[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 6. Ras AND THE CELL CYCLE CONTROL Cell cycle control is a complex process by which cells decide whether to proliferate or to stay quiescent. Many different proteins are involved in this process. Basically, the engine that produces cell cycle progression is represented by a family of protein kinases, the Cyclin-Dependent Kinases (CDK). The activity of these molecules is dependent on the presence of a stimulating subunit, the cyclins, named after their tight regulation through the cell cycle phases, and an inhibitory subunit, the CDK inhibitors (CKIs). In addition, other several kinases and phosphatases are also involved in the regulation of these molecules (249, 250). Early studies demonstrated that Ras induces DNA synthesis in the nucleus of quiescent cells (251), suggesting that the Ras signaling pathway is linked directly to the G1/S phase transition of the cell cycle. In fact, the only phase when inhibition of Ras affects cell cycle progression is G1 (251). Once cells have entered S phase, Ras become dispensable until the next cell cycle. In general, Ras plays an important role integrating mitogenic signals with the cell cycle progression. Ras mediated signals induce gene expression, as demonstrated by studies on the early-responsive transcription factors. Transient expression of oncogenic Ras induces expression of endogenous Jun and Fos, activating the AP-1 transcription factor complex (7). In addition, other transcription factors such as the PEA-3 element and the members of the ets family are also activated by Ras. The molecular basis of cell cycle control and the regulatory role of CDKs, cyclins and CKIs has been extensively studied in the last few years. The understanding of these processes has led to the analysis of the role of cell cycle regulators in Ras signaling. All this work has provided information about how Ras produces so many different responses in cells, such as cell proliferation or cell cycle arrest, and how different genetic alterations cooperate in cellular tumorigenesis. 6.1. Cyclin D-CDK4/6 complexes and the Retinoblastoma pathway Few cyclin-CDK substrates have been unequivocally identified to date. One of the most important for G1 progression is the product of the Retinoblastoma gene (Rb). Regulation of the phosphorylation state of the retinoblastoma protein is a key event in the progression of cells from G1 phase into S phase. In growth-arrested or early G1 cells, Rb is hypophosphorylated and binds to transcription factors of the E2F family, recruiting histone deacetylase (HDAC) and thus repressing transcription (252-254). Cyclin D1 is synthesized during G1, binding and activating CDK4/6, which phosphorylate Rb at multiple sites. Hyperphosphorylated Rb does not longer bind to the E2F family of transcription factors leading to the dissolution of the E2F-Rb-HDAC complexes and allowing gene transcription and the progression of the cell cycle to the DNA synthesis phase (S). Different groups have reported the induction of expression of cyclin D by Ras (255-262). The shortening of the G1 phase, detected in Ras transformed cells, can be associated to an increased expression of cyclin D1 and, in fact, can be abrogated by a cyclin D1 antisense. However, although constitutive overexpression of cyclin D1 accelerates G1 progression, cells remain untransformed, indicating that cyclin D1 may be necessary but it is not sufficient for the transforming activity of Ras (257, 260). Cyclin D1 activation by Ras seems to be dependent on the Raf/MAP kinase pathway (263, 264) and the AP-1 like sequences present in the cyclin D1 promoter are involved in this activation (256). Cyclin D induction by Ras has been shown to be necessary for Ras-induced anchorage-independent growth (257). Others have shown that Ras also up-regulates cyclin A, D3, and E, and the E2F family of transcription factors (262). Ras-dependent Cyclin A induction seems to be specially important for anchorage-independent growth, at least in some cell types (265). Inactivation of Ras by the use of dominant negative forms in cycling cells causes a decline in cyclin D1 protein levels, accumulation of the hypophosphorylated, growth-suppressive from of Rb, and G1 arrest (266, 267). This G1 arrest can be corrected by forcing the expression of cyclin D1. Interestingly, when Rb is disrupted, cells fail to arrest in G1 following Ras inactivation (266). When a neutralizing antibody directed against Ras was microinjected, cells without Rb or p16^INK4a were more resistant -although not completely- to the inhibitory effects of the anti-Ras antibody, indicating that Ras is required for Rb inactivation but has also other functions in cell-cycle progression (268). Thus, the ability to induce Rb phosphorylation through cyclin D-dependent kinases, although does not account for all aspects of Ras regulation of the cell cycle, seems to be a major part of the link between Ras and the cell-cycle machinery (269). 