in the EGFR pathway, the apparent preference for Cbp/Csk in the
regulation of Fyn within the FcεRI pathway suggests that alternative explanations are
required to provide a rationale for this result. In the EGFR pathway, SHP2 has been
shown to promote Erk signaling by also antagonizing the recruitment of RasGAP to the
EGFR131 and to Gab1132. Thus, there is precedence for SHP2 in directly
dephosphorylating RasGAP SH2 binding sites, and there are several potential sites of
RasGAP recruitment in the FcεRI pathway. These sites occur on proteins including the
SHP2 substrate Cbp150, and on Dok1 and Dok2159. SHP2 may thus play a role in
promoting Erk signaling in the FcεRI signaling cascade by antagonizing RasGAP
67
recruitment to one or more of these proteins. An additional mechanism could involve
SHP2 regulation of FcεRI-mediated NTAL phosphorylation. NTAL is a transmembrane
adapter protein that antagonizes Erk activation and its phosphorylation is very transient79,
indicating that it is likely the target of a phosphatase; however, the identity of this
phosphatase it is currently unknown. NTAL contains a predicted SHP1 recruitment site84,
which could potentially serve to recruit SHP2 as well and result in the dephosphorylation
of NTAL. We have preliminary co-IP data which suggests that both SHP1 and SHP2
bind to NTAL upon FcεRI activation, lending credence to this potential mechanism for
SHP2-mediated propagation of Erk activation (data not shown). Finally, Lyn KO leads to
reduced Erk activation119; if Csk does in fact antagonize Lyn activity, SHP2 KO could
result in reduced Lyn activity and thus reduced Erk activation.
There are a number of avenues to pursue in order to broaden our understanding of
SHP2’s role in mast cell FcεRI activation. Csk RNAi knockdown (KD) was shown to
rescue the defect in EGFR-mediated Erk activation observed in SHP2 KO MEFs124. It
may be possible to rescue the Akt or Erk MAPK activation and TNFα release defects
seen in SHP2 KO BMMCs using a similar strategy. To this end, we have obtained
lentiviral Csk shRNA KD constructs (obtained from Open Biosystems) and have
preliminary data that suggests that it is possible to reduce the cytosolic Csk protein levels
by approximately 50% in BMMCs (data not shown). In addition, if we were able to
rescue the attenuation of SFK activity seen in SHP2 KO BMMCs, it may be possible to
identify further roles of SHP2 in regulating FcεRI signaling. The pathway would no
longer be subject to widespread downregulation from the beginning of the cascade, thus
68
allowing for SHP2 substrates to become more readily hyperphosphorylated in its absence,
thereby allowing for additional ease of detection.
Recently a transgenic mouse line has been reported to allow for Cre recombinase
expression under the control of mMCP-5 promoter160. The use of this mast cell-specific
Cre mouse line, which could allow for the study of the consequences of in vivo SHP2 KO
in mast cells160. This could be accomplished by interbreeding our shp2fl mouse line with
the mast cell specific Cre mouse line to generate mice containing SHP2 KO mast cells.
However, the authors have published limited data on how far along the mast cell
differentiation pathway the mMCP-5 will drive Cre recombinase protein expression.
Thus, if the Cre recombinase protein is expressed early in the differentiation process,
SHP2’s role in hematopoiesis99 may adversely affect mast cell development. Also, the
key role of SHP2 in integrin signaling127 and cell migration161 may adversely affect mast
cell homing to target tissues.
Ultimately, one of the major goals of animal model-based cell signaling research
is to provide insight into how the human system works, so that novel therapeutics may be
designed to manipulate signaling pathways to treat human disease. Thus, it is important
to verify that the results obtained in the murine mast cell system are transferable to
human mast cells. To this end, we have obtained the LAD2 human mast cell leukemia
cell line162, along with a SHP2 targeting shRNA encoding lentivirus (kindly provided by
Matthias Eder, Hannover Medical School)163. This cell line expresses the FcεRI receptor
and has been shown to degranulate in response to FcεRI stimulation, indicating that the
signaling cascade is intact in these cells162. Thus, the LAD2 cell line could be a useful
69
tool and would allow use to verify results that we have obtained in the BMMC model. To
date, I have been able to deplete SHP2 protein levels in the LAD2 cells by between 30%
and 90% via the use of the lentiviral vector (unpublished observations), indicating that
this may be a viable experimental approach to study SHP2’s role in regulating the FcεRI
signaling cascade in human mast cells.
In conclusion, this study has provided novel insight into the role of SHP2 in
regulating FcεRI signaling. In the future, it will be important to investigate the in vivo
consequences of shp2 inactivation and to evaluate how closely the BMMC model
approximates the human mast cell system. These investigations will allow for the
observations produced by this study to be included in the considerations behind designing
strategies for the manipulation of FcεRI pathway function during the process of novel
drug design in the creation of new therapeutic immunomodulators.
