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Increasing Complexity of the Ras Signaling Pathway

Increasing Complexity of the Ras Signaling Pathway THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 32, Issue of August 7, pp. 19925–19928, 1998 Minireview © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. suggests that Ras promotes more than just membrane trans- Increasing Complexity of the location of Raf and instead may also facilitate the subsequent Ras Signaling Pathway* events that lead to Raf-1 activation. Other components that contribute to Raf-1 activation include 14-3-3 proteins, phospho- Anne B. Vojtek‡ and Channing J. Der§ lipids, and serine/threonine and tyrosine kinases (15). There- fore, the connection between Ras and Raf alone is not simply From the ‡Department of Biological Chemistry, linear and requires multicomplex formation to complete Raf University of Michigan, Ann Arbor, Michigan 48109-0636 and §Department of Pharmacology, activation. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 Ras Targets Multiple Effectors Ras is likely to act through additional proteins besides Raf. Ras is a key regulator of cell growth in all eukaryotic cells. The earliest observations that Ras has multiple effector pro- Genetic, biochemical, and molecular studies in Caenorhabditis teins came from genetic studies in the budding yeast Saccha- elegans, Drosophila, and mammalian cells have positioned Ras romyces cerevisiae and later the fission yeast Schizosaccharo- centrally in signal transduction pathways that respond to di- myces pombe. Budding yeast devoid of Ras function were verse extracellular stimuli, including peptide growth factors, inviable, but yeast lacking adenylyl cyclase, an effector of Ras cytokines, and hormones. The biological activity of Ras is con- in this organism, were often capable of forming slow growing trolled by a regulated GDP/GTP cycle. Guanine nucleotide ex- microcolonies (16). This result suggested that Ras proteins in change factors (GEFs ; RasGRF1/2 and Sos1/2) promote the S. cerevisiae have an essential function other than the activa- formation of the active, GTP-bound form of Ras (1). GTPase- tion of adenylyl cyclase. In S. pombe, Ras directly interacts activating proteins (GAPs; p120 GAP and NF1) accelerate the with two effectors: Byr2, a MAPK kinase kinase, and Scd1, a intrinsic GTP hydrolytic activity of Ras to promote formation of GEF for the Rho family protein Cdc42 (17). Three additional the inactive, GDP-bound form of Ras (1). Mutations in Ras at observations in mammalian cells indicated that the events amino acids 12, 13, or 61 make Ras insensitive to GAP action downstream of Ras are more complex than simply activating and, hence, constitutively active in transforming mammalian the Raf kinase. First, activated Raf induces only a subset of the cells (2, 3). These activating mutations in Ras are prevalent in events mediated by activated Ras. For example, activated Ras a wide spectrum of human cancers. It has been estimated that activates three distinct MAPK cascades (ERK, JNK, p38), 30% of all human tumors contain an activating mutation in whereas Raf causes direct activation only of ERK (18, 19). Ras. The frequency of Ras mutations varies depending on tu- Second, activated Raf is not sufficient to promote all functions mor type, with the highest frequencies seen in lung, colon, of Ras, such as the transformation of some epithelial cells (20). thyroid, and pancreatic carcinomas (3). The frequency of Ras Third, studies with Ras mutants that discriminate between mutations is likely to be an underestimation of the contribution effectors suggest that multiple effector-mediated pathways are of aberrant signaling through the Ras pathway to human ma- important for establishing and maintaining the transformed lignancies because chronic up-regulation of the Ras pathway state (21, 48). can occur in the absence of mutations in Ras itself (4 – 6). A plethora of candidate Ras effectors in addition to Raf have been reported. These include p120 Ras GAP (22), GEFs for the Ras Directly Binds Raf and Activates a Kinase small GTPase Ral (RalGDS, RGL, RLF/RGL2) (23), AF6/Canoe Cascade (24, 25), RIN1 (26), and phosphatidylinositol 3-kinase (PI3K) Ras mediates its effects on