[Frontiers in Bioscience 3, d887-912, August 6, 98]

	[Frontiers in Bioscience 3, d887-912, August 6, 98] Reprints PubMed CAVEAT LECTOR
	RAS PATHWAYS TO CELL CYCLE CONTROL AND CELL TRANSFORMATION Marcos Malumbres and Angel Pellicer Department of Pathology and Kaplan Comprehensive Cancer Center, New York University Medical Center, 550 First Avenue, New York Received 7/17/98 Accepted 7/25/98 4. UPSTREAM OF Ras 4.1. Signal transduction from external factors to Ras Ras is a point of convergence of many signaling pathways from a diverse array of extracellular signals, such as growth factors, cytokines, hormones and neurotransmitters that stimulate cell surface receptors. Basically, extracellular signals that activate Ras proteins can be mediated through different types of receptors: tyrosine kinase receptors (e.g. platelet derived growth factor (PDGF) receptor or epidermal growth factor (EGF) receptor), cytokine receptors (e.g. IL-2 receptor), T cell receptors, and subunits of heterotrimeric G proteins. In mammalian cells, Erk activation from tyrosine kinase receptors is the most studied route of the Ras transduced pathways. Many Src Homology 2 (SH2)-containing proteins bind to the phosphotyrosine residues of the activated tyrosine kinase receptors, including Grb2, Shc, PI3K, phospholipase C-g (PLC-g), p120-GAP and SH-PTP2 tyrosine phosphatase (Syp) (76, 77). Both Grb2 and Shc proteins have been demonstrated to contribute to Ras activation through the direct binding between Grb2 and Sos and the subsequent activation of the Ras exchange factor Sos (see below). Heterotrimeric guanine triphosphate-binding proteins (G proteins), can stimulate the Ras/Erk pathway through the Gbg subunits. This stimulation is inhibited by wortmannin (a PI3K inhibitor), whereas stimulation of this pathway by tyrosine kinase receptors such as the epidermal growth factor receptor is insensitive to wortmannin (78). This suggests a role for PI3K in Ras activation by heterotrimeric G protein-coupled receptors. This effect is dependent on PI3Kg, a PI3K isotype activated by both the a and bg subunits of heterotrimeric G proteins (78, 79). A different intriguing example is the MAP kinase pathway activation by protein kinase C. By using the Ras dominant negative, Ras17N (80), this activation has been shown to by Ras-dependent in some cell types such as NIH 3T3 cells (81) but Ras-independent in a number of other cell types (82) Recently, Raf-1 activation by PKC has been shown to be mediated through Ras activation. However, this activation differs from activation by receptor tyrosine kinases in that it is not blocked by Ras17N, the "dominant negative" form of Ras, in COS cells (82). These results indicate that Ras is a component of PKC signaling and that the absence of an effect of Ras17N expression cannot be used to conclude that Ras is not part of a signaling pathway in a specific cell type. However, since Raf-1 seems to associate In vivo preferentially with N-Ras but not H-Ras (75), the role of the three isoforms in PKC signaling can be different. In fact, cells deficient in N-Ras, but not in the other Ras isoforms, seem to have a decreased response to the PKC-dependent PMA stimulation (M. M. & A. P., unpublished data), suggesting an important role for N-Ras in PKC signaling. 4.2. Ras activation: Nucleotide exchange factors for Ras In order to facilitate the exchange of GTP for GDP, the Guanine Exchange Factor (GEF) must first form a complex with the GDP bound form of the GTPase. GDP is then released from the complex and the high GTP/GDP intracellular ratio ensures that the released nucleotide is immediately replaced with GTP. GEF then dissociates leaving the Ras in the active form (14, 83). Various GEFs have been isolated for the Ras subfamily members, such as Sos, RasGRF, RasGRF2, RasGRP, C3G, and RalGDS-related sequences (43, 84-86). A mammalian GEF, related in sequence to the Saccharomyces cerevisiae Ras exchange factor CDC25, was isolated first and named RasGRF (or Cdc25Mn). The related proteins Sos1 and Sos2 are most closely related to the Drosophila son-of-sevenless product. Sos is known to stimulate the GTP exchange on Ras (87), while GRF has