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MPHY616: Spring 2005 Ras / MAP kinase signaling

MPHY616: Spring 2005 Ras / MAP kinase signaling. Objectives: 1) Characterize known mechanisms of Ras activation. 2) Define known mechanisms of Ras-MAPK pathway coupling / activation / regulation. 3) Distinguish major MAP kinase pathways.

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MPHY616: Spring 2005 Ras / MAP kinase signaling

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  1. MPHY616: Spring 2005 Ras / MAP kinase signaling Objectives: 1) Characterize known mechanisms of Ras activation. 2) Define known mechanisms of Ras-MAPK pathway coupling / activation / regulation. 3) Distinguish major MAP kinase pathways. 4) Identify downstream MAP kinase targets / known functions. Paul Shapiro Room: 222 (Office) 269 (Lab) Pharmacy Hall Phone: 6-8522 (Office) 6-2980 (Lab) pshapiro@rx.umaryland.edu

  2. Mitogen activated protein (MAP) kinases • Biological responses regulated by MAP kinases: • Cell growth, proliferation, and senescence. • Cell differentiation and development. • Stress, injury, inflammation, immune response. • Neurological function (memory, emotion). • Most physiological systems. Lewis et al (1998) Advances in Cancer Research Vol. 74:49-139.

  3. Signal Transduction: Overview Extracellular signals (Hormones, growth factors…) SR RTK RS/TK CR Other Effects Other Effects Transcription RTK – Receptor tyrosine kinase. SR – Serpentine (seven transmembrane spanning) G-protein coupled receptors. RS/TK – Receptor serine/threonine kinase. CR – Cytokine receptor - Non-membrane cytosolic and nuclear receptors Ion-Channels.

  4. Overview: Ras / MAP kinase signaling Receptor EC Signal Ras Other pathways Adaptor proteins Exchange factors MKKKs MKKs Other signaling proteins Nuclear Txn factors MAPKs Structural proteins Kinases

  5. MAP Kinase Signaling Pathways Growth / Differentiation Stress Response Undefined Growth factors, neurotransmitters, vasoactive peptides, cytokines Inflammmatory cytokines, DNA damage, stress sensors RTK, G-protein coupled, cytokine MEKK1,2,3 MAPKKK5 Raf, Mos ??? TAK1 MLK/DLK ASK1 Tpl2 MKK1/2 MKK3/6 ??+MKK5 MKK4 ??? ?+Myoblast Diffn.

  6. Ras Family of GTPases 1) Activation mutants found in 30% of human tumors. 2) Membrane bound: C-terminal CAAX motif which is prenylated (farnesyl and geranylgeranyl modifications). 2) Human isoforms: H, K and N-ras Approx. 188-189 AAs (21 kD) 3) G protein cycle Inactive GDP-Ras Active GTP-Ras Guanine nucleotide Exchange Factors (GEFs) (GAPs) GTPase Activating Proteins

  7. RTK coupled activation of Ras EGF Ras pY pY SOS pY pY pY creates binding sites: Src homology 2 (SH2) domains. GAP SHC Grb2 Adaptor proteins: -SHC is tyrosine phosphorylated interacts with Grb2 -Grb2 and or SHC/Grb2 interact with SOS (Important for SOS function). -Grb2 Binding domains: SH2 (pY residue interactions) and SH3 domains (SOS interactions) Khosravi-Far et al. (1998) Adv Cancer Res. Vol.72:57-107.

  8. G-protein coupled receptors: MAP kinase regulation Stimuli: thrombin, endothelin, opiates, prostaglandins, epinephrine, histamine, hormones… Coupling to RTK. Gs Gq Gi Gas Gibg Gaq PKC PLCg RTKs PI3K PI3K AC PLCb PDK1 “Cell survival pathway” cAMP/PKA Ras/Raf-1 AKT1/2 B-Raf p70S6K, BAD, GSK3 ERK1/2 Daub et al. (1996) Nature. Vol. 379:557-60. Belcheva, and Coscia (2002) Neurosignals Vol.11:34-44.