cellular proliferation in part by (27). Although these candidate effectors comprise a very di- activation of a cascade of kinases: Raf (c-Raf-1, A-Raf, and verse collection of structurally and functionally distinct pro- B-Raf), MEK (MAPK/ERK kinases 1 and 2), and ERK1/2 (7). teins, they all show preferential affinity for active Ras-GTP. Upon activation, the ERKs phosphorylate cytoplasmic targets Therefore, it is not surprising that residues corresponding to (such as Rsk (8) and Mnk (9, 10)) and translocate to the nu- the switch I (Ras residues 30 –37) and II (residues 59 –76), cleus, where they stimulate the activity of various transcrip- which define the conformation differences between the GDP- tion factors that include the Elk-1 transcription factor (Fig. 1). and GTP-bound Ras, are involved in effector recognition. Spe- Ras activates this kinase cascade by directly binding to Raf (11, cifically, an intact core Ras effector domain (residues 32– 40) is 12). The binding of Ras to Raf requires active, GTP-bound Ras essential for all effector interactions. Mutation of residues in and an intact effector domain. The recent observation that Ras sequences flanking this region (spanning residues 25– 45) show interacts with two distinct NH -terminal regions of Raf-1 (RID/ differential impairment of effector interactions and provide RBS1, spanning residues 51–131 (13, 14) and Raf-CRD (14)) useful mutants to decipher the contribution of specific effectors for Ras function (28). Thus, Ras residues important for effector interaction are more extensive than originally believed. The * This minireview will be reprinted in the 1998 Minireview Compen- dium, which will be available in December, 1998. This is the first article interaction of Ras with candidate effectors is often direct (in- of five in the “Small GTPases Minireview Series.” teraction is observed in vitro using proteins purified from bac- ¶ To whom correspondence should be addressed. Tel.: 919-966-5634; teria). For some, the interaction with Ras is observed in vivo Fax: 919-966-0162. upon co-immunoprecipitation, but these experiments are often The abbreviations used are: GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; MAPK, mitogen-activated pro- done under conditions in which the Ras target is overex- tein kinase; ERK, extracellular receptor-stimulated kinase; MEK, pressed. To date, Raf is the only Ras target protein for which MAPK/ERK kinase; RID, Ras-interaction domain; RBS, Ras-binding genetic studies confirm its fundamental role in Ras signaling in site; CRD, cysteine-rich domain; JNK, Jun NH -terminal kinase; PI3K, a normal cellular context. Nonetheless, the interaction of Ras phosphatidylinositol 3-kinase; PH, pleckstrin homology; CAAX, cys- teine, aliphatic, aliphatic, terminal amino acid. with at least some of these target proteins is likely to be critical This paper is available on line at http://www.jbc.org 19925 This is an Open Access article under the CC BY license. 19926 Minireview: Ras Signaling cDNAs that were capable of suppressing the phenotypes asso- ciated with constitutive activation of the Ras pathway in S. cerevisiae (38). RIN1 interacts directly with Ras in a GTP- and effector domain-dependent fashion and localizes to the plasma membrane (26). Subsequently, RIN1 was shown to interact with Abl and Bcr/Abl in vitro and in vivo through a domain distinct from the Ras binding domain (39, 40). Moreover, RIN1 can enhance the transforming activity of Bcr/Abl and rescue several transformation-defective mutants of Bcr/Abl (40). The aspects of Ras function mediated by RIN1 are still the subject of investigation, but one possibility is that RIN1 coordinates signals from Ras and Abl. Biochemically, AF6/Canoe are candidate Ras effectors (25). In addition, genetic studies in Drosophila have linked Canoe to Ras in eye development (41). Canoe/AF6 have a GLG(F/D)HR motif, a conserved sequence found in proteins that associate with cellular junctions, so perhaps Canoe/AF6 coordinate sig- naling events at the plasma membrane to remodeling of the actin cytoskeleton. Finally, activation of PI3K, via a direct interaction between Ras and the catalytic subunit of the protein, is necessary for actin cytoskeletal rearrangements associated with the trans- formed