been shown to activate both Ras (88) and R-Ras (89). C3G has recently been shown to possess exchange activity on both K-rev-1/Rap1A and R-ras (89). Finally, RalGDS-related sequences include a growing family of proteins with exchange activities for the Ral GTPases. Interestingly, most of these RalGDS proteins have been shown to be putative Ras effectors (see below). Only some of these proteins (Sos, RasGRF, RasGRF2, RasGRP, Vav, C3G and SmgGDS) have been reported to act as exchange factors for the true Ras proteins, although Vav and C3G rather seem to act in vivo as exchange factors for other GTPases than Ras. Their properties are discussed below. 4.2.1. Sos The Sos1 and Sos2 proteins are widely expressed in mouse and human tissues and are related to the Drosophila son-of-sevenless product, which functions upstream of Ras in the R7 cell development of the Drosophila eye (90, 91). A central region of about 200 amino acids in the mSos1 protein is similar to a comparable region of Cdc25, Sdc25 and Ste6 (43). This domain is known to catalyze exchange activity on H-Ras and N-Ras, but not RalA or Cdc42 (92). The C-terminal proline-rich region of Sos binds to the Src Homology 3 (SH3) domains of Grb2 and the Sos-Grb2 complex interacts with the activated tyrosine kinase receptors, as the epidermal growth factor (EGF) receptor, through the SH2 domain of Grb2, thus linking external growth factor-tyrosine kinases signaling and Ras activation (93-97). In addition to the Cdc25 domain, responsible for the Ras-GEF activity, and the Grb2-binding domain, Sos proteins possess Dbl homology (DH) and pleckstrin homology (PH) domains arranged in tandem in the N-terminal region. DH domains have been demonstrated to promote guanine nucleotide exchange on Rho family GTPases, whereas PH domains participate in protein-protein and protein-lipid interactions, binding tightly and specifically to phosphatidylinositol derivatives. Both the Grb2- and the DH-binding domains have been recently shown to have autoinhibitory effects on the intrinsic GEF activity of the Cdc25 domain (98). The DH domain of Sos is able to stimulate guanine nucleotide exchange on Rac but not Cdc42. Both the exchange activity on Rac and Ras are inhibited by binding of the PH domain to phosphatidylinositol-4,5-biphosphate (PtdIns[4,5]P2), a substrate of the phosphatidylinositide 3-kinase (PI3K) and this activity is regained when coexpressed with activated Ras (99, 100). Thus, Sos may couple Ras and Rac activation through its different domains and this interactions may be dependent on PI3K signaling. In addition to tyrosine kinase-dependent activation of the Sos-Ras pathway, a different mechanism seems to be involved in the PKC-dependent activation of Ras. In NIH 3T3 cells, the PKC-dependent activation of the Ras-Erk signaling pathway by PMA is suppressed by the dominant negative Raf1 and, in some cell types, also by the dominant negative forms of Ras and Sos. The PMA signal seems to be fed at or upstream of Shc and involves serine (but not tyrosine, as it occurs in the growth factor-induced Shc phosphorylation) residues (81). 4.2.2. RasGRF This protein was identified as homologous to the Saccharomyces cerevisiae Ras exchange factor encoded by CDC25 (101, 102). RasGRF, also named Cdc25Mm, contains Dbl homology (DH) and pleckstrin homology (PH) domains and is expressed specifically in brain. Both GRF and Sos are expressed in several different protein forms by alternative splicing. The heterogeneity of GRF and Sos products may therefore contribute to a fine regulation of Ras activation in different tissues or at different stages of development (103). RasGRF has been recently shown to activate H-Ras but not N-Ras or K-RasB proteins In vivo (64). 4.2.3. RasGRF2 RasGRF2 is a new multidomain protein catalyzing the specific release of GDP from Ras but not other GTPases (85). This protein contains a Cdc25-related domain in the COOH terminus of the protein. In the NH₂-terminal region, it contains several domains with homology to various signaling proteins including two pleckstrin homology domains, a Dbl homology region, and an IQ motif required for its apparent constitutive association with calmodulin. Accordingly, calcium influx caused a shift of RasGRF2 subcellular localization from cytosolic to the cell periphery, stimulating GTP binding by Ras and potentiating calcium ionophore-induced activation of Erk (85). 