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The Ras-Raf relationship: an unfinished puzzle
Advan. Enzyme Regul., Vol. 41, pp. 261–267, 2001 # 2001 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0065-2571/01/$ - see front matter
PII: S0065-2571(00)00023-6
THE RAS-RAF RELATIONSHIP: AN UNFINISHED PUZZLE EUGEN KERKHOFF and ULF R. RAPP* Institut fu¨r Medizinische Strahlenkunde und Zellforschung (MSZ), Versbacher Str. 5, 97078 Wu¨rzburg, Germany INTRODUCTION
The Raf serine/threonine kinase, originally identified as an onco-protein, is a key element in cell fate determination (1, 2). Raf function is involved in the regulation of cell proliferation, differentiation and programmed cell death. The Raf kinase is a component of signal transduction cascades of a variety of different stimuli such as growth factors, cytokines and hormones (1, 3). It is well established that Raf transmits its signal via the phosphorylationdependent activation of Mek, which subsequently phosphorylates and activates the Erk MAP kinase (1). Despite 15 years of intensive research, resulting in more than 3000 publications, the mechanism of Raf activation is still an unfocused picture. Purification of the full length Raf kinase is one of the major problems. The lack of this essential tool makes biochemical studies on the protein structure very difficult. The function of the Ras GTPase is closely related to Raf and the Raf kinase was in fact the first effector element identified for Ras (4). Raf proteins have an N-terminal regulatory domain and a C-terminal kinase domain. Deletion of the N-terminal half of the proteins results in constitutively active oncogenic kinases (5–7). The GTP-dependent interaction of the Raf N-terminal regulatory domain with Ras was found to be necessary but not sufficient for Raf activation (30, 16, 17). Additional interaction partners and modifying enzymes are required for the induction of the enzymatic activity. Raf proteins are phosphorylated on serine, threonine and tyrosine amino acids (1, 17). Mutational analyses have shown that the phosphorylation status of the protein determines its activity. Here we summarize the recent progress of Raf research and discuss these results in respect to the increasing knowledge of the regulation mechanisms of related GTPase/effector interactions.
*Corresponding author. Fax: +49-931-201-3835; e-mail: [email protected] (U.R. Rapp).
262
E. KERKHOFF and U.R. RAPP THE RAS/RAF INTERACTION
A central question is the role of the Ras GTPase in the Raf activation process. Raf binds Ras in a GTP-dependent manner (4). The Ras/Raf interaction is mediated by two distinct contact regions (8, 9). The first contact is made by the so-called Raf RBD (Ras binding domain) encompassing amino acids 51–131 of the human c-Raf-1 protein (10). The Raf-RBD binds to the switch I region of Ras-GTP. The switch I and switch II regions of the Ras family of small GTPases change their conformation upon GDP/GTP exchange and thereby regulate the GTP-dependent binding of the effectors (9). The structure of the isolated Raf-RBD in complex with the Ras family protein Rap1A has been resolved by X-ray diffraction (10). Rap1A is a close homologue of Ras and has the same core effector region. The interaction is mediated by an inter-protein -sheet, which is formed by two anti-parallel strands from the associated proteins. The second contact is mediated by the Raf cysteine-rich domain (Raf-CRD) (8). The CRD encompasses amino acids 139–184 of the human c-Raf-1 protein and forms a zinc finger motif related to the C1 domains of protein kinase C (PKC) family proteins (11, 12). The structure of the isolated c-Raf-1 atypical C1 domain has been resolved by heteronuclear multidimensional NMR (12). A surface-scanning mutagenesis based on the structural data indicates that an epitope consisting of S177, T182 and M183 of the human c-Raf-1 protein contributes to the Ras/Raf interaction (8). The regions of the Ras protein involved in the interaction with the Raf-CRD are still unknown. Contact sites in addition to the switch region–effector interaction have also been found in the interaction of the Cdc42 small GTPase with its effectors Pak, Ack and WASP (25).
MEMBRANE LOCALIZATION AND ACTIVATION OF THE RAF KINASE
The Ras GTPase is membrane localized by a C-terminal farnesyl-anchor (13). Farnelysation of the Ras proteins is directed by the so-called CAAX motif. A fusion protein of the Ras-CAAX motif and the Raf kinase generates a constitutively active membrane localized kinase (14, 15). The activity of the Raf-CAAX protein was found to be independent of Ras function in those studies. These data in combination with experiments showing that the Ras/Raf interaction by itself is insufficient in activating the kinase (16, 17, 30) led to a model, in which the major function of Ras is the recruitment of Raf to the cellular membrane. More recent studies analyzing the mechanism of membrane localization and the Ras/Raf interaction in combination with novel structural data of the Raf-related Pak kinase indicate a more sophisticated role of Ras in the Raf activation process.