70
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Metz, M. et al. Mast cells in the promotion and limitation of chronic
inflammation. Immunol Rev 217, 304-28 (2007).
Iwasaki, H. & Akashi, K. Myeloid lineage commitment from the hematopoietic
stem cell. Immunity 26, 726-40 (2007).
Rivera, J. & Gilfillan, A.M. Molecular regulation of mast cell activation. J Allergy
Clin Immunol 117, 1214-25; quiz 1226 (2006).
Dawicki, W. & Marshall, J.S. New and emerging roles for mast cells in host
defence. Curr Opin Immunol 19, 31-8 (2007).
Okayama, Y. & Kawakami, T. Development, migration, and survival of mast
cells. Immunol Res 34, 97-115 (2006).
Hallgren, J. & Gurish, M.F. Pathways of murine mast cell development and
trafficking: tracking the roots and routes of the mast cell. Immunol Rev 217, 8-18
(2007).
Gurish, M.F. et al. Differential expression of secretory granule proteases in mouse
mast cells exposed to interleukin 3 and c-kit ligand. J Exp Med 175, 1003-12
(1992).
Beil, W.J., Schulz, M. & Wefelmeyer, U. Mast cell granule composition and
tissue location--a close correlation. Histol Histopathol 15, 937-46 (2000).
Hu, Z.Q., Zhao, W.H. & Shimamura, T. Regulation of mast cell development by
inflammatory factors. Curr Med Chem 14, 3044-50 (2007).
Ihle, J.N. et al. Biologic properties of homogeneous interleukin 3. I.
Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity,
p cell-stimulating factor activity, colony-stimulating factor activity, and
histamine-producing cell-stimulating factor activity. J Immunol 131, 282-7
(1983).
Razin, E. et al. Interleukin 3: A differentiation and growth factor for the mouse
mast cell that contains chondroitin sulfate E proteoglycan. J Immunol 132, 147986 (1984).
Lantz, C.S. et al. Role for interleukin-3 in mast-cell and basophil development
and in immunity to parasites. Nature 392, 90-3 (1998).
Valent, P. et al. Failure to detect IL-3-binding sites on human mast cells. J
Immunol 145, 3432-7 (1990).
Hamaguchi, Y. et al. Interleukin 4 as an essential factor for in vitro clonal growth
of murine connective tissue-type mast cells. J Exp Med 165, 268-73 (1987).
Hu, Z.Q., Kobayashi, K., Zenda, N. & Shimamura, T. Tumor necrosis factoralpha- and interleukin-6-triggered mast cell development from mouse spleen cells.
Blood 89, 526-33 (1997).
Kirshenbaum, A.S. et al. Demonstration that human mast cells arise from a
progenitor cell population that is CD34(+), c-kit(+), and expresses
aminopeptidase N (CD13). Blood 94, 2333-42 (1999).
71
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Oskeritzian, C.A. et al. Recombinant human (rh)IL-4-mediated apoptosis and
recombinant human IL-6-mediated protection of recombinant human stem cell
factor-dependent human mast cells derived from cord blood mononuclear cell
progenitors. J Immunol 163, 5105-15 (1999).
Eklund, K.K., Ghildyal, N., Austen, K.F. & Stevens, R.L. Induction by IL-9 and
suppression by IL-3 and IL-4 of the levels of chromosome 14-derived transcripts
that encode late-expressed mouse mast cell proteases. J Immunol 151, 4266-73
(1993).
Velin, D., Bachmann, D., Bouzourene, H. & Michetti, P. Mast cells are critical
mediators of vaccine-induced Helicobacter clearance in the mouse model.
Gastroenterology 129, 142-55 (2005).
Dimitriadou, V. et al. Expression of functional major histocompatibility complex
class II molecules on HMC-1 human mast cells. J Leukoc Biol 64, 791-9 (1998).
Fox, C.C., Jewell, S.D. & Whitacre, C.C. Rat peritoneal mast cells present antigen
to a PPD-specific T cell line. Cell Immunol 158, 253-64 (1994).
El Behi, M. et al. New insights into cell responses involved in experimental
autoimmune encephalomyelitis and multiple sclerosis. Immunol Lett 96, 11-26
(2005).
Yarovinsky, F., Kanzler, H., Hieny, S., Coffman, R.L. & Sher, A. Toll-like
receptor recognition regulates immunodominance in an antimicrobial CD4+ T cell
response. Immunity 25, 655-64 (2006).
Anderson, K.V. Toll signaling pathways in the innate immune response. Curr
Opin Immunol 12, 13-9 (2000).