phenotype (36). PI3K is a lipid kinase with specificity for the 3-position of the inositol ring. Activation of PI3K by a variety of extracellular stimuli leads to the accumulation of the second messenger phosphatidylinositol 3,4,5-trisphosphate. What are the downstream targets of this second messenger? FIG.1. Ras regulates a cascade of kinases. Ras is a GDP/GTP- One target is the serine/threonine kinase Akt/PKB. Binding of regulated binary switch that resides at the inner surface of the plasma Akt/PKB via its PH domain to phosphatidylinositol 3,4,5- membrane and acts to relay extracellular ligand-stimulated signals to trisphosphate localizes Akt/PKB to the plasma membrane and cytoplasmic signaling cascades. A linear pathway where Ras functions downstream of receptor tyrosine kinases (RTK) and upstream of a leads to a partial activation of its kinase activity (42). Akt/PKB cascade of serine/threonine kinases (Raf . MEK . ERK) provides a activity is further increased by phosphorylation on 2 residues complete link between the cell surface and the nucleus. Activated ERKs by two different kinases, one of which, PDK1, is itself a lipid- can translocate into the nucleus to phosphorylate and activate tran- regulated kinase (43). The events downstream of Akt/PKB are scription factors, such as Elk-1. Activated ERKs also phosphorylate substrates in the cytoplasm, including the Mnk kinase, and thus con- the subject of intense investigation in many laboratories. Akt/ tribute to translation initiation of mRNAs with structured 59-untrans- PKB phosphorylates and inactivates the pro-apoptotic protein lated regions. BAD (44, 45) but is likely to have additional substrates. Other targets of the products of PI3K include the PH domains of Vav for mediating the role of oncogenic Ras in malignant (46), SOS (37), and GRP1 (47). transformation. One of the more elegant approaches to understanding the contribution of each of the effector pathways to Ras-mediated Multiple Effector Pathways Contribute to transformation has been the use of Ras effector mutants that Ras-mediated Transformation are impaired in binding a specific target (21, 36, 48, 49). For What is the contribution of each of the known effector-medi- example, studies with effector domain mutants have revealed a ated pathways to malignant transformation? The current state bifurcation of the signaling pathways downstream of Ras lead- of affairs is depicted in Fig. 2. As described earlier, activation of ing to remodeling of the actin cytoskeleton and DNA synthesis. the Raf/ERK pathway, with its concomitant activation of tran- RasV12C40, an activated mutant of Ras with an alteration of scription factors, is essential for cell proliferation. The Ras tyrosine to cysteine at position 40 in the effector domain, is GTPase-activating protein, p120 GAP, in addition to negatively unable to bind Raf. This mutant fails to activate the ERK regulating Ras function may impinge on the Rho family via its cascade and cannot activate a Ras-responsive reporter con- association with p190, a GAP for Rho family members (29). struct, but it is capable of inducing membrane ruffling to the Activation of members of the Rho family of GTPases is likely to same extent as an activated Ras with an intact effector domain contribute significantly to the Ras-transformed phenotype (re- (49). These results suggest that stimulation of membrane ruf- viewed in Ref. 30). fling and activation of the ERK cascade are mediated by dis- The family of GEFs for Ral have also been implicated as tinct Ras effector proteins. Subsequently, RasV12C40 was target proteins for Ras (31, 32). A role for Ral in regulation of shown to bind to and activate PI3K, suggesting that Ras- phospholipase D and in actin cytoskeletal rearrangements (via induced morphological alterations may be mediated in part interaction with RalBP1) has been suggested (33, 34). In one through activation of PI3K (36). In addition to binding PI3K, report, RalA has been reported to cooperate with Ras for trans- formation (35), but others have not seen this cooperativity (36). RasV12C40 will also interact with AF6 (48), and a role for AF6 in modulation of the actin cytoskeleton by RasV12C40 cannot Perhaps the RalGDS targets other proteins in addition to Ral that can influence