THE RAS-RAF RELATIONSHIP
263
The Raf kinase was shown to interact with two different phospholipids, phosphatidic acid and phosphatidylserine (19). By deletion mutagenesis, the phosphatidic acid binding site was mapped to the N-terminal end of the c-Raf1 kinase domain (human c-Raf-1 amino acids 389–423) (19). In a recent publication it has been shown that a Raf mutant unable to bind the Ras GTPase was recruited to the cellular membrane following insulin stimulation of cells (20). The membrane localization was dependent on the integrity of the phosphatidic acid binding motif. In those studies a dominant negative Ras protein did not prevent insulin-induced membrane localization but inhibited the activation of the Erk MAP kinase (20). These experiments indicate that Raf is translocated to the cellular membrane by its interaction with phosphatidic acid and that the Ras protein is necessary for the subsequent activation of the kinase. A mutant Ras GTPase (Ras12V, 37G), which cannot interact with cRaf-1, but with the c-Raf-1-257L mutant, is able to activate membrane targeted c-Raf-1-257L-CAAX but not the c-Raf-1-CAAX kinase (21). Since Ras 12V, 37G activates interaction dependent the membrane targeted c-Raf-1-257L kinase, these experiments indicate a function of Ras/Raf interaction in the activation process subsequent to the membrane localization. The binding site of phosphatidylserine is located in the atypical C1 zinc finger domain (CRD) of the Raf kinase. A cluster of basic amino acids within the zinc finger domain (human c-Raf-1 R143, K144 and K148) was shown to be necessary for phosphatidylserine binding (22). The interaction region corresponds to the diacyl-glycerol/phorbol esther binding region of the PKC family proteins (11, 12, 18). The phorbol binding promotes the insertion of the PKC proteins into membranes. The phosphatidylserine associated c-Raf-1 zinc finger domain might therefore be involved in the membrane association of the kinase. Rabphilin3A is an effector of the Ras related small GTPase Rab3A. In Rabphilin3A, the Rab3A switch-I and switch-II interaction regions are located N-terminal to a zinc finger motif closely related to FYVE domains (11, 23). The zinc finger domain of Raf is also adjacent to the Ras switch region interaction site (1, 17). The structure of the Rab3A/Rabphilin3A complex has been resolved by X-ray diffraction (23). The hydrophobic tip of the Rabphilin3A zinc finger is proposed to penetrate cellular membranes and by this orient the effector in the right direction for the interaction with the GTPase. In a similar scenario, the Raf zinc finger might also orient the Raf kinase to the plasma membrane to facilitate and enable the interaction with Ras.
REGULATION OF THE RAF RELATED P21-ACTIVATED KINASES
The p21-activated kinases are induced by their interaction with the small GTPase Cdc42 (24). Similar to Raf, the Pak kinases have a N-terminal
264
E. KERKHOFF and U.R. RAPP
FIG. 1. Model of Raf activation by Ras. We favor a model in which the Raf kinase is targeted by its interaction with phospholipids to cellular membranes. At the membrane the Raf kinase is attracted to activated Ras-GTP proteins. By the interaction with Ras the kinase domain becomes exposed and can interact with substrate proteins. Raf proteins bind phosphorylationdependent 14-3-3 proteins (17). 14-3-3 has been shown to be essential for Raf kinase activation but is also found in inactive Raf complexes. The 14-3-3 proteins might be necessary to stabilize the Raf conformations. Chemical-induced oligomerization activates Raf proteins (27, 28). We found that c-Raf-1 and B-Raf kinases can form heterodimers following Ras activation (C. K. Weber, J. R. Slupsky, H. A. Kalmes and U. R. Rapp, submitted). In addition, the Ras GTPase has been shown to dimerize (29). (RBD: Ras binding domain, CRD: cysteine-rich domain).