Supajatura, V. et al. Protective roles of mast cells against enterobacterial infection
are mediated by Toll-like receptor 4. J Immunol 167, 2250-6 (2001).
King, C.A., Marshall, J.S., Alshurafa, H. & Anderson, R. Release of vasoactive
cytokines by antibody-enhanced dengue virus infection of a human mast
cell/basophil line. J Virol 74, 7146-50 (2000).
McDermott, J.R. et al. Mast cells disrupt epithelial barrier function during enteric
nematode infection. Proc Natl Acad Sci U S A 100, 7761-6 (2003).
Li, E., Zhou, P., Petrin, Z. & Singer, S.M. Mast cell-dependent control of Giardia
lamblia infections in mice. Infect Immun 72, 6642-9 (2004).
Lawrence, C.E., Paterson, Y.Y., Wright, S.H., Knight, P.A. & Miller, H.R. Mouse
mast cell protease-1 is required for the enteropathy induced by gastrointestinal
helminth infection in the mouse. Gastroenterology 127, 155-65 (2004).
Mallen-St Clair, J., Pham, C.T., Villalta, S.A., Caughey, G.H. & Wolters, P.J.
Mast cell dipeptidyl peptidase I mediates survival from sepsis. J Clin Invest 113,
628-34 (2004).
Rivera, J. Snake bites and bee stings: the mast cell strikes back. Nat Med 12, 9991000 (2006).
Galli, S.J. et al. Mast cells as "tunable" effector and immunoregulatory cells:
recent advances. Annu Rev Immunol 23, 749-86 (2005).
72
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
Hernandez-Hansen, V. et al. Increased expression of genes linked to FcepsilonRI
Signaling and to cytokine and chemokine production in Lyn-deficient mast cells.
J Immunol 175, 7880-8 (2005).
Cao, L. et al. Quantitative time-resolved phosphoproteomic analysis of mast cell
signaling. J Immunol 179, 5864-76 (2007).
Blank, U. & Rivera, J. The ins and outs of IgE-dependent mast-cell exocytosis.
Trends Immunol 25, 266-73 (2004).
Lin, S., Cicala, C., Scharenberg, A.M. & Kinet, J.P. The Fc(epsilon)RIbeta
subunit functions as an amplifier of Fc(epsilon)RIgamma-mediated cell activation
signals. Cell 85, 985-95 (1996).
Field, K.A., Holowka, D. & Baird, B. Compartmentalized activation of the high
affinity immunoglobulin E receptor within membrane domains. J Biol Chem 272,
4276-80 (1997).
Vonakis, B.M., Chen, H., Haleem-Smith, H. & Metzger, H. The unique domain as
the site on Lyn kinase for its constitutive association with the high affinity
receptor for IgE. J Biol Chem 272, 24072-80 (1997).
Furumoto, Y., Nunomura, S., Terada, T., Rivera, J. & Ra, C. The FcepsilonRIbeta
immunoreceptor tyrosine-based activation motif exerts inhibitory control on
MAPK and IkappaB kinase phosphorylation and mast cell cytokine production. J
Biol Chem 279, 49177-87 (2004).
Leung, W.H. & Bolland, S. The inositol 5'-phosphatase SHIP-2 negatively
regulates IgE-induced mast cell degranulation and cytokine production. J
Immunol 179, 95-102 (2007).
Parravicini, V. et al. Fyn kinase initiates complementary signals required for IgEdependent mast cell degranulation. Nat Immunol 3, 741-8 (2002).
Yu, M., Lowell, C.A., Neel, B.G. & Gu, H. Scaffolding adapter Grb2-associated
binder 2 requires Syk to transmit signals from FcepsilonRI. J Immunol 176, 24219 (2006).
Costello, P.S. et al. Critical role for the tyrosine kinase Syk in signalling through
the high affinity IgE receptor of mast cells. Oncogene 13, 2595-605 (1996).
Pasquier, B. et al. Identification of FcalphaRI as an inhibitory receptor that
controls inflammation: dual role of FcRgamma ITAM. Immunity 22, 31-42
(2005).
Nishida, K. et al. Fc{epsilon}RI-mediated mast cell degranulation requires
calcium-independent microtubule-dependent translocation of granules to the
plasma membrane. J Cell Biol 170, 115-26 (2005).
Oka, T., Hori, M. & Ozaki, H. Microtubule disruption suppresses allergic
response through the inhibition of calcium influx in the mast cell degranulation
pathway. J Immunol 174, 4584-9 (2005).
Gilfillan, A.M. & Tkaczyk, C. Integrated signalling pathways for mast-cell
activation. Nat Rev Immunol 6, 218-30 (2006).
Abramson, J. & Pecht, I. Regulation of the mast cell response to the type 1 Fc
epsilon receptor. Immunol Rev 217, 231-54 (2007).