the transformed phenotype. There is prece- be excluded. Finally, the ability of this mutant to cause tumor- dence for multiple functions residing in GEFs; SOS facilitates igenic transformation demonstrates that Raf-independent the exchange of nucleotides on Ras and couples Ras to Rac pathways alone are sufficient to promote Ras transformation. through its Dbl and pleckstrin homology (PH) domains in a Two additional approaches to dissect the contributions of PI3K-dependent manner (37). this surfeit of candidate effector proteins to Ras function have RIN1 was identified in a genetic selection for mammalian been to overexpress or membrane-target a specific effector to Minireview: Ras Signaling 19927 FIG.2. A surfeit of candidate Ras effectors. Multiple effector pathways contribute to Ras function. Our current understanding of the downstream targets of each of the Ras effectors is shown in the figure (see text for details). PLD, phos- pholipase D; PIP3, phosphatidylinositol trisphosphate; MEKK, MEK kinase; SEK, SAPK/JNK kinase; SRF, serum response factor. see if this mimics any aspect of Ras function. Targeting to the origin, a pharmacological intervention that could switch a Ras- plasma membrane is often achieved by adding the sequence dependent survival signal into an apoptotic signal might be of containing the CAAX box of Ras to the effector of interest. considerable value in the treatment of human malignancies. Membrane targeting has been shown to cause constitutive ac- Summary tivation of Raf and PI3K. If membrane targeting of the candi- The last 5 years have seen an impressive expansion in the date effector does not reproduce any aspect of Ras function, it number of candidate Ras effectors. Much progress has been may be that the target protein under study is already consti- made toward deciphering the aspects of Ras function mediated tutively localized in this cellular compartment. Although Raf by each of these proteins, and many studies, in particular those and PI3K reside in the cytoplasm and become associated with with effector domain mutants, have convincingly demonstrated the plasma membrane upon receipt of stimulatory signal(s), that Ras must target at least three different pathways for other Ras target proteins, such as adenylyl cyclase in yeast and transformation. The corruption of the signaling pathways that the RalGDS, are constitutively membrane localized. Mem- lie downstream of Ras is a recurring theme in the initiation brane-targeted RalGDS did not exhibit any transforming po- and/or progression of human malignancies. Pharmacological tential. Finally, a powerful (but as yet not common) approach interventions have directly impeded Ras function by interfer- to decipher the role of a Ras target protein is to determine ing with its farnesylation and membrane targeting or have whether fibroblasts derived from mice deficient in a target blocked activation of components of the kinase cascade down- protein are impaired in transformation (for example, see Ref. stream of Ras (53). However, the function of Ras and its down- 50). stream kinase cascade is central to many cellular processes, Ras Mediates Life and Death Decisions by and this may limit the usefulness of these approaches. The Distinct Effector Pathways diversity of Ras target proteins and the necessity for activation One perplexing aspect of the Ras signaling pathway is that of multiple effector pathways for malignant transformation by Ras can promote both cell death and cell survival through Ras open new directions for the design of additional therapeu- interactions with distinct effector proteins. Using Ras mutants, tic interventions that may negate Ras transformation without Kauffmann-Zeh et al. (51) demonstrated that activation of Raf abolishing all of Ras function. by Ras promotes apoptosis in fibroblasts containing an induc- Acknowledgments—We thank Adrienne Cox and John Colicelli for ible c-Myc oncoprotein, whereas activation of PI3K by Ras helpful comments and Jennifer Parrish for assistance in figure and promotes cell survival. In this assay, oncogenic Ras enhanced manuscript preparations. apoptosis. 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Increasing Complexity of the Ras Signaling Pathway

Vojtek, Anne B.; Der, Channing J.