6.2. Cyclin E-CDK2 complexes and the p27^Kip1 inhibitor Cyclin D, while necessary, is not sufficient for G1 progression. Cyclin E, which protein peaks at the G1/S phase border, binds to CDK2, and is also required for G1-S progression. Thus, Ras signals should also activate these cyclin E-CDK2 complexes to enter in cell proliferation. Some reports indicate that Raf weakly induces the expression of cyclin E and cyclin A (258, 270); however, the activation of the cyclin E-CDK2 complexes is mainly achieved through the degradation of the cell cycle inhibitor p27^Kip1 (260-262, 267, 271). Conditional expression of oncogenic Ras induced the G1-S transition in rat fibroblasts and significantly reduced the synthesis and shortened the half-life of p27^Kip1 protein (261, 271). Although the p27^Kip1 protein levels are tightly regulated via the ubiquitin pathway (272), the Ras-dependent protein degradation was found to occur in a proteosome-independent way (261). The lack of p27^Kip1 degradation can be sufficient to stop Ras-induced proliferation; thus, in BALB c/3T3 cells, but not in NIH 3T3, expression of activated Ras failed to increase the cyclin dependent kinase activity on Rb, because of the constant presence of the CDK inhibitor p27^Kip1 (258). Several reports indicate that Ras causes induction of p27^Kip1 degradation through the MAP kinase signaling pathway (260, 261, 267, 271). This degradation is dependent on the MEK/MAP kinase activities as shown by the use of cyclic AMP-elevating agents and a MEK inhibitor (261). Furthermore, p27^Kip1 has been shown to be phosphorylated directly by MAP kinase in vitro and the phosphorylated protein cannot bind to and inhibit CDK2 (261). The specific activation of the MEK1-Erk pathway, however, is not sufficient to trigger degradation of p27^Kip1 (264). The Raf-MEK-Erk pathway is involved in the induction of cyclin D and the levels of cyclin D-CDK complexes could sequester p27^Kip1. Thus, in cells overexpressing cyclin D1 and CDK4 subunits, these complexes have been reported to sequester p27^Kip1 and reduce its effective inhibitory threshold. Therefore, the MEK-Erk pathway could function posttranslationally to regulate cyclin D1 assembly with CDK4 and thereby to help cancel p27^Kip1-mediated inhibition (264). 6.3. Senescence response to Ras signals (p16^INK4a, p15^INK4b, p53 and p21^Cip1) Recently, several studies have found an apparently contradictory function of activated Ras proteins. Expression of Ras in primary cells is able to strongly induce expression of cell cycle inhibitory molecules such as p53 and p16^INK4a (273). Activated Ras up-regulated p53, correlating with enhanced p53 transactivation and up-regulation of p21^Cip1 and Gadd 45, two p53 effectors and negative cell regulators (262). This activated Ras-dependent response causes an arrest in the cell cycle progression of rodent and human primary fibroblasts and shows characteristics indistinguishable from cellular senescence. Cells develop a flat morphology and express specific markers of senescence (273). Cell transformation by Ras is hence inhibited since forcing the expression of high levels of the CDK inhibitors p16^INK4a (274) and p21^Cip1 (275) blocks the ability of oncogenic Ras to cause proliferation and transformation in murine fibroblast cell lines. In primary rat Schwann cells, however, Ras-specific induction of the p53-dependent inhibitor p21^Cip1 results in cells that retain certain properties of transformation -refractile morphology and increased motility- but growth arrested in the G1 phase of the cell cycle (276). Activated Raf has also been shown to lead to a G1-specific cell cycle arrest through induction of p21^Cip1, inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb (277). Importantly, only very high levels of activated Raf are able to induce this response, whereas a less strong Raf signal induces cyclin D1 and the progression of the cell cycle (270, 278). These results are in concordance with previous work with a series of activated Raf molecules showing that Raf proteins able to induce proliferation activate Erk kinases only weakly, whereas Raf molecules strongly activating Erks inhibit