4.2.4. RasGRP This recently isolated member of the Ras nucleotide exchange factor family is characterized by an atypical pair of "EF hands" that bind calcium and a diacylglycerol (DAG)-binding domain. RasGRP activates Ras and the Erk pathway in response to DAG through the DAG-binding domain. This exchange factor is expressed in the nervous system suggesting a role in the coupling of changes in DAG and calcium concentrations to Ras activation (86). 4.2.5. Some considerations on SmgGDS, Vav and C3G Some other exchange factors have been thought to participate on Ras activation. Although Vav has similar domains to the Dbl family proteins, unexpectedly, a GEF activity specific for Ras rather than Rho subfamily members was described (reviewed in Ref. 104). Several observations have shown, however, that Vav is not a true exchange factor for Ras. The morphological changes that are observed in Vav-expressing NIH 3T3 cells are distinct from those seen when the same cells are expressing other Ras GEFs such as RasGRF, suggesting that the Vav-associated oncogenic activity may not be a consequence of Ras GEF activity (105). Moreover, no increase in the level of Ras-GTP is detected in cells transformed with Vav (106). Recently, Vav protein has been shown to bind and stimulate GDP release from Rac1 and Cdc42, and to a lesser degree RhoA, but not from Ras (107, 108). This lack of Vav protein exchange on Ras is in agreement with the earlier results indicating a Ras-independent transformation by oncogenic Vav (105). Importantly, Vav has been shown to play an important role on Rac activation regulated by the substrates and products of phosphoinositide 3-kinase (PI3K) (109). C3G is an exchange factor which contains a Cdc25 homology domain and complements yeast cells with the cdc25 mutation; however, it has been shown to be a specific GEF for the Ras-related protein Rap1. On the other hand, SmgGDS is a protein that catalyzes exchange on K-Ras4B and other Ras-related GTPases, but not H-Ras (63). The relative contribution of these proteins to Ras regulation remains to be clarified. 4.3. Ras inactivation by GAPs The Ras GTPase activity results in the slow hydrolysis of bound GTP, leaving the protein complexed with GDP. However, the intrinsic rate of dissociation of the Ras-GTP complex is very low, approximately 10^-5 moles per sec dissociating per mole of complex, and since the intracellular GTP concentration is much greater than that of GDP, it seemed clear that additional regulatory proteins should be involved in the Ras GTPase activity. A cytosolic protein fraction was found to be able to stimulate the hydrolysis of GTP bound to Ras (110, 111). A 120 kDa protein, named p120-GAP for GTPase Activating Protein, was initially purified and the corresponding cDNA cloned. This protein binds preferentially to Ras-GTP and this binding is impaired in some Ras effector mutants. At least six different GAPs have been found to date in mammals: p120-GAP, the neurofibromatosis type 1 protein NF1 (112, 113), GAP1m, GAP1IP4BP (114), IQGAP1 (115, 116) and SynGAP, a Ras GAP selectively expressed at excitatory synapses in the brain (117, 118). All these proteins negatively regulate Ras through their GTPase stimulatory activities. Accordingly, overexpression of GAP is associated with a reduction of endogenous Ras in the GTP-bound form and can prevent transformation by overexpression of normal Ras or by other oncogenes whose transforming activities depend on endogenous Ras (42, 119). However, GAP does not affect to the transforming potential of some Ras mutants, such as those with mutations in codons 12, 13 or 61. Analysis of these mutants showed that mutations in the effector domain of Ras render the protein unable to respond to GAP, and they are therefore constitutively in the GTP-bound form (7, 44). In addition to their GTPase stimulating activity, a putative effector role for p120GAP and NF1 has been suggested (see below).