Journal of Biological Chemistry – Aug 1, 1998
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 32, Issue of August 7, pp. 19925–19928, 1998 Minireview © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. suggests that Ras promotes more than just membrane trans- Increasing Complexity of the location of Raf and instead may also facilitate the subsequent Ras Signaling Pathway* events that lead to Raf-1 activation. Other components that contribute to Raf-1 activation include 14-3-3 proteins, phospho- Anne B. Vojtek‡ and Channing J. Der§ lipids, and serine/threonine and tyrosine kinases (15). There- fore, the connection between Ras and Raf alone is not simply From the ‡Department of Biological Chemistry, linear and requires multicomplex formation to complete Raf University of Michigan, Ann Arbor, Michigan 48109-0636 and §Department of Pharmacology, activation. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 Ras Targets Multiple Effectors Ras is likely to act through additional proteins besides Raf. Ras is a key regulator of cell growth in all eukaryotic cells. The earliest observations that Ras has multiple effector pro- Genetic, biochemical, and molecular studies in Caenorhabditis teins came from genetic studies in the budding yeast Saccha- elegans, Drosophila, and mammalian cells have positioned Ras romyces cerevisiae and later the fission yeast Schizosaccharo- centrally in signal transduction pathways that respond to di- myces pombe. Budding yeast devoid of Ras function were verse extracellular stimuli, including peptide growth factors, inviable, but yeast lacking adenylyl cyclase, an effector of Ras cytokines, and hormones. The biological activity of Ras is con- in this organism, were often capable of forming slow growing trolled by a regulated GDP/GTP cycle. Guanine nucleotide ex- microcolonies (16). This result suggested that Ras proteins in change factors (GEFs ; RasGRF1/2 and Sos1/2) promote the S. cerevisiae have an essential function other than the activa- formation of the active, GTP-bound form of Ras (1). GTPase- tion of adenylyl cyclase. In S. pombe, Ras directly interacts activating proteins (GAPs; p120 GAP and NF1) accelerate the with two effectors: Byr2, a MAPK kinase kinase, and Scd1, a intrinsic GTP hydrolytic activity of Ras to promote formation of GEF for the Rho family protein Cdc42 (17). Three additional the inactive, GDP-bound form of Ras (1). Mutations in Ras at observations in mammalian cells indicated that the events amino acids 12, 13, or 61 make Ras insensitive to GAP action downstream of Ras are more complex than simply activating and, hence, constitutively active in transforming mammalian the Raf kinase. First, activated Raf induces only a subset of the cells (2, 3). These activating mutations in Ras are prevalent in events mediated by activated Ras. For example, activated Ras a wide spectrum of human cancers. It has been estimated that activates three distinct MAPK cascades (ERK, JNK, p38), 30% of all human tumors contain an activating mutation in whereas Raf causes direct activation only of ERK (18, 19). Ras. The frequency of Ras mutations varies depending on tu- Second, activated Raf is not sufficient to promote all functions mor type, with the highest frequencies seen in lung, colon, of Ras, such as the transformation of some epithelial cells (20). thyroid, and pancreatic carcinomas (3). The frequency of Ras Third, studies with Ras mutants that discriminate between mutations is likely to be an underestimation of the contribution effectors suggest that multiple effector-mediated pathways are of aberrant signaling through the Ras pathway to human ma- important for establishing and maintaining the transformed lignancies because chronic up-regulation of the Ras pathway state (21, 48). can occur in the absence of mutations in Ras itself (4 – 6). A plethora of candidate Ras effectors in addition to Raf have been reported. These include p120 Ras GAP (22), GEFs for the Ras Directly Binds Raf and Activates a Kinase small GTPase Ral (RalGDS, RGL, RLF/RGL2) (23), AF6/Canoe Cascade (24, 25), RIN1 (26), and phosphatidylinositol 3-kinase (PI3K) Ras mediates its effects on cellular proliferation in part by (27). Although these candidate effectors comprise a