regulatory domain, mediating the GTPase interaction, and a C-terminal kinase domain. It has been shown by NMR studies, employing purified Cdc42 Q61L (1–184) and Pak (75–118) proteins, that Pak contacts the switch region of Cdc42 via its CRIB (Cdc and Rac interactive binding) motif (25). As the Ras/Raf switch/RBD interaction, the Pak CRIB/Cdc42 interaction surface forms an intermolecular -sheet. Next to the GTPdependent interaction, Pak makes an additional contact with the GTPase. Recently, the isolated N-terminal autoregulatory and the C-terminal kinase domain fragments of Pak1 have been co-crystallized (26). The structure analysis revealed that Pak1 forms an autoinhibited dimer. The dimerization is mediated via the CRIB domains. The N-terminal inhibitory loop switch domain associates tightly with the C loop of the kinase domain and thereby positions the kinase inhibitory segment to the cleft of the kinase domain. It has been proposed that the binding of the kinase to Cdc42 will trigger a
THE RAS-RAF RELATIONSHIP
265
series of conformational changes (26). These changes include the disruption of the dimer and the rearrangement of the kinase active site into a catalytically competent state. The structure and oligomerization status of the inactive Raf kinase are unknown. Based on early mutagenesis studies, showing that the deletion of the N-terminal regulatory part of the protein generates a constitutively active kinase (5, 6), the N-terminal Raf regulatory domain might fold in such a way that it inhibits the kinase activity. A model in which the Ras GTPase drives the structural rearrangement of the inhibited Raf kinase into its activated state seems to be very tempting (Fig. 1).
SUMMARY
Raf kinases interact with GTP-loaded Ras proteins. The Ras/Raf interaction is essential for the activation of the kinase. Based on recent data we favor a model in which the Raf kinase is located to cellular membranes by its interaction with phosphatidic acid and phosphatidylserine. At the cellular membrane Raf proteins are attracted to GTP loaded Ras GTPases which might trigger profound structural changes similar to those found in Cdc42-associated Pak1 kinases (Fig. 1).
1. 2. 3. 4. 5. 6. 7. 8.
9.
REFERENCES G. DAUM, I. EISENMANN-TAPPE, H. W. FRIES, J. TROPPMAIR and U. R. RAPP, The ins and outs of Raf-1 kinases, Trends Biochem. Sci. 19, 474–480 (1994). E. KERKHOFF and U. R. RAPP, Cell cycle targets of Ras/Raf signalling, Oncogene 17, 1457–1462 (1998). U. R. RAPP, Role of Raf-1 serine/threonine kinase in growth factor signal transduction, Oncogene 6, 495–500 (1991). J. AVRUCH, X. F. ZHANG and J. M. KYRIAKIS, Raf meets Ras: completing the framework of a signal transduction pathway, Trends Biochem. Sci. 19, 279–283 (1994). G. HEIDECKER, M. HULEIHEL, J. L. CLEVELAND, W. KO¨LCH, T. W. BECK, P. LLOYD, T. PAWSON and U. R. RAPP, Mutational activation of c-raf-1 and the definition of the minimal transforming sequence, Mol. Cell. Biol. 10, 2503–2512 (1990). V. P. STANTON, D. W. NICHOLAS, A. P. LAUDANO and G. M. COOPER, Definition of the human Raf amino-terminal regulatory region by deletion mutagenesis, Mol. Cell. Biol. 9, 639–647 (1989). C. WASYLYK, B. WASYLYK, G. HEIDECKER, M. HULEIHEL and U. R. RAPP, Expression of Raf oncogenes activates the PEA1 transcription factor motif, Mol. Cell. Biol. 5, 2247–2250 (1989). M. DAUB, J. JO¨CKEL, T. QUACK, C. K. WEBER, F. SCHMITZ, U. R. RAPP, A. WITTINGHOFER and C. BLOCK, The Raf C1 Cysteine-rich domain contains multiple distinct regulatory epitopes which control Ras-dependent Raf activation, Mol. Cell. Biol. 18, 6698–6710 (1998). A. WITTINGHOFER and N. NASSAR, How Ras related proteins talk to their effectors, Trends Biochem. Sci. 21, 488–491 (1996).