  9. Ras coupling to MEKKs Ras/Raf Ras/MEKK Ras interactions with Raf: *Ras binding domain (RBD). (N-terminal region:residues 50-150) Requirement for Zinc finger domain *Membrane localization. Ras interactions with MEKK1: *Kinase domain (C-terminal) of MEKK1. *GTP dependent. MKK1/2 MKK3,4,6,7 MAPK (ERK1/2) MAPK (JNK,p38) Pumiglia et al. (1995) Mol Cell Biol. Vol. 15:398-406. Russell et al. (1995) J. Biol. Chem. Vol. 270:11757-60.

  10. Mechanisms of Raf Regulation / Activation • Ras interactions • Protein phosphorylation 1 648 Regulatory Catalytic N C S259,268,T269 S338 Y340,341 S499, S621 (+) (+) (+) (-) (+) (+) Pak1 Src, PKCa, PKA PKCa, CAP kinase (Ceramide Activating Protein kinase) CAP kinase = KSR (Kinase Suppressor of Ras) Raf/MKK/ERK scaffold? Morrison and Cutler (1997) Curr Opin Cell Biol. Vol. 9:174-179.

  11. Summary: Raf-1 phosphorylation sites PKC-a: S259, 268 (+?) CAP kinase? T269 (+) Pak1 S338 (+) Src Y340,341 (+) PKC-a S499 (+?) PKA S621 (-)

  12. Raf isoforms: c-Raf-1, A-Raf, B-Raf. Catalytic Regulatory 1 648 C N -66% of malignant melanomas contain B-Raf mutations. Of these mutations 80% are a single V599E mutation. -V599 located in kinase domain. Mutation increases Raf catalytic activity. • Davies et al. Nature (2002) Vol. 417(6892):949-54.

  13. Mechanisms of MKK1/2 Regulation and Activation. ERK binding (1-32) (ERK, Cdc2) T286, T292, T386 MKK1 392 S218MANS222 (Raf) K97 (ATP) E44QQKKRLE51 Proline rich 262-307 MKK2 400 K101 (ATP) S222MANS226 (Raf) N C E48QQKKRLE55 Proline rich 266-315 NES: ~residues 30-45 (see next page) Lewis et al (1998) Advances in Cancer Research Vol. 74:49-139.

  14. Subcellular Localization: Cytosolic vs. Nuclear NLS:Nuclear Localization Sequence Consensus: PKKKRKV (basic residues) NES:Nuclear Export Sequence Consensus: LXXLXXLXXL (Leucine rich) *MKKs have NES but no NLS* MKK1: L29EALQKKLEELEL41 MKK2: L34VDLQKKLEELEL46 NLS: Kalderon et al. (1984) Cell, Vol.39:499-509. NES: Fukuda et al. (1996) J.Biol.Chem. 271:20024-8.

  15. Mechanisms of MAP kinase (ERK) activation/regulation Cyt. Retention / Nuc. Trans. (MKK1/2 only) T183EY185 312-320 321-327 ERK2 358 C N K52 (ATP) L333,336,341,344 (Putative NES) Dimerization? Kinase Domain (23-311) ERK Localization to nucleus: 1. Observed following activation. 2. Requires pTpY but not activity. 3. Dimerization? 4. Cytosolic retention (MKK) or nuclear translocation sequence? (E312QYYDPSDE320PIAEAPF327) TXY activation motif: TPY - JNKs TGY - p38s Pearson et al. (2001) Endocr Rev. Vol. 22:153-83.

  16. Stress-Activated Protein Kinases: JNK / SAPK and p38 families of MAP kinases: (c-Jun N-terminal Kinase / Stress Activated Protein Kinase) Stress Inflammatory cytokines Growth factors LPS, IL-1, TNF, ionizing or UV irradiation, translation inhibitors, chemo. drugs, heat shock, osmolarity. MEKK1,4 MLKs ASK1 TAK MLKs ASK1 Coupled to RTKs or cytokine receptors via GCK. Regulated by Rac and Cdc42 cytoskeletal regulating G-proteins MKK4/7 MKK3/6 Phosphorylation regulation, TXY motif JNK1/2/3 p38a,b,g,d Tibbles and Woodgett (1999) Cell Mol Life Sci. Vol.55:1230-54.