73
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
Sulimenko, V. et al. Regulation of microtubule formation in activated mast cells
by complexes of gamma-tubulin with Fyn and Syk kinases. J Immunol 176, 724353 (2006).
Lewis, R.A. et al. Prostaglandin D2 generation after activation of rat and human
mast cells with anti-IgE. J Immunol 129, 1627-31 (1982).
Razin, E. et al. IgE-mediated release of leukotriene C4, chondroitin sulfate E
proteoglycan, beta-hexosaminidase, and histamine from cultured bone marrowderived mouse mast cells. J Exp Med 157, 189-201 (1983).
Alessi, D.R. et al. Characterization of a 3-phosphoinositide-dependent protein
kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7,
261-9 (1997).
Thurmond, R.L., Gelfand, E.W. & Dunford, P.J. The role of histamine H1 and H4
receptors in allergic inflammation: the search for new antihistamines. Nat Rev
Drug Discov 7, 41-53 (2008).
Martini, F., Timmons, M.J. & Tallitsch, R.B. Human anatomy, xxviii, 868 p.
(Prentice Hall, Upper Saddle River, N.J., 2003).
Thorlacius, H., Raud, J., Xie, X., Hedqvist, P. & Lindbom, L. Microvascular
mechanisms of histamine-induced potentiation of leukocyte adhesion evoked by
chemoattractants. Br J Pharmacol 116, 3175-80 (1995).
O'Byrne, P.M. Leukotrienes in the pathogenesis of asthma. Chest 111, 27S-34S
(1997).
Newcombe, D.S. Leukotrienes: regulation of biosynthesis, metabolism, and
bioactivity. J Clin Pharmacol 28, 530-49 (1988).
Bjermer, L. Time for a paradigm shift in asthma treatment: from relieving
bronchospasm to controlling systemic inflammation. J Allergy Clin Immunol 120,
1269-75 (2007).
Hirano, T. et al. Complementary DNA for a novel human interleukin (BSF-2) that
induces B lymphocytes to produce immunoglobulin. Nature 324, 73-6 (1986).
Romano, M. et al. Role of IL-6 and its soluble receptor in induction of
chemokines and leukocyte recruitment. Immunity 6, 315-25 (1997).
Kishimoto, T. Interleukin-6: discovery of a pleiotropic cytokine. Arthritis Res
Ther 8 Suppl 2, S2 (2006).
Ravetch, J.V. & Lanier, L.L. Immune inhibitory receptors. Science 290, 84-9
(2000).
Mertsching, E. et al. A mouse Fcgamma-Fcepsilon protein that inhibits mast cells
through activation of FcgammaRIIB, SH2 domain-containing inositol
phosphatase 1, and SH2 domain-containing protein tyrosine phosphatases. J
Allergy Clin Immunol 121, 441-447 e5 (2008).
Kawakami, T. & Galli, S.J. Regulation of mast-cell and basophil function and
survival by IgE. Nat Rev Immunol 2, 773-86 (2002).
Ravetch, J.V. & Bolland, S. IgG Fc receptors. Annu Rev Immunol 19, 275-90
(2001).
74
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
Strait, R.T., Morris, S.C. & Finkelman, F.D. IgG-blocking antibodies inhibit IgEmediated anaphylaxis in vivo through both antigen interception and Fc gamma
RIIb cross-linking. J Clin Invest 116, 833-41 (2006).
Ohtake, H., Ichikawa, N., Okada, M. & Yamashita, T. Cutting Edge:
Transmembrane phosphoprotein Csk-binding protein/phosphoprotein associated
with glycosphingolipid-enriched microdomains as a negative feedback regulator
of mast cell signaling through the FcepsilonRI. J Immunol 168, 2087-90 (2002).
Kovarova, M. et al. Cholesterol deficiency in a mouse model of Smith-LemliOpitz syndrome reveals increased mast cell responsiveness. J Exp Med 203, 116171 (2006).
Kitaura, J., Kawakami, Y., Maeda-Yamamoto, M., Horejsi, V. & Kawakami, T.
Dysregulation of Src family kinases in mast cells from epilepsy-resistant ASK
versus epilepsy-prone EL mice. J Immunol 178, 455-62 (2007).
Odom, S. et al. Negative regulation of immunoglobulin E-dependent allergic
responses by Lyn kinase. J Exp Med 199, 1491-502 (2004).
Hong, H. et al. The Src family kinase Hck regulates mast cell activation by
suppressing an inhibitory Src family kinase Lyn. Blood 110, 2511-9 (2007).
Ingley, E. Src family kinases: regulation of their activities, levels and
identification of new pathways. Biochim Biophys Acta 1784, 56-65 (2008).