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E. KERKHOFF and U.R. RAPP
10. N. NASSAR, G. HORN, C. HERMANN, A. SCHERER, F. MCCORMICK and A. WITTINGHOFER, The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf-1 in complex with Rap1A and a GTP analogue, Nature 375, 554–560 (1995). 11. J. H. HURLEY and S. MISRA, Signalling and subcellular targeting by membranebinding domains, Annu. Rev. Biophys. Biomol. Struct. 29, 49–79 (2000). 12. H. R. MOTT, J. W. CARPENTER, S. ZHONG, S. GHOSH, R. M. BELL and S. L. CAMPBELL, The solution structure of the Raf-1 cysteine-rich domain, a novel Ras and phospholipid binding site, Proc. Natl. Acad. Sci. USA 93, 8312–8317 (1996). 13. M. D. SCHABER, M. B. O’HARA, V. M. GARSKY, S. C. MOSSER, J. D. BERGSTROM, S. L. MOORES, M. S. MARSHALL, P. A. FRIEDMAN, R. A. DIXON and J. B. GIBBS, Polyisoprenylation of Ras in vitro by a farnesyl-protein transferase, J. Biol. Chem. 265, 14701–14704 (1990). 14. S. J. LEEVERS, H. F. PATERSON and C. J. MARSHALL, Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane, Nature 369, 411–414 (1994). 15. D. STOKOE, S. G. MACDONALD, K. CADWALLADER, M. SYMONS and J. F. HANCOCK, Activation of Raf as a result of recruitment to the plasma membrane, Science 264, 1463–1467 (1994). 16. S. MIZUTANI, H. KOIDE and Y. KAZIRO, Isolation of a new protein factor required for activation of Raf-1 by Ha-Ras: partial purification from rat brain cytosols, Oncogene 16, 2781–2786 (1998). 17. D. K. MORRISON and R. E. CUTLER Jr., The complexity of Raf-1 regulation, Current Opinion Cell Biol. 9, 174–179 (1997). 18. G. ZHANG, M. G. KAZANIETZ, P. M. BLUMBERG and J. H. HURLEY, Crystal structure of the Cys2 activator-binding domain of protein kinase Cd in complex with phorbol ester, Cell 81, 917–924 (1995). 19. S. GHOSH, J. C. STRUM, V. A. SCIORRA, L. DANIEL and R. M. BELL, Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13acetate-stimulated Madin-Darby canine kidney cells, J. Biol. Chem. 271, 8472–8480 (1996). 20. M. A. RIZZO, K. SHOME, S. C. WATKINS and G. ROMERO, The recruitment of Raf1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras, J. Biol. Chem. 275, 23911–23918 (2000). 21. C. MINEO, R. G. ANDERSON and M. A. WHITE, Physical association with Ras enhances activation of membrane bound raf (RafCAAX), J. Biol. Chem. 272, 10345–10348 (1997). 22. T. IMPROTA-BREARS, S. GHOSH and R. M. BELL, Mutational analysis of Raf-1 cysteine rich domain: requirement for a cluster of basic aminoacids for interaction with phosphatidylserine, Mol. Cell. Biochem. 198, 171–178 (1999). 23. C. OSTERMEIER and A. T. BRUNGER, Structural basis of Rab effector specifity: crystal structure of the small G protein Rab3A complexed with the effector domain of Rabphilin-3A, Cell 96, 363–374 (1999). 24. R. H. DANIELS and G. M. BOKOCH, p21-activated protein kinase: a crucial component of morphological signalling?, Trends Biochem. Sci. 24, 350–355 (1999). 25. A. MORREALE, M. VENKATESAN, H. R. MOTT, D. OWEN, D. NIETLISPACH, P. N. LOWE and E. D. LAUE, Structure of Cdc42 bound to the GTPase binding domain of PAK1, Nature Struct. Bio. 7, 384–388 (2000). 26. M. LEI, W. LU, W. MENG, M. PARRINI, M. J. ECK, B. J. MAYER and S. C. HARRISON, Structure of PAK in an autoinhibited conformation reveals a multistage activation switch, Cell 102, 387–397 (2000). 27. M. A. FARRAR, J. ALBEROLA-ILA and R. M. PERLMUTTER, Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization, Nature 383, 178–181 (1996). 28. Z. LUO, G. TZIVION, P. J. BELSHAW, D. VAVVAS, M. MARSHALL and J. AVRUCH, Oligomerization activates c-Raf-1 through a Ras-dependent mechanism, Nature 383, 181–185 (1996).