[Frontiers in Bioscience 3, d887-912, August 6, 98]
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CAVEAT LECTOR

RAS PATHWAYS TO CELL CYCLE CONTROL AND CELL TRANSFORMATION

Marcos Malumbres and Angel Pellicer

Department of Pathology and Kaplan Comprehensive Cancer Center, New York University Medical Center, 550 First Avenue, New York

Received 7/17/98 Accepted 7/25/98

4. UPSTREAM OF Ras

4.1. Signal transduction from external factors to Ras

Ras is a point of convergence of many signaling pathways from a diverse array of extracellular signals, such as growth factors, cytokines, hormones and neurotransmitters that stimulate cell surface receptors. Basically, extracellular signals that activate Ras proteins can be mediated through different types of receptors: tyrosine kinase receptors (e.g. platelet derived growth factor (PDGF) receptor or epidermal growth factor (EGF) receptor), cytokine receptors (e.g. IL-2 receptor), T cell receptors, and subunits of heterotrimeric G proteins. In mammalian cells, Erk activation from tyrosine kinase receptors is the most studied route of the Ras transduced pathways. Many Src Homology 2 (SH2)-containing proteins bind to the phosphotyrosine residues of the activated tyrosine kinase receptors, including Grb2, Shc, PI3K, phospholipase C-g (PLC-g), p120-GAP and SH-PTP2 tyrosine phosphatase (Syp) (76, 77). Both Grb2 and Shc proteins have been demonstrated to contribute to Ras activation through the direct binding between Grb2 and Sos and the subsequent activation of the Ras exchange factor Sos (see below). Heterotrimeric guanine triphosphate-binding proteins (G proteins), can stimulate the Ras/Erk pathway through the Gbg subunits. This stimulation is inhibited by wortmannin (a PI3K inhibitor), whereas stimulation of this pathway by tyrosine kinase receptors such as the epidermal growth factor receptor is insensitive to wortmannin (78). This suggests a role for PI3K in Ras activation by heterotrimeric G protein-coupled receptors. This effect is dependent on PI3Kg, a PI3K isotype activated by both the a and bg subunits of heterotrimeric G proteins (78, 79).

A different intriguing example is the MAP kinase pathway activation by protein kinase C. By using the Ras dominant negative, Ras17N (80), this activation has been shown to by Ras-dependent in some cell types such as NIH 3T3 cells (81) but Ras-independent in a number of other cell types (82) Recently, Raf-1 activation by PKC has been shown to be mediated through Ras activation. However, this activation differs from activation by receptor tyrosine kinases in that it is not blocked by Ras17N, the "dominant negative" form of Ras, in COS cells (82). These results indicate that Ras is a component of PKC signaling and that the absence of an effect of Ras17N expression cannot be used to conclude that Ras is not part of a signaling pathway in a specific cell type. However, since Raf-1 seems to associate In vivo preferentially with N-Ras but not H-Ras (75), the role of the three isoforms in PKC signaling can be different. In fact, cells deficient in N-Ras, but not in the other Ras isoforms, seem to have a decreased response to the PKC-dependent PMA stimulation (M. M. & A. P., unpublished data), suggesting an important role for N-Ras in PKC signaling.

4.2. Ras activation: Nucleotide exchange factors for Ras

In order to facilitate the exchange of GTP for GDP, the Guanine Exchange Factor (GEF) must first form a complex with the GDP bound form of the GTPase. GDP is then released from the complex and the high GTP/GDP intracellular ratio ensures that the released nucleotide is immediately replaced with GTP. GEF then dissociates leaving the Ras in the active form (14, 83). Various GEFs have been isolated for the Ras subfamily members, such as Sos, RasGRF, RasGRF2, RasGRP, C3G, and RalGDS-related sequences (43, 84-86). A mammalian GEF, related in sequence to the Saccharomyces cerevisiae Ras exchange factor CDC25, was isolated first and named RasGRF (or Cdc25Mn). The related proteins Sos1 and Sos2 are most closely related to the Drosophila son-of-sevenless product. Sos is known to stimulate the GTP exchange on Ras (87), while GRF has been shown to activate both Ras (88) and R-Ras (89). C3G has recently been shown to possess exchange activity on both K-rev-1/Rap1A and R-ras (89). Finally, RalGDS-related sequences include a growing family of proteins with exchange activities for the Ral GTPases. Interestingly, most of these RalGDS proteins have been shown to be putative Ras effectors (see below).