Hernandez-Hansen, V. et al. Dysregulated FcepsilonRI signaling and altered Fyn
and SHIP activities in Lyn-deficient mast cells. J Immunol 173, 100-12 (2004).
Wong, M.X., Roberts, D., Bartley, P.A. & Jackson, D.E. Absence of platelet
endothelial cell adhesion molecule-1 (CD31) leads to increased severity of local
and systemic IgE-mediated anaphylaxis and modulation of mast cell activation. J
Immunol 168, 6455-62 (2002).
Udell, C.M., Samayawardhena, L.A., Kawakami, Y., Kawakami, T. & Craig,
A.W. Fer and Fps/Fes participate in a Lyn-dependent pathway from FcepsilonRI
to platelet-endothelial cell adhesion molecule 1 to limit mast cell activation. J Biol
Chem 281, 20949-57 (2006).
Craig, A.W. & Greer, P.A. Fer kinase is required for sustained p38 kinase
activation and maximal chemotaxis of activated mast cells. Mol Cell Biol 22,
6363-74 (2002).
Ilan, N. et al. Pecam-1 is a modulator of stat family member phosphorylation and
localization: lessons from a transgenic mouse. Dev Biol 232, 219-32 (2001).
Sangrar, W., Gao, Y., Zirngibl, R.A., Scott, M.L. & Greer, P.A. The fps/fes protooncogene regulates hematopoietic lineage output. Exp Hematol 31, 1259-67
(2003).
Tkaczyk, C. et al. NTAL phosphorylation is a pivotal link between the signaling
cascades leading to human mast cell degranulation following Kit activation and
Fc epsilon RI aggregation. Blood 104, 207-14 (2004).
Volna, P. et al. Negative regulation of mast cell signaling and function by the
adaptor LAB/NTAL. J Exp Med 200, 1001-13 (2004).
75
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
Zhu, M., Liu, Y., Koonpaew, S., Granillo, O. & Zhang, W. Positive and negative
regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL.
J Exp Med 200, 991-1000 (2004).
Herzog, S. & Jumaa, H. The N terminus of the non-T cell activation linker
(NTAL) confers inhibitory effects on pre-B cell differentiation. J Immunol 178,
2336-43 (2007).
Brdicka, T. et al. Non-T cell activation linker (NTAL): a transmembrane adaptor
protein involved in immunoreceptor signaling. J Exp Med 196, 1617-26 (2002).
Iwaki, S., Jensen, B.M. & Gilfillan, A.M. Ntal/Lab/Lat2. Int J Biochem Cell Biol
39, 868-73 (2007).
Draberova, L. et al. Regulation of Ca2+ signaling in mast cells by tyrosinephosphorylated and unphosphorylated non-T cell activation linker. J Immunol
179, 5169-80 (2007).
Roget, K., Malissen, M., Malbec, O., Malissen, B. & Daeron, M. Non-T cell
activation linker promotes mast cell survival by dampening the recruitment of
SHIP1 by linker for activation of T cells. J Immunol 180, 3689-98 (2008).
Bischoff, S.C. & Dahinden, C.A. c-kit ligand: a unique potentiator of mediator
release by human lung mast cells. J Exp Med 175, 237-44 (1992).
Hundley, T.R. et al. Kit and FcepsilonRI mediate unique and convergent signals
for release of inflammatory mediators from human mast cells. Blood 104, 2410-7
(2004).
Montero Vega, M.T. New aspects on inflammation in allergic diseases. Allergol
Immunopathol (Madr) 34, 156-70 (2006).
Bradding, P., Walls, A.F. & Holgate, S.T. The role of the mast cell in the
pathophysiology of asthma. J Allergy Clin Immunol 117, 1277-84 (2006).
Bradding, P. & Holgate, S.T. Immunopathology and human mast cell cytokines.
Crit Rev Oncol Hematol 31, 119-33 (1999).
Tuomisto, L., Kilpelainen, H. & Riekkinen, P. Histamine and histamine-Nmethyltransferase in the CSF of patients with multiple sclerosis. Agents Actions
13, 255-7 (1983).
Rozniecki, J.J., Hauser, S.L., Stein, M., Lincoln, R. & Theoharides, T.C. Elevated
mast cell tryptase in cerebrospinal fluid of multiple sclerosis patients. Ann Neurol
37, 63-6 (1995).
Kruger, P.G. Mast cells and multiple sclerosis: a quantitative analysis.
Neuropathol Appl Neurobiol 27, 275-80 (2001).
Zhuang, X., Silverman, A.J. & Silver, R. Brain mast cell degranulation regulates
blood-brain barrier. J Neurobiol 31, 393-403 (1996).
Johnson, D., Seeldrayers, P.A. & Weiner, H.L. The role of mast cells in
demyelination. 1. Myelin proteins are degraded by mast cell proteases and myelin
basic protein and P2 can stimulate mast cell degranulation. Brain Res 444, 195-8
(1988).