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29. K. INOUYE, S. MIZUTANI, K. KOIDE and Y. KAZIRO, Formation of the Ras dimer is essential for Raf-activation, J. Biol. Chem. 275, 3737–3740 (2000). 30. X. -F. ZHANG, J. SETTLEMAN, J. M. KYRIAKIS, E. TAKEUCHI-SUZUKI, S. J. ELLEDGE, M. S. MARSHALL, J. T. BRUDER, U. R. RAPP and J. AVRUCH, Normal and oncogenic p21-Ras proteins bind to the aminoterminal regulatory domain of c-Raf-1, Nature 364, 308–313 (1993).
PII: S0065-2571(00)00023-6
THE RAS-RAF RELATIONSHIP: AN UNFINISHED PUZZLE EUGEN KERKHOFF and ULF R. RAPP* Institut fu¨r Medizinische Strahlenkunde und Zellforschung (MSZ), Versbacher Str. 5, 97078 Wu¨rzburg, Germany INTRODUCTION
The Raf serine/threonine kinase, originally identified as an onco-protein, is a key element in cell fate determination (1, 2). Raf function is involved in the regulation of cell proliferation, differentiation and programmed cell death. The Raf kinase is a component of signal transduction cascades of a variety of different stimuli such as growth factors, cytokines and hormones (1, 3). It is well established that Raf transmits its signal via the phosphorylationdependent activation of Mek, which subsequently phosphorylates and activates the Erk MAP kinase (1). Despite 15 years of intensive research, resulting in more than 3000 publications, the mechanism of Raf activation is still an unfocused picture. Purification of the full length Raf kinase is one of the major problems. The lack of this essential tool makes biochemical studies on the protein structure very difficult. The function of the Ras GTPase is closely related to Raf and the Raf kinase was in fact the first effector element identified for Ras (4). Raf proteins have an N-terminal regulatory domain and a C-terminal kinase domain. Deletion of the N-terminal half of the proteins results in constitutively active oncogenic kinases (5–7). The GTP-dependent interaction of the Raf N-terminal regulatory domain with Ras was found to be necessary but not sufficient for Raf activation (30, 16, 17). Additional interaction partners and modifying enzymes are required for the induction of the enzymatic activity. Raf proteins are phosphorylated on serine, threonine and tyrosine amino acids (1, 17). Mutational analyses have shown that the phosphorylation status of the protein determines its activity. Here we summarize the recent progress of Raf research and discuss these results in respect to the increasing knowledge of the regulation mechanisms of related GTPase/effector interactions.
*Corresponding author. Fax: +49-931-201-3835; e-mail: [email protected] (U.R. Rapp).
262
E. KERKHOFF and U.R. RAPP THE RAS/RAF INTERACTION
A central question is the role of the Ras GTPase in the Raf activation process. Raf binds Ras in a GTP-dependent manner (4). The Ras/Raf interaction is mediated by two distinct contact regions (8, 9). The first contact is made by the so-called Raf RBD (Ras binding domain) encompassing amino acids 51–131 of the human c-Raf-1 protein (10). The Raf-RBD binds to the switch I region of Ras-GTP. The switch I and switch II regions of the Ras family of small GTPases change their conformation upon GDP/GTP exchange and thereby regulate the GTP-dependent binding of the effectors (9). The structure of the isolated Raf-RBD in complex with the Ras family protein Rap1A has been resolved by X-ray diffraction (10). Rap1A is a close homologue of Ras and has the same core effector region. The interaction is mediated by an inter-protein -sheet, which is formed by two anti-parallel strands from the associated proteins. The second contact is mediated by the Raf cysteine-rich domain (Raf-CRD) (8). The CRD encompasses amino acids 139–184 of the human c-Raf-1 protein and forms a zinc finger motif related to the C1 domains of protein kinase C (PKC) family proteins (11, 12). The structure of the isolated c-Raf-1 atypical C1 domain has been resolved by heteronuclear multidimensional NMR (12). A surface-scanning mutagenesis based on the structural data indicates that an epitope consisting of S177, T182 and M183 of the human c-Raf-1 protein contributes to the Ras/Raf interaction (8). The regions of the Ras protein involved in the interaction with the Raf-CRD are still unknown. Contact sites in addition to the switch region–effector interaction have also been found in the interaction of the Cdc42 small GTPase with its effectors Pak, Ack and WASP (25).