Only some of these proteins (Sos, RasGRF, RasGRF2, RasGRP, Vav, C3G and SmgGDS) have been reported to act as exchange factors for the true Ras proteins, although Vav and C3G rather seem to act in vivo as exchange factors for other GTPases than Ras. Their properties are discussed below.

4.2.1. Sos

The Sos1 and Sos2 proteins are widely expressed in mouse and human tissues and are related to the Drosophila son-of-sevenless product, which functions upstream of Ras in the R7 cell development of the Drosophila eye (90, 91). A central region of about 200 amino acids in the mSos1 protein is similar to a comparable region of Cdc25, Sdc25 and Ste6 (43). This domain is known to catalyze exchange activity on H-Ras and N-Ras, but not RalA or Cdc42 (92). The C-terminal proline-rich region of Sos binds to the Src Homology 3 (SH3) domains of Grb2 and the Sos-Grb2 complex interacts with the activated tyrosine kinase receptors, as the epidermal growth factor (EGF) receptor, through the SH2 domain of Grb2, thus linking external growth factor-tyrosine kinases signaling and Ras activation (93-97).

In addition to the Cdc25 domain, responsible for the Ras-GEF activity, and the Grb2-binding domain, Sos proteins possess Dbl homology (DH) and pleckstrin homology (PH) domains arranged in tandem in the N-terminal region. DH domains have been demonstrated to promote guanine nucleotide exchange on Rho family GTPases, whereas PH domains participate in protein-protein and protein-lipid interactions, binding tightly and specifically to phosphatidylinositol derivatives. Both the Grb2- and the DH-binding domains have been recently shown to have autoinhibitory effects on the intrinsic GEF activity of the Cdc25 domain (98). The DH domain of Sos is able to stimulate guanine nucleotide exchange on Rac but not Cdc42. Both the exchange activity on Rac and Ras are inhibited by binding of the PH domain to phosphatidylinositol-4,5-biphosphate (PtdIns[4,5]P2), a substrate of the phosphatidylinositide 3-kinase (PI3K) and this activity is regained when coexpressed with activated Ras (99, 100). Thus, Sos may couple Ras and Rac activation through its different domains and this interactions may be dependent on PI3K signaling.

In addition to tyrosine kinase-dependent activation of the Sos-Ras pathway, a different mechanism seems to be involved in the PKC-dependent activation of Ras. In NIH 3T3 cells, the PKC-dependent activation of the Ras-Erk signaling pathway by PMA is suppressed by the dominant negative Raf1 and, in some cell types, also by the dominant negative forms of Ras and Sos. The PMA signal seems to be fed at or upstream of Shc and involves serine (but not tyrosine, as it occurs in the growth factor-induced Shc phosphorylation) residues (81).

4.2.2. RasGRF

This protein was identified as homologous to the Saccharomyces cerevisiae Ras exchange factor encoded by CDC25 (101, 102). RasGRF, also named Cdc25Mm, contains Dbl homology (DH) and pleckstrin homology (PH) domains and is expressed specifically in brain. Both GRF and Sos are expressed in several different protein forms by alternative splicing. The heterogeneity of GRF and Sos products may therefore contribute to a fine regulation of Ras activation in different tissues or at different stages of development (103). RasGRF has been recently shown to activate H-Ras but not N-Ras or K-RasB proteins In vivo (64).