proliferation (279). Although activation of the Raf-Erk pathway leads to the induction, in a p53-dependent manner, of the CKI p21^Cip1 (276, 277), a more complex signaling seems to be required for the induction of p16^INK4a. Multiple Ras downstream pathways are required for the induction not only of p16^INK4a but also of the cell cycle inhibitor p15^INK4b, a member of the INK family involved in G1 control and able to inhibit cell transformation by Ras (M. M. & A. P., submitted). Both p16^INK4a and p15^INK4b are likely to cooperate in the senescence response to activated Ras signals. As discussed in the next heading, Ras may need the cooperation of other molecules, mainly nuclear oncogenes, to overcome the inhibitory effect of these cell cycle inhibitors. 6.4. Cooperation between oncogenes to overdrive the cell cycle Carcinogenesis is clearly a multistep process and to reach the malignant phenotype requires multiple alterations affecting several levels of growth control. In contrast to established lines, primary cells are not usually susceptible to full transformation by mutant ras alone. Early gene transfer experiments demonstrated that two or more cooperating oncogenes are required to convert normal cells to a tumorigenic state (280-282). Transformation of primary fibroblasts by ras requires cooperation with immortalizing oncogenes such as c-myc, N-myc, E1A, or polyoma large T. These observations indicated that ras oncogenes can drive transformation but they are highly inefficient in overcoming senescence. However, establishment as continuous cell lines is not sufficient for transformation by ras (283) and, for instance, transformation of rat REF52 cells with ras oncogenes requires complementation with E1A or SV40 (284). This resistance of primary cells to be transformed by Ras is now explained by the effect of strong Ras signaling on the cell cycle inhibitors (see above). The ability of oncogenic Ras to induce the expression of the CKIs is thought to be a protective or stress response of the cell when receiving a Ras signal at an inappropriate stage in the cell cycle (269, 285). This protective response, therefore, has to be overcome by cooperating oncogenes in order to transform cells. This effect can be obtained either by the loss of function of the inhibitory proteins or through the expression of cooperating molecules. Thus, in primary rat Schwann cells, the attenuation of p21^Cip1 induction, either by antisense expression or by inhibiting p53 activity, results in a loss of the growth inhibitory signal from Ras (276). Cooperation between loss of function of many of the CKIs and Ras has been studied In vivo in knockout mice. Primary cells from p16-, p21-, or p53-deficient mice can be transformed by Ras alone (273, 286). Since the abrogation of the negative growth signal from Ras is sufficient to unleash its transforming activity, many of the proteins that cooperate with Ras in primary embryonic fibroblast transformation have the ability to counteract the cell-cycle inhibitory effects of CKIs (285). For instance, overexpression of cyclin D, cyclin E, or cdc25 cooperates with Ras in rat embryonic fibroblasts (REF) transformation presumably by increasing the activity of cyclin/CDK complexes over the threshold imposed by the CKIs (287-289). Deregulation of the E2F transcription factors, which act downstream of Retinoblastoma, also leads to transformation of rat embryo fibroblasts in cooperation with Ras and E2F1 itself induces anchorage-independent growth in immortalized 3T3 or REF cells (290). Viral oncogenes cooperate with Ras by blocking either the effects of p53 (human papilloma E6, E1A, SV40LT), p16^INK4a by binding Rb (human papilloma E7, SV40LT) or p21^Cip1 (E7) (285). The 'classic' transforming cooperation between Ras and Myc has been widely analyzed in vitro (291) and In vivo (292), and, recently, this cooperation is being understood as a result of the effect of these proteins on the cell cycle regulators. Whereas Ras induces cell cycle progression activating cyclin D proteins, Myc is essentially a positive regulator of G1-specific CDKs and, in particular, of cyclin E/CDK2 complexes. Myc acts via at least three distinct pathways which can enhance CDK function: (1) functional inactivation of the CDK inhibitor p27^Kip1, (2) induction of the CDK-activating phosphatase Cdc25A and (3) deregulation of cyclin E expression (293). Thus, Ras and Myc collaborate in several different points on the cell cycle regulators. All these activities produce a synergistic enhancement of cyclin E/CDK2 and E2F activity (267).