MEMBRANE LOCALIZATION AND ACTIVATION OF THE RAF KINASE
The Ras GTPase is membrane localized by a C-terminal farnesyl-anchor (13). Farnelysation of the Ras proteins is directed by the so-called CAAX motif. A fusion protein of the Ras-CAAX motif and the Raf kinase generates a constitutively active membrane localized kinase (14, 15). The activity of the Raf-CAAX protein was found to be independent of Ras function in those studies. These data in combination with experiments showing that the Ras/Raf interaction by itself is insufficient in activating the kinase (16, 17, 30) led to a model, in which the major function of Ras is the recruitment of Raf to the cellular membrane. More recent studies analyzing the mechanism of membrane localization and the Ras/Raf interaction in combination with novel structural data of the Raf-related Pak kinase indicate a more sophisticated role of Ras in the Raf activation process.
THE RAS-RAF RELATIONSHIP
263
The Raf kinase was shown to interact with two different phospholipids, phosphatidic acid and phosphatidylserine (19). By deletion mutagenesis, the phosphatidic acid binding site was mapped to the N-terminal end of the c-Raf1 kinase domain (human c-Raf-1 amino acids 389–423) (19). In a recent publication it has been shown that a Raf mutant unable to bind the Ras GTPase was recruited to the cellular membrane following insulin stimulation of cells (20). The membrane localization was dependent on the integrity of the phosphatidic acid binding motif. In those studies a dominant negative Ras protein did not prevent insulin-induced membrane localization but inhibited the activation of the Erk MAP kinase (20). These experiments indicate that Raf is translocated to the cellular membrane by its interaction with phosphatidic acid and that the Ras protein is necessary for the subsequent activation of the kinase. A mutant Ras GTPase (Ras12V, 37G), which cannot interact with cRaf-1, but with the c-Raf-1-257L mutant, is able to activate membrane targeted c-Raf-1-257L-CAAX but not the c-Raf-1-CAAX kinase (21). Since Ras 12V, 37G activates interaction dependent the membrane targeted c-Raf-1-257L kinase, these experiments indicate a function of Ras/Raf interaction in the activation process subsequent to the membrane localization. The binding site of phosphatidylserine is located in the atypical C1 zinc finger domain (CRD) of the Raf kinase. A cluster of basic amino acids within the zinc finger domain (human c-Raf-1 R143, K144 and K148) was shown to be necessary for phosphatidylserine binding (22). The interaction region corresponds to the diacyl-glycerol/phorbol esther binding region of the PKC family proteins (11, 12, 18). The phorbol binding promotes the insertion of the PKC proteins into membranes. The phosphatidylserine associated c-Raf-1 zinc finger domain might therefore be involved in the membrane association of the kinase. Rabphilin3A is an effector of the Ras related small GTPase Rab3A. In Rabphilin3A, the Rab3A switch-I and switch-II interaction regions are located N-terminal to a zinc finger motif closely related to FYVE domains (11, 23). The zinc finger domain of Raf is also adjacent to the Ras switch region interaction site (1, 17). The structure of the Rab3A/Rabphilin3A complex has been resolved by X-ray diffraction (23). The hydrophobic tip of the Rabphilin3A zinc finger is proposed to penetrate cellular membranes and by this orient the effector in the right direction for the interaction with the GTPase. In a similar scenario, the Raf zinc finger might also orient the Raf kinase to the plasma membrane to facilitate and enable the interaction with Ras.
REGULATION OF THE RAF RELATED P21-ACTIVATED KINASES
The p21-activated kinases are induced by their interaction with the small GTPase Cdc42 (24). Similar to Raf, the Pak kinases have a N-terminal
264
E. KERKHOFF and U.R. RAPP
FIG. 1. Model of Raf activation by Ras. We favor a model in which the Raf kinase is targeted by its interaction with phospholipids to cellular membranes. At the membrane the Raf kinase is attracted to activated Ras-GTP proteins. By the interaction with Ras the kinase domain becomes exposed and can interact with substrate proteins. Raf proteins bind phosphorylationdependent 14-3-3 proteins (17). 14-3-3 has been shown to be essential for Raf kinase activation but is also found in inactive Raf complexes. The 14-3-3 proteins might be necessary to stabilize the Raf conformations. Chemical-induced oligomerization activates Raf proteins (27, 28). We found that c-Raf-1 and B-Raf kinases can form heterodimers following Ras activation (C. K. Weber, J. R. Slupsky, H. A. Kalmes and U. R. Rapp, submitted). In addition, the Ras GTPase has been shown to dimerize (29). (RBD: Ras binding domain, CRD: cysteine-rich domain).