  17. Cross talk between MAP kinase signaling pathways. Frost et al.(1997) The Embo J.Vol. 16:6426-6438. Coles and Shaw (2002) Oncogene. Vol.21:2236-2241. Mitogens, growth factors Cytokines, cellular stress? Ras Rac,Cdc42 Pak1 ? Raf-1 MEKK S338 MEK1/2 MKK S298 ERK1/2 JNK (+/-)Transcription

  18. MAP kinase pathway inactivation: (Mammalian) MKKKs 1) Regulation by phosphatases A. Tyrosine phosphatases: DSP-subfamily Dual Specificity Phosphatases (eg. MKPs) B. Serine/Threonine phosphatases PP1, PP2A etc. D. Cytosol / Nuclear / Cell type PP2A: Cyt. (MKK/ERK) MKP1,2: Nuc. MKP3: Cyt. 2) Negative feedback example: (SOS) MKKs (-) MAPKs

  19. Dual Specificity Phosphatases: MAP kinase regulation 1) MKP1 (CL100, 3CH134, hVH-1) ERK, p38, JNK 2) MKP2 (hVH-2, TYP-1) ERK, p38, JNK 3) MKP3 (rVH-6, PYST1) ERK 4) MKP4 ERK, p38, JNK 5) PAC1 ERK, p38 6) M3/6 p38, JNK DSP characteristics -Regulation by mitogen or stress stimuli. -Immediate early gene. -MKP1 promoter contains Sp1, AP1, AP2, CRE sites -ERKs target AP1 -JNK/p38 target CRE Camps et al. (2000) Faseb J. Vol. 14:6-16.

  20. MAP kinase targets 1) Consensus phosphorylation motif: PX(T/S)P -minimum (T/S)P 2) In vitro (in vivo?) targets: -Transcription factors -Other kinases -Cytoskeletal proteins -Other signaling proteins 3) Gain of function mutants: activate pathways. -Raf (CAAX) -MKK (D or E substitution of activating Serines and N-terminal deletions) 4) Catalytically inactive mutants: inhibit pathways.

  21. MEK1/2 inhibitors: Most common: PD98059, PD184352, U0126, PD98059PD184352U0126 PD184352 shown to be effective in inhibiting colon tumor growth in mouse xenografts. In phase I and II clinical trials. Type of inhibition? Non-competitive in respect to substrates. Favata et al., (1998) J. Biol. Chem. 273: 18623-32. Davies et al. (2000) Biochem. J. Vol. 351:95-105.

  22. Other MAP kinase pathway inhibitors: p38: SB202190 and SB 203580. PKC isoforms: Bisindolylmaleimides, rottlerin, etc... Raf-1: Geldanamycin Tyrosine kinases (growth factor receptors): many Major concern: Inhibitor specificity. Davies et al. (2000) Biochem J. Vol. 351:95-105.

  23. Kinases: Structure / function relationship ERK2 PKA

  24. Kinase – Substrate interactions (Docking domains) What determines substrate specificity and efficiency of phosphorylation? ERK2 and p38 docking domains: (shown for ERK2) Red: activating sites Blue: Asp316 and 319 (common docking, CD, domain). Green: Thr157 and 158 (ED domain) Tanoue et al. (2001) EMBO J. Vol. 20:466-79

  25. ERK docking domains: Common docking (CD) residues Asp316 and Asp319 as blue spheres and the ED residues Thr157 and Thr158 as green spheres and the sphere set defining the putative binding pocket as purple spheres.