[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

6. Ras AND THE CELL CYCLE CONTROL

Cell cycle control is a complex process by which cells decide whether to proliferate or to stay quiescent. Many different proteins are involved in this process. Basically, the engine that produces cell cycle progression is represented by a family of protein kinases, the Cyclin-Dependent Kinases (CDK). The activity of these molecules is dependent on the presence of a stimulating subunit, the cyclins, named after their tight regulation through the cell cycle phases, and an inhibitory subunit, the CDK inhibitors (CKIs). In addition, other several kinases and phosphatases are also involved in the regulation of these molecules (249, 250).

Early studies demonstrated that Ras induces DNA synthesis in the nucleus of quiescent cells (251), suggesting that the Ras signaling pathway is linked directly to the G1/S phase transition of the cell cycle. In fact, the only phase when inhibition of Ras affects cell cycle progression is G1 (251). Once cells have entered S phase, Ras become dispensable until the next cell cycle.

In general, Ras plays an important role integrating mitogenic signals with the cell cycle progression. Ras mediated signals induce gene expression, as demonstrated by studies on the early-responsive transcription factors. Transient expression of oncogenic Ras induces expression of endogenous Jun and Fos, activating the AP-1 transcription factor complex (7). In addition, other transcription factors such as the PEA-3 element and the members of the ets family are also activated by Ras.

The molecular basis of cell cycle control and the regulatory role of CDKs, cyclins and CKIs has been extensively studied in the last few years. The understanding of these processes has led to the analysis of the role of cell cycle regulators in Ras signaling. All this work has provided information about how Ras produces so many different responses in cells, such as cell proliferation or cell cycle arrest, and how different genetic alterations cooperate in cellular tumorigenesis.

6.1. Cyclin D-CDK4/6 complexes and the Retinoblastoma pathway

Few cyclin-CDK substrates have been unequivocally identified to date. One of the most important for G1 progression is the product of the Retinoblastoma gene (Rb). Regulation of the phosphorylation state of the retinoblastoma protein is a key event in the progression of cells from G1 phase into S phase. In growth-arrested or early G1 cells, Rb is hypophosphorylated and binds to transcription factors of the E2F family, recruiting histone deacetylase (HDAC) and thus repressing transcription (252-254). Cyclin D1 is synthesized during G1, binding and activating CDK4/6, which phosphorylate Rb at multiple sites. Hyperphosphorylated Rb does not longer bind to the E2F family of transcription factors leading to the dissolution of the E2F-Rb-HDAC complexes and allowing gene transcription and the progression of the cell cycle to the DNA synthesis phase (S). Different groups have reported the induction of expression of cyclin D by Ras (255-262). The shortening of the G1 phase, detected in Ras transformed cells, can be associated to an increased expression of cyclin D1 and, in fact, can be abrogated by a cyclin D1 antisense. However, although constitutive overexpression of cyclin D1 accelerates G1 progression, cells remain untransformed, indicating that cyclin D1 may be necessary but it is not sufficient for the transforming activity of Ras (257, 260). Cyclin D1 activation by Ras seems to be dependent on the Raf/MAP kinase pathway (263, 264) and the AP-1 like sequences present in the cyclin D1 promoter are involved in this activation (256). Cyclin D induction by Ras has been shown to be necessary for Ras-induced anchorage-independent growth (257). Others have shown that Ras also up-regulates cyclin A, D3, and E, and the E2F family of transcription factors (262). Ras-dependent Cyclin A induction seems to be specially important for anchorage-independent growth, at least in some cell types (265).

Inactivation of Ras by the use of dominant negative forms in cycling cells causes a decline in cyclin D1 protein levels, accumulation of the hypophosphorylated, growth-suppressive from of Rb, and G1 arrest (266, 267). This G1 arrest can be corrected by forcing the expression of cyclin D1. Interestingly, when Rb is disrupted, cells fail to arrest in G1 following Ras inactivation (266). When a neutralizing antibody directed against Ras was microinjected, cells without Rb or p16^INK4a were more resistant -although not completely- to the inhibitory effects of the anti-Ras antibody, indicating that Ras is required for Rb inactivation but has also other functions in cell-cycle progression (268). Thus, the ability to induce Rb phosphorylation through cyclin D-dependent kinases, although does not account for all aspects of Ras regulation of the cell cycle, seems to be a major part of the link between Ras and the cell-cycle machinery (269).