very di- activation of a cascade of kinases: Raf (c-Raf-1, A-Raf, and verse collection of structurally and functionally distinct pro- B-Raf), MEK (MAPK/ERK kinases 1 and 2), and ERK1/2 (7). teins, they all show preferential affinity for active Ras-GTP. Upon activation, the ERKs phosphorylate cytoplasmic targets Therefore, it is not surprising that residues corresponding to (such as Rsk (8) and Mnk (9, 10)) and translocate to the nu- the switch I (Ras residues 30 –37) and II (residues 59 –76), cleus, where they stimulate the activity of various transcrip- which define the conformation differences between the GDP- tion factors that include the Elk-1 transcription factor (Fig. 1). and GTP-bound Ras, are involved in effector recognition. Spe- Ras activates this kinase cascade by directly binding to Raf (11, cifically, an intact core Ras effector domain (residues 32– 40) is 12). The binding of Ras to Raf requires active, GTP-bound Ras essential for all effector interactions. Mutation of residues in and an intact effector domain. The recent observation that Ras sequences flanking this region (spanning residues 25– 45) show interacts with two distinct NH -terminal regions of Raf-1 (RID/ differential impairment of effector interactions and provide RBS1, spanning residues 51–131 (13, 14) and Raf-CRD (14)) useful mutants to decipher the contribution of specific effectors for Ras function (28). Thus, Ras residues important for effector interaction are more extensive than originally believed. The * This minireview will be reprinted in the 1998 Minireview Compen- dium, which will be available in December, 1998. This is the first article interaction of Ras with candidate effectors is often direct (in- of five in the “Small GTPases Minireview Series.” teraction is observed in vitro using proteins purified from bac- ¶ To whom correspondence should be addressed. Tel.: 919-966-5634; teria). For some, the interaction with Ras is observed in vivo Fax: 919-966-0162. upon co-immunoprecipitation, but these experiments are often The abbreviations used are: GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; MAPK, mitogen-activated pro- done under conditions in which the Ras target is overex- tein kinase; ERK, extracellular receptor-stimulated kinase; MEK, pressed. To date, Raf is the only Ras target protein for which MAPK/ERK kinase; RID, Ras-interaction domain; RBS, Ras-binding genetic studies confirm its fundamental role in Ras signaling in site; CRD, cysteine-rich domain; JNK, Jun NH -terminal kinase; PI3K, a normal cellular context. Nonetheless, the interaction of Ras phosphatidylinositol 3-kinase; PH, pleckstrin homology; CAAX, cys- teine, aliphatic, aliphatic, terminal amino acid. with at least some of these target proteins is likely to be critical This paper is available on line at http://www.jbc.org 19925 This is an Open Access article under the CC BY license. 19926 Minireview: Ras Signaling cDNAs that were capable of suppressing the phenotypes asso- ciated with constitutive activation of the Ras pathway in S. cerevisiae (38). RIN1 interacts directly with Ras in a GTP- and effector domain-dependent fashion and localizes to the plasma membrane (26). Subsequently, RIN1 was shown to interact with Abl and Bcr/Abl in vitro and in vivo through a domain distinct from the Ras binding domain (39, 40). Moreover, RIN1 can enhance the transforming activity of Bcr/Abl and rescue several transformation-defective mutants of Bcr/Abl (40). The aspects of Ras function mediated by RIN1 are still the subject of investigation, but one possibility is that RIN1 coordinates signals from Ras and Abl. Biochemically, AF6/Canoe are candidate Ras effectors (25). In addition, genetic studies in Drosophila have linked Canoe to Ras in eye development (41). Canoe/AF6 have a GLG(F/D)HR motif, a conserved sequence found in proteins that associate with cellular junctions, so perhaps Canoe/AF6 coordinate sig- naling events at the plasma membrane to remodeling of the actin cytoskeleton. Finally, activation of PI3K, via a direct interaction between Ras and the catalytic subunit of the protein, is necessary for actin cytoskeletal rearrangements associated with the trans- formed phenotype (36). PI3K is a lipid kinase with specificity