  26. References: Belcheva, M.M., and Coscia, C.J. (2002). Diversity of G protein-coupled receptor signaling pathways to ERK/MAP kinase. Neurosignals 11, 34-44. Camps, M., Nichols, A., and Arkinstall, S. (2000). Dual specificity phosphatases: a gene family for control of MAP kinase function. Faseb J 14, 6-16. Coles, L.C., and Shaw, P.E. (2002). PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway. Oncogene 21, 2236-2244. Daub, H., Weiss, F.U., Wallasch, C., and Ullrich, A. (1996). Role of transactivation of the EGF receptor in signalling by G-protein- coupled receptors. Nature 379, 557-560. Davies, H., Bignell, G.R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin, H., Garnett, M.J., Bottomley, W., Davis, N., Dicks, E., Ewing, R., Floyd, Y., Gray, K., Hall, S., Hawes, R., Hughes, J., Kosmidou, V., Menzies, A., Mould, C., Parker, A., Stevens, C., Watt, S., Hooper, S., Wilson, R., Jayatilake, H., Gusterson, B.A., Cooper, C., Shipley, J., Hargrave, D., Pritchard-Jones, K., Maitland, N., Chenevix-Trench, G., Riggins, G.J., Bigner, D.D., Palmieri, G., Cossu, A., Flanagan, A., Nicholson, A., Ho, J.W., Leung, S.Y., Yuen, S.T., Weber, B.L., Seigler, H.F., Darrow, T.L., Paterson, H., Marais, R., Marshall, C.J., Wooster, R., Stratton, M.R., and Futreal, P.A. (2002). Mutations of the BRAF gene in human cancer. Nature 417, 949-954. Davies, S.P., Reddy, H., Caivano, M., and Cohen, P. (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351, 95-105. Favata, M.F., Horiuchi, K.Y., Manos, E.J., Daulerio, A.J., Stradley, D.A., Feeser, W.S., Van Dyk, D.E., Pitts, W.J., Earl, R.A., Hobbs, F., Copeland, R.A., Magolda, R.L., Scherle, P.A., and Trzaskos, J.M. (1998). Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273, 18623-18632. Frost, J.A., Steen, H., Shapiro, P., Lewis, T., Ahn, N., Shaw, P.E., and Cobb, M.H. (1997). Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins. Embo J 16, 6426-6438.

  27. References: Fukuda, M., Gotoh, I., Gotoh, Y., and Nishida, E. (1996). Cytoplasmic localization of mitogen-activated protein kinase kinase directed by its NH2-terminal, leucine-rich short amino acid sequence, which acts as a nuclear export signal. J Biol Chem 271, 20024-20028. Kalderon, D., Roberts, B.L., Richardson, W.D., and Smith, A.E. (1984). A short amino acid sequence able to specify nuclear location. Cell 39, 499-509. Khosravi-Far, R., Campbell, S., Rossman, K.L., and Der, C.J. (1998). Increasing complexity of Ras signal transduction: involvement of Rho family proteins. Adv Cancer Res 72, 57-107. Lewis, T.S., Shapiro, P.S., Ahn, N.G. (1998). Signal tranduction through MAP Kinase Cascades. Advances in Cancer Research 74, 49-139. Morrison, D.K., and Cutler, R.E. (1997). The complexity of Raf-1 regulation. Curr Opin Cell Biol 9, 174-179. Pearson, G., Robinson, F., Beers Gibson, T., Xu, B.E., Karandikar, M., Berman, K., and Cobb, M.H. (2001). Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22, 153-183. Pumiglia, K., Chow, Y.H., Fabian, J., Morrison, D., Decker, S., and Jove, R. (1995). Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction. Mol Cell Biol 15, 398-406. Russell, M., Lange-Carter, C.A., and Johnson, G.L. (1995). Direct interaction between Ras and the kinase domain of mitogen- activated protein kinase kinase kinase (MEKK1). J Biol Chem 270, 11757-11760. Tanoue, T., Adachi, M., Moriguchi, T., and Nishida, E. (2000). A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat Cell Biol 2, 110-116. Tibbles, L.A., and Woodgett, J.R. (1999). The stress-activated protein kinase pathways. Cell Mol Life Sci 55, 1230-1254.

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