6.2. Cyclin E-CDK2 complexes and the p27^Kip1 inhibitor

Cyclin D, while necessary, is not sufficient for G1 progression. Cyclin E, which protein peaks at the G1/S phase border, binds to CDK2, and is also required for G1-S progression. Thus, Ras signals should also activate these cyclin E-CDK2 complexes to enter in cell proliferation. Some reports indicate that Raf weakly induces the expression of cyclin E and cyclin A (258, 270); however, the activation of the cyclin E-CDK2 complexes is mainly achieved through the degradation of the cell cycle inhibitor p27^Kip1 (260-262, 267, 271). Conditional expression of oncogenic Ras induced the G1-S transition in rat fibroblasts and significantly reduced the synthesis and shortened the half-life of p27^Kip1 protein (261, 271). Although the p27^Kip1 protein levels are tightly regulated via the ubiquitin pathway (272), the Ras-dependent protein degradation was found to occur in a proteosome-independent way (261). The lack of p27^Kip1 degradation can be sufficient to stop Ras-induced proliferation; thus, in BALB c/3T3 cells, but not in NIH 3T3, expression of activated Ras failed to increase the cyclin dependent kinase activity on Rb, because of the constant presence of the CDK inhibitor p27^Kip1 (258).

Several reports indicate that Ras causes induction of p27^Kip1 degradation through the MAP kinase signaling pathway (260, 261, 267, 271). This degradation is dependent on the MEK/MAP kinase activities as shown by the use of cyclic AMP-elevating agents and a MEK inhibitor (261). Furthermore, p27^Kip1 has been shown to be phosphorylated directly by MAP kinase in vitro and the phosphorylated protein cannot bind to and inhibit CDK2 (261). The specific activation of the MEK1-Erk pathway, however, is not sufficient to trigger degradation of p27^Kip1 (264). The Raf-MEK-Erk pathway is involved in the induction of cyclin D and the levels of cyclin D-CDK complexes could sequester p27^Kip1. Thus, in cells overexpressing cyclin D1 and CDK4 subunits, these complexes have been reported to sequester p27^Kip1 and reduce its effective inhibitory threshold. Therefore, the MEK-Erk pathway could function posttranslationally to regulate cyclin D1 assembly with CDK4 and thereby to help cancel p27^Kip1-mediated inhibition (264).

6.3. Senescence response to Ras signals (p16^INK4a, p15^INK4b, p53 and p21^Cip1)

Recently, several studies have found an apparently contradictory function of activated Ras proteins. Expression of Ras in primary cells is able to strongly induce expression of cell cycle inhibitory molecules such as p53 and p16^INK4a (273). Activated Ras up-regulated p53, correlating with enhanced p53 transactivation and up-regulation of p21^Cip1 and Gadd 45, two p53 effectors and negative cell regulators (262). This activated Ras-dependent response causes an arrest in the cell cycle progression of rodent and human primary fibroblasts and shows characteristics indistinguishable from cellular senescence. Cells develop a flat morphology and express specific markers of senescence (273). Cell transformation by Ras is hence inhibited since forcing the expression of high levels of the CDK inhibitors p16^INK4a (274) and p21^Cip1 (275) blocks the ability of oncogenic Ras to cause proliferation and transformation in murine fibroblast cell lines. In primary rat Schwann cells, however, Ras-specific induction of the p53-dependent inhibitor p21^Cip1 results in cells that retain certain properties of transformation -refractile morphology and increased motility- but growth arrested in the G1 phase of the cell cycle (276).

Activated Raf has also been shown to lead to a G1-specific cell cycle arrest through induction of p21^Cip1, inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb (277). Importantly, only very high levels of activated Raf are able to induce this response, whereas a less strong Raf signal induces cyclin D1 and the progression of the cell cycle (270, 278). These results are in concordance with previous work with a series of activated Raf molecules showing that Raf proteins able to induce proliferation activate Erk kinases only weakly, whereas Raf molecules strongly activating Erks inhibit proliferation (279).

