Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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)