4.2.3. RasGRF2

RasGRF2 is a new multidomain protein catalyzing the specific release of GDP from Ras but not other GTPases (85). This protein contains a Cdc25-related domain in the COOH terminus of the protein. In the NH₂-terminal region, it contains several domains with homology to various signaling proteins including two pleckstrin homology domains, a Dbl homology region, and an IQ motif required for its apparent constitutive association with calmodulin. Accordingly, calcium influx caused a shift of RasGRF2 subcellular localization from cytosolic to the cell periphery, stimulating GTP binding by Ras and potentiating calcium ionophore-induced activation of Erk (85).

4.2.4. RasGRP

This recently isolated member of the Ras nucleotide exchange factor family is characterized by an atypical pair of "EF hands" that bind calcium and a diacylglycerol (DAG)-binding domain. RasGRP activates Ras and the Erk pathway in response to DAG through the DAG-binding domain. This exchange factor is expressed in the nervous system suggesting a role in the coupling of changes in DAG and calcium concentrations to Ras activation (86).

4.2.5. Some considerations on SmgGDS, Vav and C3G

Some other exchange factors have been thought to participate on Ras activation. Although Vav has similar domains to the Dbl family proteins, unexpectedly, a GEF activity specific for Ras rather than Rho subfamily members was described (reviewed in Ref. 104). Several observations have shown, however, that Vav is not a true exchange factor for Ras. The morphological changes that are observed in Vav-expressing NIH 3T3 cells are distinct from those seen when the same cells are expressing other Ras GEFs such as RasGRF, suggesting that the Vav-associated oncogenic activity may not be a consequence of Ras GEF activity (105). Moreover, no increase in the level of Ras-GTP is detected in cells transformed with Vav (106). Recently, Vav protein has been shown to bind and stimulate GDP release from Rac1 and Cdc42, and to a lesser degree RhoA, but not from Ras (107, 108). This lack of Vav protein exchange on Ras is in agreement with the earlier results indicating a Ras-independent transformation by oncogenic Vav (105). Importantly, Vav has been shown to play an important role on Rac activation regulated by the substrates and products of phosphoinositide 3-kinase (PI3K) (109).

C3G is an exchange factor which contains a Cdc25 homology domain and complements yeast cells with the cdc25 mutation; however, it has been shown to be a specific GEF for the Ras-related protein Rap1. On the other hand, SmgGDS is a protein that catalyzes exchange on K-Ras4B and other Ras-related GTPases, but not H-Ras (63). The relative contribution of these proteins to Ras regulation remains to be clarified.

4.3. Ras inactivation by GAPs

The Ras GTPase activity results in the slow hydrolysis of bound GTP, leaving the protein complexed with GDP. However, the intrinsic rate of dissociation of the Ras-GTP complex is very low, approximately 10^-5 moles per sec dissociating per mole of complex, and since the intracellular GTP concentration is much greater than that of GDP, it seemed clear that additional regulatory proteins should be involved in the Ras GTPase activity. A cytosolic protein fraction was found to be able to stimulate the hydrolysis of GTP bound to Ras (110, 111). A 120 kDa protein, named p120-GAP for GTPase Activating Protein, was initially purified and the corresponding cDNA cloned. This protein binds preferentially to Ras-GTP and this binding is impaired in some Ras effector mutants.

At least six different GAPs have been found to date in mammals: p120-GAP, the neurofibromatosis type 1 protein NF1 (112, 113), GAP1m, GAP1IP4BP (114), IQGAP1 (115, 116) and SynGAP, a Ras GAP selectively expressed at excitatory synapses in the brain (117, 118). All these proteins negatively regulate Ras through their GTPase stimulatory activities. Accordingly, overexpression of GAP is associated with a reduction of endogenous Ras in the GTP-bound form and can prevent transformation by overexpression of normal Ras or by other oncogenes whose transforming activities depend on endogenous Ras (42, 119). However, GAP does not affect to the transforming potential of some Ras mutants, such as those with mutations in codons 12, 13 or 61. Analysis of these mutants showed that mutations in the effector domain of Ras render the protein unable to respond to GAP, and they are therefore constitutively in the GTP-bound form (7, 44). In addition to their GTPase stimulating activity, a putative effector role for p120GAP and NF1 has been suggested (see below).