Download Linder-web

Protein Modifications
in Signaling
Maurine Linder
College of Veterinary Medicine
Cornell University
APS Refresher Course
Experimental Biology
Posttranslational Modification of Proteins
Expanding Nature’s Inventory
“chemical alterations of protein side chains
and main-chain peptide–bond connectivity
that occur after the messenger RNA code
has been translated into the amino acid
sequence of nascent polypeptide”
Christopher T. Walsh
Roberts and Co. Publishers 2006
Increase in complexity
Human Genome
~ 21,000 genes
Transcriptome
~100,000 transcripts
alternative splicing, RNA editing, etc.
Proteome
~1,000,000? proteins
Post-translational modifications
http://ca.expasy.org/sprot/hpi/hpi_desc.html
Expanding the proteome
• >200 kinds of posttranslational modifications
• ~5% of the human genome is dedicated to
covalent modifications
–
–
–
–
–
–
–
~500 proteases
~500 protein kinases
~150 protein phosphatases
>100 ubiquitinligases
>50 deubiquitinating enzymes
>20 palmitoyltransferases
etc.
Walsh 2006
Two broad categories of protein modifications
• Proteolytic processing
• Addition of small molecule or small protein
– Phosphorylation
– Lipidation
– Glycosylation
– Ubiquitination
– Sumoylation
– And many more
Functional Consequences
•
•
•
•
•
Modulate enzyme activity
Change protein conformation
Alter protein-protein interactions
Regulate protein lifetime
Regulate protein localization
Functional Consequences
Protein Phosphorylation
•
•
•
•
•
Modulate enzyme activity
Change protein conformation
Alter protein-protein interactions
Regulate protein lifetime
Regulate protein localization
Protein modifications as binding sites recognized
by modular domains
Figure 1a.
From: Deribe et al. Nat Struct Mol Biol. (6): 666-672, 2010.
Available at
http://www.nature.com/nsmb/journal/v17/n6/full/nsmb.1842.
html
Tyrosine phosphorylation
Functional Consequences
•
•
•
•
•
Modulate enzyme activity
Change protein conformation
Alter protein-protein interactions
Regulate protein lifetime - ubiquitination
Regulate protein localization
Regulate protein half-life:
Ubiquitination targets proteins for degradation
• E1 – Ubiquitin-activating enzyme
• E2 – Ubiquitin-conjugating enzyme
• E3 – Ubiquitinligase
Adapted from Nakayama and
Nakayama. Nat. Rev. Cancer 6(5):
369-381, 2006.
Developmental signaling pathway regulated by
proteolysis of latent gene regulatory proteins
• Wnt proteins are secreted signaling molecules
that control many aspects of development
• Wnt regulates multiple signaling pathways
– Canonical Wnt signaling operates through bcatenin, a transcriptional co-activator
The Wnt/b-catenin signaling pathway
Figure 15-77
From: Molecular Biology of the Cell
Alberts et al. (Garland Science 2008)
Mechanisms of Regulated Destruction
Figure 6-94b
From: Molecular Biology of the Cell
Alberts et al. (Garland Science 2008)
b-catenin
N-end rule
N-end Rule Pathway in Mammals
Figure 1.
From: Varshavsky. Nature Cell Biol. 5: 373-376, 2003.
Available at
http://www.nature.com/ncb/journal/v5/n5/full/ncb0503-373.html
(With red outline around primary destabilizing residues
RKHLFWYIAST)
Specific N-terminal residues and an internal lysine = N-terminal degron
Tertiary destabilizing residues are deamidated, then arginylated for recognition by
ubiquitinating enzymes
N-end Rule Pathway in Mammals
Figure 1.
From: Varshavsky. Nature Cell Biol. 5: 373-376, 2003.