for the 3-position of the inositol ring. Activation of PI3K by a variety of extracellular stimuli leads to the accumulation of the second messenger phosphatidylinositol 3,4,5-trisphosphate. What are the downstream targets of this second messenger? FIG.1. Ras regulates a cascade of kinases. Ras is a GDP/GTP- One target is the serine/threonine kinase Akt/PKB. Binding of regulated binary switch that resides at the inner surface of the plasma Akt/PKB via its PH domain to phosphatidylinositol 3,4,5- membrane and acts to relay extracellular ligand-stimulated signals to trisphosphate localizes Akt/PKB to the plasma membrane and cytoplasmic signaling cascades. A linear pathway where Ras functions downstream of receptor tyrosine kinases (RTK) and upstream of a leads to a partial activation of its kinase activity (42). Akt/PKB cascade of serine/threonine kinases (Raf . MEK . ERK) provides a activity is further increased by phosphorylation on 2 residues complete link between the cell surface and the nucleus. Activated ERKs by two different kinases, one of which, PDK1, is itself a lipid- can translocate into the nucleus to phosphorylate and activate tran- regulated kinase (43). The events downstream of Akt/PKB are scription factors, such as Elk-1. Activated ERKs also phosphorylate substrates in the cytoplasm, including the Mnk kinase, and thus con- the subject of intense investigation in many laboratories. Akt/ tribute to translation initiation of mRNAs with structured 59-untrans- PKB phosphorylates and inactivates the pro-apoptotic protein lated regions. BAD (44, 45) but is likely to have additional substrates. Other targets of the products of PI3K include the PH domains of Vav for mediating the role of oncogenic Ras in malignant (46), SOS (37), and GRP1 (47). transformation. One of the more elegant approaches to understanding the contribution of each of the effector pathways to Ras-mediated Multiple Effector Pathways Contribute to transformation has been the use of Ras effector mutants that Ras-mediated Transformation are impaired in binding a specific target (21, 36, 48, 49). For What is the contribution of each of the known effector-medi- example, studies with effector domain mutants have revealed a ated pathways to malignant transformation? The current state bifurcation of the signaling pathways downstream of Ras lead- of affairs is depicted in Fig. 2. As described earlier, activation of ing to remodeling of the actin cytoskeleton and DNA synthesis. the Raf/ERK pathway, with its concomitant activation of tran- RasV12C40, an activated mutant of Ras with an alteration of scription factors, is essential for cell proliferation. The Ras tyrosine to cysteine at position 40 in the effector domain, is GTPase-activating protein, p120 GAP, in addition to negatively unable to bind Raf. This mutant fails to activate the ERK regulating Ras function may impinge on the Rho family via its cascade and cannot activate a Ras-responsive reporter con- association with p190, a GAP for Rho family members (29). struct, but it is capable of inducing membrane ruffling to the Activation of members of the Rho family of GTPases is likely to same extent as an activated Ras with an intact effector domain contribute significantly to the Ras-transformed phenotype (re- (49). These results suggest that stimulation of membrane ruf- viewed in Ref. 30). fling and activation of the ERK cascade are mediated by dis- The family of GEFs for Ral have also been implicated as tinct Ras effector proteins. Subsequently, RasV12C40 was target proteins for Ras (31, 32). A role for Ral in regulation of shown to bind to and activate PI3K, suggesting that Ras- phospholipase D and in actin cytoskeletal rearrangements (via induced morphological alterations may be mediated in part interaction with RalBP1) has been suggested (33, 34). In one through activation of PI3K (36). In addition to binding PI3K, report, RalA has been reported to cooperate with Ras for trans- formation (35), but others have not seen this cooperativity (36). RasV12C40 will also interact with AF6 (48), and a role for AF6 in modulation of the actin cytoskeleton by RasV12C40 cannot Perhaps the RalGDS targets other proteins in addition to Ral that can influence the transformed