Neel, B.G., Gu, H. & Pao, L. The 'Shp'ing news: SH2 domain-containing tyrosine
phosphatases in cell signaling. Trends Biochem Sci 28, 284-93 (2003).
76
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
Mohi, M.G. et al. Prognostic, therapeutic, and mechanistic implications of a
mouse model of leukemia evoked by Shp2 (PTPN11) mutations. Cancer Cell 7,
179-91 (2005).
Salmond, R.J. & Alexander, D.R. SHP2 forecast for the immune system: fog
gradually clearing. Trends Immunol 27, 154-60 (2006).
Chan, R.J. & Feng, G.S. PTPN11 is the first identified proto-oncogene that
encodes a tyrosine phosphatase. Blood (2006).
Bentires-Alj, M. et al. Activating mutations of the noonan syndrome-associated
SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous
leukemia. Cancer Res 64, 8816-20 (2004).
Loh, M.L. et al. Mutations in PTPN11 implicate the SHP-2 phosphatase in
leukemogenesis. Blood 103, 2325-31 (2004).
Chan, R.J. et al. Human somatic PTPN11 mutations induce hematopoietic-cell
hypersensitivity to granulocyte-macrophage colony-stimulating factor. Blood 105,
3737-42 (2005).
Xu, R. et al. Overexpression of Shp2 tyrosine phosphatase is implicated in
leukemogenesis in adult human leukemia. Blood 106, 3142-9 (2005).
Tartaglia, M. et al. Mutations in PTPN11, encoding the protein tyrosine
phosphatase SHP-2, cause Noonan syndrome. Nat Genet 29, 465-8 (2001).
Yang, W. et al. An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast
stem cell survival. Dev Cell 10, 317-27 (2006).
Siminovitch, K.A. & Neel, B.G. Regulation of B cell signal transduction by SH2containing protein-tyrosine phosphatases. Semin Immunol 10, 329-47 (1998).
Pei, D., Lorenz, U., Klingmuller, U., Neel, B.G. & Walsh, C.T. Intramolecular
regulation of protein tyrosine phosphatase SH-PTP1: a new function for Src
homology 2 domains. Biochemistry 33, 15483-93 (1994).
Paling, N.R. & Welham, M.J. Tyrosine phosphatase SHP-1 acts at different stages
of development to regulate hematopoiesis. Blood 105, 4290-7 (2005).
Paling, N.R. & Welham, M.J. Role of the protein tyrosine phosphatase SHP-1
(Src homology phosphatase-1) in the regulation of interleukin-3-induced survival,
proliferation and signalling. Biochem J 368, 885-94 (2002).
Wheadon, H., Paling, N.R. & Welham, M.J. Molecular interactions of SHP1 and
SHP2 in IL-3-signalling. Cell Signal 14, 219-29 (2002).
Matthews, R.J., Bowne, D.B., Flores, E. & Thomas, M.L. Characterization of
hematopoietic intracellular protein tyrosine phosphatases: description of a
phosphatase containing an SH2 domain and another enriched in proline-, glutamic
acid-, serine-, and threonine-rich sequences. Mol Cell Biol 12, 2396-405 (1992).
Yi, T., Gilbert, D.J., Jenkins, N.A., Copeland, N.G. & Ihle, J.N. Assignment of a
novel protein tyrosine phosphatase gene (Hcph) to mouse chromosome 6.
Genomics 14, 793-5 (1992).
Lorenz, U. et al. Lck-dependent tyrosyl phosphorylation of the phosphotyrosine
phosphatase SH-PTP1 in murine T cells. Mol Cell Biol 14, 1824-34 (1994).
77
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
Kozlowski, M. et al. Expression and catalytic activity of the tyrosine phosphatase
PTP1C is severely impaired in motheaten and viable motheaten mice. J Exp Med
178, 2157-63 (1993).
Zhang, J., Somani, A.K. & Siminovitch, K.A. Roles of the SHP-1 tyrosine
phosphatase in the negative regulation of cell signalling. Semin Immunol 12, 36178 (2000).
Tenev, T. et al. Both SH2 domains are involved in interaction of SHP-1 with the
epidermal growth factor receptor but cannot confer receptor-directed activity to
SHP-1/SHP-2 chimera. J Biol Chem 272, 5966-73 (1997).
Hellmuth, K. et al. Specific inhibitors of the protein tyrosine phosphatase Shp2
identified by high-throughput docking. Proc Natl Acad Sci U S A 105, 7275-80
(2008).
Xiao, W. et al. Positive and negative regulation of mast cell activation by Lyn via
the FcepsilonRI. J Immunol 175, 6885-92 (2005).