Available at
http://www.nature.com/ncb/journal/v5/n5/full/ncb0503-373.html
Examples
1. R4-family RGS proteins
2. DIAP – apoptosis inhibitor, cleavage by caspase leads to degradation
Mechanisms of Regulated Destruction
Figure 6-94a
From: Molecular Biology of the Cell
Alberts et al. (Garland Science 2008)
Mechanisms of Regulated Destruction: Cell Cycle
Box 2, left
From: Bardin and Amon
Nat. Rev. Mol. Cell Biol.
2(11):815-826, 2001.
Available at
http://www.nature.com/nr
m/journal/v2/n11/full/nrm1
101-815a.html
Image courtesy C. Rieder
http://www.wadsworth.org/bms/SCBlinks/web_mit2/res_mit.htm
Mechanisms of Regulated Destruction: Cell Cycle
Box 2
From: Bardin and Amon. Nat. Rev. Mol. Cell Biol.
2(11):815-826, 2001.
Available at
http://www.nature.com/nrm/journal/v2/n11/full/nrm1101815a.html
Ubiquitin as a signal for
receptor internalization and
lysosomal degradation
Figure 2a.
From: Deribe et al. Nat. Struct. Mol.
Biol. (6): 666-672, 2010.
Available at
http://www.nature.com/nsmb/journal/
v17/n6/full/nsmb.1842.html
• Binding EGF initiates receptor
dimerization and
autophosphorylation
• C-Cbl binds to phosphorylated
tyrosine through its SH2 domain
• C-cbl is a ubiquitinligase that
modifies the receptor
• Trafficking proteins (eps15/HRS)
bind to ubiquitin through
ubiquitin interaction motifs
• Recruitment of the escort protein
complexes facilitates
incorporation of the receptor in
multivesicular bodies and then
lysosomes for degradation
Summary
• Ubiquitin serves as a targeting signal for protein destruction
– 26S proteasome in the cytoplasm and nucleus
– Trafficking to the lysosome by proteins containing ubiquitin-binding
domains
• Large family of E2-E3 ligases participate in the regulation of
numerous cellular processes – diversity allows for specificity
• Recognition motif in a substrate for ubiquitination is called a degron
• Regulation of the process occurs through the availability of the
degron on the substrate or through activation of the E3 ligase
• Frequent interplay between ubiquitination and other protein
modifications
– Phosphorylation
– Proteolytic cleavage by caspases
Functional Consequences
•
•
•
•
•
Modulate enzyme activity
Change protein conformation
Alter protein-protein interactions
Regulate protein lifetime - ubiquitination
Regulate protein localization - lipidation
Protein Localization
Figure 2B.
From: Gonzalo et al. J. Biol.
Chem. 274: 21313-21318,
1999
SNAP-25-GFP
Figure 9.
From: Gonzalo et al. J. Biol.
Chem. 274: 21313-21318,
1999
Freely accessible at
http://www.jbc.org/content/
274/30/21313.full
Figure 4e.
From: Loranger & Linder. J.
Biol. Chem. 277: 3430334309, 2002
Freely accessible at
http://www.jbc.org/content/
277/37/34303.full
SNAP-25-GFP
cysteine cluster
mutant
Freely accessible at
http://www.jbc.org/content/
274/30/21313.full
SNAP-25 is anchored to the membrane by
palmitate attachment to a central cluster of
cysteine residues.
Signaling through
Heterotrimeric G Proteins
Figure 2.
From: Oldham & Hamm. Nature Rev.
