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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 ii 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. iii 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 iv 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 xi 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. 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