Xie, Z.H., Zhang, J. & Siraganian, R.P. Positive regulation of c-Jun N-terminal
kinase and TNF-alpha production but not histamine release by SHP-1 in RBL2H3 mast cells. J Immunol 164, 1521-8 (2000).
Kamata, T. et al. src homology 2 domain-containing tyrosine phosphatase SHP-1
controls the development of allergic airway inflammation. J Clin Invest 111, 10919 (2003).
Yu, M. et al. The scaffolding adapter Gab2, via Shp-2, regulates kit-evoked mast
cell proliferation by activating the Rac/JNK pathway. J Biol Chem 281, 28615-26
(2006).
Xu, R. & Pecht, I. The protein tyrosine kinase syk activity is reduced by
clustering the mast cell function-associated antigen. Eur J Immunol 31, 1571-81
(2001).
Zhang, S.Q. et al. Shp2 regulates SRC family kinase activity and Ras/Erk
activation by controlling Csk recruitment. Mol Cell 13, 341-55 (2004).
Kontaridis, M.I. et al. SHP-2 positively regulates myogenesis by coupling to the
Rho GTPase signaling pathway. Mol Cell Biol 24, 5340-52 (2004).
Dance, M., Montagner, A., Salles, J.P., Yart, A. & Raynal, P. The molecular
functions of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway.
Cell Signal 20, 453-9 (2008).
Fornaro, M. et al. SHP-2 activates signaling of the nuclear factor of activated T
cells to promote skeletal muscle growth. J Cell Biol 175, 87-97 (2006).
Zhang, S.Q. et al. Receptor-specific regulation of phosphatidylinositol 3'-kinase
activation by the protein tyrosine phosphatase Shp2. Mol Cell Biol 22, 4062-72
(2002).
Wu, C.J. et al. The tyrosine phosphatase SHP-2 is required for mediating
phosphatidylinositol 3-kinase/Akt activation by growth factors. Oncogene 20,
6018-25 (2001).
Nyga, R. et al. Activated STAT5 proteins induce activation of the PI 3-kinase/Akt
and Ras/MAPK pathways via the Gab2 scaffolding adapter. Biochem J 390, 35966 (2005).
78
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
Agazie, Y.M. & Hayman, M.J. Molecular mechanism for a role of SHP2 in
epidermal growth factor receptor signaling. Mol Cell Biol 23, 7875-86 (2003).
Montagner, A. et al. A novel role for Gab1 and SHP2 in epidermal growth factorinduced Ras activation. J Biol Chem 280, 5350-60 (2005).
Kalesnikoff, J. et al. Monomeric IgE stimulates signaling pathways in mast cells
that lead to cytokine production and cell survival. Immunity 14, 801-11 (2001).
Wong, C.K., Tsang, C.M., Ip, W.K. & Lam, C.W. Molecular mechanisms for the
release of chemokines from human leukemic mast cell line (HMC)-1 cells
activated by SCF and TNF-alpha: roles of ERK, p38 MAPK, and NF-kappaB.
Allergy 61, 289-97 (2006).
Blanchetot, C., Chagnon, M., Dube, N., Halle, M. & Tremblay, M.L. Substratetrapping techniques in the identification of cellular PTP targets. Methods 35, 4453 (2005).
Agazie, Y.M. & Hayman, M.J. Development of an efficient "substrate-trapping"
mutant of Src homology phosphotyrosine phosphatase 2 and identification of the
epidermal growth factor receptor, Gab1, and three other proteins as target
substrates. J Biol Chem 278, 13952-8 (2003).
Jia, Z., Barford, D., Flint, A.J. & Tonks, N.K. Structural basis for phosphotyrosine
peptide recognition by protein tyrosine phosphatase 1B. Science 268, 1754-8
(1995).
Sun, H., Charles, C.H., Lau, L.F. & Tonks, N.K. MKP-1 (3CH134), an immediate
early gene product, is a dual specificity phosphatase that dephosphorylates MAP
kinase in vivo. Cell 75, 487-93 (1993).
Furukawa, T., Itoh, M., Krueger, N.X., Streuli, M. & Saito, H. Specific interaction
of the CD45 protein-tyrosine phosphatase with tyrosine-phosphorylated CD3 zeta
chain. Proc Natl Acad Sci U S A 91, 10928-32 (1994).
Wu, L. & Zhang, Z.Y. Probing the function of Asp128 in the lower molecular
weight protein-tyrosine phosphatase-catalyzed reaction. A pre-steady-state and
steady-state kinetic investigation. Biochemistry 35, 5426-34 (1996).
Denu, J.M., Lohse, D.L., Vijayalakshmi, J., Saper, M.A. & Dixon, J.E.