regulatory domain, mediating the GTPase interaction, and a C-terminal kinase domain. It has been shown by NMR studies, employing purified Cdc42 Q61L (1–184) and Pak (75–118) proteins, that Pak contacts the switch region of Cdc42 via its CRIB (Cdc and Rac interactive binding) motif (25). As the Ras/Raf switch/RBD interaction, the Pak CRIB/Cdc42 interaction surface forms an intermolecular -sheet. Next to the GTPdependent interaction, Pak makes an additional contact with the GTPase. Recently, the isolated N-terminal autoregulatory and the C-terminal kinase domain fragments of Pak1 have been co-crystallized (26). The structure analysis revealed that Pak1 forms an autoinhibited dimer. The dimerization is mediated via the CRIB domains. The N-terminal inhibitory loop switch domain associates tightly with the C loop of the kinase domain and thereby positions the kinase inhibitory segment to the cleft of the kinase domain. It has been proposed that the binding of the kinase to Cdc42 will trigger a
THE RAS-RAF RELATIONSHIP
265
series of conformational changes (26). These changes include the disruption of the dimer and the rearrangement of the kinase active site into a catalytically competent state. The structure and oligomerization status of the inactive Raf kinase are unknown. Based on early mutagenesis studies, showing that the deletion of the N-terminal regulatory part of the protein generates a constitutively active kinase (5, 6), the N-terminal Raf regulatory domain might fold in such a way that it inhibits the kinase activity. A model in which the Ras GTPase drives the structural rearrangement of the inhibited Raf kinase into its activated state seems to be very tempting (Fig. 1).
SUMMARY
Raf kinases interact with GTP-loaded Ras proteins. The Ras/Raf interaction is essential for the activation of the kinase. Based on recent data we favor a model in which the Raf kinase is located to cellular membranes by its interaction with phosphatidic acid and phosphatidylserine. At the cellular membrane Raf proteins are attracted to GTP loaded Ras GTPases which might trigger profound structural changes similar to those found in Cdc42-associated Pak1 kinases (Fig. 1).
1. 2. 3. 4. 5. 6. 7. 8.
9.
REFERENCES G. DAUM, I. EISENMANN-TAPPE, H. W. FRIES, J. TROPPMAIR and U. R. RAPP, The ins and outs of Raf-1 kinases, Trends Biochem. Sci. 19, 474–480 (1994). E. KERKHOFF and U. R. RAPP, Cell cycle targets of Ras/Raf signalling, Oncogene 17, 1457–1462 (1998). U. R. RAPP, Role of Raf-1 serine/threonine kinase in growth factor signal transduction, Oncogene 6, 495–500 (1991). J. AVRUCH, X. F. ZHANG and J. M. KYRIAKIS, Raf meets Ras: completing the framework of a signal transduction pathway, Trends Biochem. Sci. 19, 279–283 (1994). G. HEIDECKER, M. HULEIHEL, J. L. CLEVELAND, W. KO¨LCH, T. W. BECK, P. LLOYD, T. PAWSON and U. R. RAPP, Mutational activation of c-raf-1 and the definition of the minimal transforming sequence, Mol. Cell. Biol. 10, 2503–2512 (1990). V. P. STANTON, D. W. NICHOLAS, A. P. LAUDANO and G. M. COOPER, Definition of the human Raf amino-terminal regulatory region by deletion mutagenesis, Mol. Cell. Biol. 9, 639–647 (1989). C. WASYLYK, B. WASYLYK, G. HEIDECKER, M. HULEIHEL and U. R. RAPP, Expression of Raf oncogenes activates the PEA1 transcription factor motif, Mol. Cell. Biol. 5, 2247–2250 (1989). M. DAUB, J. JO¨CKEL, T. QUACK, C. K. WEBER, F. SCHMITZ, U. R. RAPP, A. WITTINGHOFER and C. BLOCK, The Raf C1 Cysteine-rich domain contains multiple distinct regulatory epitopes which control Ras-dependent Raf activation, Mol. Cell. Biol. 18, 6698–6710 (1998). A. WITTINGHOFER and N. NASSAR, How Ras related proteins talk to their effectors, Trends Biochem. Sci. 21, 488–491 (1996).
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