Mol. Cell Biol. 9: 60-71, 2008
Available from
http://www.nature.com/nrm/journal/
v9/n1/abs/nrm2299.html
G protein Lipid Modifications
MYRISTOYLATION of Gai
*14-Carbon fatty acid
*N-terminal amide linkage to glycine
*Stable
PRENYLATION OF Gg
*C15 farnesyl or C20 geranylgeranyl
*C-terminal thioether linkage to cysteine
*Stable
PALMITOYLATION of Gai, Gas, Gaq, Ga12/13 – not transducin
*16-Carbon fatty acid
*Thioester linkage to cysteine
*Reversible
The Pheromone Response Pathway
a-factor
Ste2p
g
b
a
b g
a
GTP
GDP
MAP Kinase Cascade
Gpa1 – alpha subunit
N-terminal sequence – Met-Gly-CysN-myristoylation – essential; cells die
S-palmitoylation – partial loss of function
Ste18 – gamma subunit
C-terminal sequence – ---Cys-Thr-Leu-Met
Farnesyl modification – required for function;
cells are sterile
Ste12p
FUS 1
PRE
Cell Cycle Arrest
Polarity and Morphology
Cell and Nuclear Fusion
Adaptation and Recovery
Palmitoylation of Ga12 and Ga13
• Palmitoylated at Nterminal cysteine residues
– G12 H2N-MSGVVRTLSRC11
– G13 H2N-MAD--14CFPGC18
• Palmitoylation is
necessary for function in
cell migration,
proliferation, and
morphology
• G12/G13 regulates RhoA
by activating RhoGEFs
Figure 6 a-f.
From: Bhattacharyya &
Wedegaertner. J. Biol. Chem.
275: 14992-14999, 2000.
Freely available at
http://www.jbc.org/content/275
/20/14992.full?sid=be08e687d1d2-41df-9605-d1f355b120d4
Stress fiber formation assay
Lipid modifications of Ras Proteins
• All Ras proteins are
farnesylated
• H-Ras and N-Ras proteins
are farnesylated and
palmitoylated
• 2 signals for plasma
membrane targeting
• Yeast Ras is dually
lipidated
– -CCIIS motif at the Cterminus
Figure 1.
From: Hancock. Nat. Rev. Mol. Cell.
Biol. 4(5): 373-384, 2003.
Available at
http://www.nature.com/nrm/journal/
v4/n5/full/nrm1105.html
Post-translational Processing of Ras
Figure 2.
From: Linder & Deschenes. J. Cell Sci. 117: 521526, 2004
Freely accessible at
http://jcs.biologists.org/content/117/4/521.full
Ram1/Ram2; Farnesyltransferase in mammals (Ftase)
Rce1; Ras-converting enzyme in mammals (RCE1)
Ste14; isoprenylcarboxylmethyltransferase in mammals (ICMT)
Mouse knockouts of Ftase subunits, RCE1, and ICMT are embryonic lethal
Palmitoylation vs Phosphorylation
PAT
RasPalmitoylation
Figure 2.
From: Linder & Deschenes. J. Cell Sci. 117: 521526, 2004
Freely accessible at
http://jcs.biologists.org/content/117/4/521.full
• Genetic screen to identify factors required for Raspalmitoylation
identified ERF2 and ERF4
• Two hypotheses
• Trafficking factors that get Ras to the plasma membrane where
it’s palmitoylated
• An Erf2/Erf4 complex is a PAT
Bartels et al. Mol. Cell Biol. 19: 6775-6787, 1999
Available at http://mcb.asm.org/cgi/content/full/19/10/6775
Yeast RasPalmitoylation
Figure 2, right-hand portion
From: Linder & Deschenes.
J. Cell Sci. 117: 521-526, 2004
Figure 8A.
From: Bartels et al. Mol. Cell Biol.
19: 6775-6787, 1999
Freely accessible at
http://jcs.biologists.org/content/117/
4/521.full
Available at
http://mcb.asm.org/cgi/content/full
/19/10/6775
Figure 1C.