Visualization of intermediate and transition-state structures in protein-tyrosine
phosphatase catalysis. Proc Natl Acad Sci U S A 93, 2493-8 (1996).
Xie, L., Zhang, Y.L. & Zhang, Z.Y. Design and characterization of an improved
protein tyrosine phosphatase substrate-trapping mutant. Biochemistry 41, 4032-9
(2002).
Kos, C.H. Cre/loxP system for generating tissue-specific knockout mouse models.
Nutr Rev 62, 243-6 (2004).
Zhang, E.E., Chapeau, E., Hagihara, K. & Feng, G.S. Neuronal Shp2 tyrosine
phosphatase controls energy balance and metabolism. Proc Natl Acad Sci U S A
101, 16064-9 (2004).
Krajewska, M. et al. Development of diabesity in mice with neuronal deletion of
Shp2 tyrosine phosphatase. Am J Pathol 172, 1312-24 (2008).
Bard-Chapeau, E.A. et al. Concerted functions of Gab1 and Shp2 in liver
regeneration and hepatoprotection. Mol Cell Biol 26, 4664-74 (2006).
79
147.
148.
149.
150.
151.
152.
153.
154.
155.
156.
157.
158.
159.
160.
161.
162.
Hayashi, S. & McMahon, A.P. Efficient recombination in diverse tissues by a
tamoxifen-inducible form of Cre: a tool for temporally regulated gene
activation/inactivation in the mouse. Dev Biol 244, 305-18 (2002).
Metzger, D., Clifford, J., Chiba, H. & Chambon, P. Conditional site-specific
recombination in mammalian cells using a ligand-dependent chimeric Cre
recombinase. Proc Natl Acad Sci U S A 92, 6991-5 (1995).
Picard, D. Regulation of protein function through expression of chimaeric
proteins. Curr Opin Biotechnol 5, 511-5 (1994).
Smida, M., Posevitz-Fejfar, A., Horejsi, V., Schraven, B. & Lindquist, J.A. A
novel negative regulatory function of the phosphoprotein associated with
glycosphingolipid-enriched microdomains: blocking Ras activation. Blood 110,
596-615 (2007).
Choi, O.H. et al. Antigen and carbachol mobilize calcium by similar mechanisms
in a transfected mast cell line (RBL-2H3 cells) that expresses ml muscarinic
receptors. J Immunol 151, 5586-95 (1993).
Wang, Z., Gluck, S., Zhang, L. & Moran, M.F. Requirement for phospholipase Cgamma1 enzymatic activity in growth factor-induced mitogenesis. Mol Cell Biol
18, 590-7 (1998).
Watanabe, D. et al. Four tyrosine residues in phospholipase C-gamma 2,
identified as Btk-dependent phosphorylation sites, are required for B cell antigen
receptor-coupled calcium signaling. J Biol Chem 276, 38595-601 (2001).
Ozes, O.N. et al. NF-kappaB activation by tumour necrosis factor requires the Akt
serine-threonine kinase. Nature 401, 82-5 (1999).
Chen, J. et al. SHP-2 phosphatase is required for hematopoietic cell
transformation by Bcr-Abl. Blood 109, 778-85 (2007).
Welch, H.C., Coadwell, W.J., Stephens, L.R. & Hawkins, P.T. Phosphoinositide
3-kinase-dependent activation of Rac. FEBS Lett 546, 93-7 (2003).
Lambert, J.M. et al. Tiam1 mediates Ras activation of Rac by a PI(3)Kindependent mechanism. Nat Cell Biol 4, 621-5 (2002).
Gomez, G. et al. Impaired FcepsilonRI-dependent gene expression and defective
eicosanoid and cytokine production as a consequence of Fyn deficiency in mast
cells. J Immunol 175, 7602-10 (2005).
Abramson, J., Rozenblum, G. & Pecht, I. Dok protein family members are
involved in signaling mediated by the type 1 Fcepsilon receptor. Eur J Immunol
33, 85-91 (2003).
Scholten, J. et al. Mast cell-specific Cre/loxP-mediated recombination in vivo.
Transgenic Res 17, 307-15 (2008).
Yu, D.H., Qu, C.K., Henegariu, O., Lu, X. & Feng, G.S. Protein-tyrosine
phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion. J Biol
Chem 273, 21125-31 (1998).
Kirshenbaum, A.S. et al. Characterization of novel stem cell factor responsive
human mast cell lines LAD 1 and 2 established from a patient with mast cell
sarcoma/leukemia; activation following aggregation of FcepsilonRI or
FcgammaRI. Leuk Res 27, 677-82 (2003).
80
163.
Scherr, M. et al. Enhanced sensitivity to inhibition of SHP2, STAT5, and Gab2
expression in chronic myeloid leukemia (CML). Blood 107, 3279-87 (2006).
81