phenotype. There is prece- be excluded. Finally, the ability of this mutant to cause tumor- dence for multiple functions residing in GEFs; SOS facilitates igenic transformation demonstrates that Raf-independent the exchange of nucleotides on Ras and couples Ras to Rac pathways alone are sufficient to promote Ras transformation. through its Dbl and pleckstrin homology (PH) domains in a Two additional approaches to dissect the contributions of PI3K-dependent manner (37). this surfeit of candidate effector proteins to Ras function have RIN1 was identified in a genetic selection for mammalian been to overexpress or membrane-target a specific effector to Minireview: Ras Signaling 19927 FIG.2. A surfeit of candidate Ras effectors. Multiple effector pathways contribute to Ras function. Our current understanding of the downstream targets of each of the Ras effectors is shown in the figure (see text for details). PLD, phos- pholipase D; PIP3, phosphatidylinositol trisphosphate; MEKK, MEK kinase; SEK, SAPK/JNK kinase; SRF, serum response factor. see if this mimics any aspect of Ras function. Targeting to the origin, a pharmacological intervention that could switch a Ras- plasma membrane is often achieved by adding the sequence dependent survival signal into an apoptotic signal might be of containing the CAAX box of Ras to the effector of interest. considerable value in the treatment of human malignancies. Membrane targeting has been shown to cause constitutive ac- Summary tivation of Raf and PI3K. If membrane targeting of the candi- The last 5 years have seen an impressive expansion in the date effector does not reproduce any aspect of Ras function, it number of candidate Ras effectors. Much progress has been may be that the target protein under study is already consti- made toward deciphering the aspects of Ras function mediated tutively localized in this cellular compartment. Although Raf by each of these proteins, and many studies, in particular those and PI3K reside in the cytoplasm and become associated with with effector domain mutants, have convincingly demonstrated the plasma membrane upon receipt of stimulatory signal(s), that Ras must target at least three different pathways for other Ras target proteins, such as adenylyl cyclase in yeast and transformation. The corruption of the signaling pathways that the RalGDS, are constitutively membrane localized. Mem- lie downstream of Ras is a recurring theme in the initiation brane-targeted RalGDS did not exhibit any transforming po- and/or progression of human malignancies. Pharmacological tential. Finally, a powerful (but as yet not common) approach interventions have directly impeded Ras function by interfer- to decipher the role of a Ras target protein is to determine ing with its farnesylation and membrane targeting or have whether fibroblasts derived from mice deficient in a target blocked activation of components of the kinase cascade down- protein are impaired in transformation (for example, see Ref. stream of Ras (53). However, the function of Ras and its down- 50). stream kinase cascade is central to many cellular processes, Ras Mediates Life and Death Decisions by and this may limit the usefulness of these approaches. The Distinct Effector Pathways diversity of Ras target proteins and the necessity for activation One perplexing aspect of the Ras signaling pathway is that of multiple effector pathways for malignant transformation by Ras can promote both cell death and cell survival through Ras open new directions for the design of additional therapeu- interactions with distinct effector proteins. Using Ras mutants, tic interventions that may negate Ras transformation without Kauffmann-Zeh et al. (51) demonstrated that activation of Raf abolishing all of Ras function. by Ras promotes apoptosis in fibroblasts containing an induc- Acknowledgments—We thank Adrienne Cox and John Colicelli for ible c-Myc oncoprotein, whereas activation of PI3K by Ras helpful comments and Jennifer Parrish for assistance in figure and promotes cell survival. In this assay, oncogenic Ras enhanced manuscript preparations. apoptosis. 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Published: Aug 1, 1998

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