From: Lobo et al. J. Biol. Chem. 277:
41268-41273, 2002
Freely accessible at
http://www.jbc.org/content/277/43/
41268.full
• erf2D and erf4D cells have
reduced Ras2p
palmitoylation and plasma
membrane localization
• Erf2/Erf4 complex has PAT
activity for Ras in vitro
DHHC Proteins arePalmitoyltransferases
• Erf2 - Erf4(Shr5) complex palmitoylates Ras2
– CPalm-CFarn-OCH3
(Lobo et al. 2002)
• Akr1 palmitoylates Yck2 (Roth et al. JCB 2002)
– CPalm-CPalm
Cx2Cx3(R/K)PxRx2HCx2Cx4DHHCxW(V/I)xNC(I/V)Gx2Nx3F
Adapted from Mitchell et al. J. Lipid Res. 47: 1118-1127, 2006
Yeast DHHC Proteins
Erf2
Swf1
Pfa3
Pfa4
Pfa5
Akr1
Akr2
Ankyrin Repeat
DHHC-CRD
Human and Yeast DHHC Palmitoyltransferases
Figure 1b
From: Ohno, Y. et al. Biochim Biophys
Acta 1761(4):474-483, 2006
Available at
http://www.sciencedirect.com/science
/article/pii/S1388198106000709
Identification of Erf4 homologs
Human E4-1 = GCP16
Golgi complex protein of 16 kDa
51957-51967, 2003
Ohta et al. J. Biol. Chem. 278:
Purification of DHHC9/GCP16
Figure 6.
From: Swarthout et al. J. Biol. Chem. 280:31141–31148, 2005
Freely accessible at
http://www.jbc.org/content/280/35/31141.full
DHHC9/Gcp16 PalmitoylatesRas in vitro
Figure 7.
From: Swarthout et al. J. Biol. Chem. 280:31141–31148, 2005
Freely accessible at
http://www.jbc.org/content/280/35/31141.full
DHHC9/GCP16 - a human Ras PAT
• DHHC9/GCP16 has Ras PAT activity in vitro
• Localization of DHHC9/GCP16 in Golgi is consistent
with models of Ras trafficking
• In vivo? Likely to be other PATs
• Mutations in DHHC9 cause X-linked mental
retardation associated with a Marfanoidhabitus
– (Am. J. Hum. Genet. 80:982-987, 2007)
An Acylation and Deacylation Cycle Regulates Ras Localization
Figure 4.
From: Brunsveld et al. Biochim Biophys Acta
1788(1):273-288, 2009.
Available at
http://www.sciencedirect.com/science/article/pii/
S0005273608002447
1) Ras is palmitoylated at the Golgi apparatus by DHHC9/GCP16, other PATs?
2) Ras is transported to the plasma membrane by vesicular transport
3) At the plasma membrane, Ras is depalmitoylated by APT1/other thioesterases?
4) Retrograde transport of depalmitoylatedRas is diffusion-mediated
Scope of Protein Palmitoylation
•Receptors
–GPCRs
–Ion channels
•Transducers
–G protein a subunits
–Ras-family GTPases
–Src family kinases
•Effectors
–Adenylyl cyclase
–eNOS
•Regulators
–RGS proteins
–G protein receptor kinases
•Scaffolds/Anchoring proteins
–PSD-95
–AKAP18
–R7 binding protein
•Transporters
–Amino acid transporters
•Cell surface proteins
–Adhesion molecules
–Tetraspannins
•Protein trafficking
–SNAREs
–Synaptotagmins
–Caveolin
DHHC Proteins
Learning and memory
Neuronal deficits
X-linked Mental Retardation
Mental Retardation?
Huntingtin
Fukata and Fukata. Nature Rev. Neurosci. 11:
161-175, 2010
DHHC Proteins
Bladder cancer
Learning and memory
Neuronal deficits
Lymphoma
Colorectal cancer
Mental Retardation
Hair follicle differentiation
Metastasis?
Mental Retardation?
Huntingtin
Oncogenic
Alopecia, osteoporosis,
systemic amyloidosis
Pro-apototic
Summary of Protein Lipidation
• Prenylation and N-myristoylation are stable lipid modifications of
proteins
• Prenylation and N-myristoylation promote transient interactions
with membranes; in conjunction with palmitate, stable membrane
association ensues
• Palmitoylation is a reversible modification of integral and peripheral
membrane proteins
• Cycles of acylation and deacylation underlie Ras trafficking between
between the Golgi and plasma membrane
• There is no universal function for palmitoylation of integral
membrane proteins; palmitoylation can affect trafficking, stability,
or interaction with other proteins
• Proteins with a DHHC domain are palmitoylacyltransferases (PATs)