Although activation of the Raf-Erk pathway leads to the induction, in a p53-dependent manner, of the CKI p21^Cip1 (276, 277), a more complex signaling seems to be required for the induction of p16^INK4a. Multiple Ras downstream pathways are required for the induction not only of p16^INK4a but also of the cell cycle inhibitor p15^INK4b, a member of the INK family involved in G1 control and able to inhibit cell transformation by Ras (M. M. & A. P., submitted). Both p16^INK4a and p15^INK4b are likely to cooperate in the senescence response to activated Ras signals. As discussed in the next heading, Ras may need the cooperation of other molecules, mainly nuclear oncogenes, to overcome the inhibitory effect of these cell cycle inhibitors.

6.4. Cooperation between oncogenes to overdrive the cell cycle

Carcinogenesis is clearly a multistep process and to reach the malignant phenotype requires multiple alterations affecting several levels of growth control. In contrast to established lines, primary cells are not usually susceptible to full transformation by mutant ras alone. Early gene transfer experiments demonstrated that two or more cooperating oncogenes are required to convert normal cells to a tumorigenic state (280-282). Transformation of primary fibroblasts by ras requires cooperation with immortalizing oncogenes such as c-myc, N-myc, E1A, or polyoma large T. These observations indicated that ras oncogenes can drive transformation but they are highly inefficient in overcoming senescence. However, establishment as continuous cell lines is not sufficient for transformation by ras (283) and, for instance, transformation of rat REF52 cells with ras oncogenes requires complementation with E1A or SV40 (284). This resistance of primary cells to be transformed by Ras is now explained by the effect of strong Ras signaling on the cell cycle inhibitors (see above). The ability of oncogenic Ras to induce the expression of the CKIs is thought to be a protective or stress response of the cell when receiving a Ras signal at an inappropriate stage in the cell cycle (269, 285).

This protective response, therefore, has to be overcome by cooperating oncogenes in order to transform cells. This effect can be obtained either by the loss of function of the inhibitory proteins or through the expression of cooperating molecules. Thus, in primary rat Schwann cells, the attenuation of p21^Cip1 induction, either by antisense expression or by inhibiting p53 activity, results in a loss of the growth inhibitory signal from Ras (276). Cooperation between loss of function of many of the CKIs and Ras has been studied In vivo in knockout mice. Primary cells from p16-, p21-, or p53-deficient mice can be transformed by Ras alone (273, 286).

Since the abrogation of the negative growth signal from Ras is sufficient to unleash its transforming activity, many of the proteins that cooperate with Ras in primary embryonic fibroblast transformation have the ability to counteract the cell-cycle inhibitory effects of CKIs (285). For instance, overexpression of cyclin D, cyclin E, or cdc25 cooperates with Ras in rat embryonic fibroblasts (REF) transformation presumably by increasing the activity of cyclin/CDK complexes over the threshold imposed by the CKIs (287-289). Deregulation of the E2F transcription factors, which act downstream of Retinoblastoma, also leads to transformation of rat embryo fibroblasts in cooperation with Ras and E2F1 itself induces anchorage-independent growth in immortalized 3T3 or REF cells (290). Viral oncogenes cooperate with Ras by blocking either the effects of p53 (human papilloma E6, E1A, SV40LT), p16^INK4a by binding Rb (human papilloma E7, SV40LT) or p21^Cip1 (E7) (285).

The 'classic' transforming cooperation between Ras and Myc has been widely analyzed in vitro (291) and In vivo (292), and, recently, this cooperation is being understood as a result of the effect of these proteins on the cell cycle regulators. Whereas Ras induces cell cycle progression activating cyclin D proteins, Myc is essentially a positive regulator of G1-specific CDKs and, in particular, of cyclin E/CDK2 complexes. Myc acts via at least three distinct pathways which can enhance CDK function: (1) functional inactivation of the CDK inhibitor p27^Kip1, (2) induction of the CDK-activating phosphatase Cdc25A and (3) deregulation of cyclin E expression (293). Thus, Ras and Myc collaborate in several different points on the cell cycle regulators. All these activities produce a synergistic enhancement of cyclin E/CDK2 and E2F activity (267).