[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 7. PERSPECTIVE 7.1. Multiple pathways to control transcription, morphology and other cellular processes Ras proteins act at a central point of many signal transduction pathways from extracellular signals to the cytoplasm and the nucleus. Ras activation results in two main cellular changes: reorganization of cytoskeleton and mitogenesis. The analysis of these cellular processes using different effector loop mutants of Ras (129, 131, 204) and the study of different Ras effectors has allowed the dissection of the different pathways downstream of Ras. Although the Raf-Erk pathway has been largely thought to be the main pathway for Ras response, the use of these mutants showed that at least another pathway is responsible for the morphological changes associated with the actin cytoskeleton and is also required, together with the Raf-Erk pathway, for a complete mitogenesis response (204). This pathway is dependent on the Rho family proteins and is frequently mediated by PI3K regulating polymerization of actin, stress fiber formation and focal adhesion (9, 131, 194, 294). Using different downstream pathways, Ras proteins are involved in many cellular processes including transcription, translation, control of cytoskeleton, vesicle formation, cell-cell junctions, etc. Several of these pathways converge in the regulation of transcription. Transcription of the early-response gene c-fos is regulated by different promoter elements. Among these, the serum response element (SRE) is believed to play a central regulatory role (295) (figure 3). Induction through the SRE requires binding of two cellular proteins, the serum response factor (p67^SRF) and one from a family of proteins able to form a ternary complex with SRE and SRF, therefore termed ternary complex factors (TCF: Elk-1, SAP-1). The ets-domain transcription factor Elk-1 is a substrate for three distinct classes of MAP kinases. Elk-1 has been shown to regulate SRE activity in response to the activation of the Ras-Raf-Erk pathway (144). Furthermore, Elk-1 can also be phosphorylated by JNK (296, 297) and the p38 MAP kinases (146, 298). Figure 3. Ras mediates control of c-fos expression through different pathways. Rac/Cdc42 and Rho proteins activate the serum response factor (SRF), whereas the JNK and Erk kinases transactivate Elk-1 (TCF). RalGDS family proteins also activate c-fos expression by a mechanism which is not clear. In addition, SRE can also be regulated in a TCF-independent manner through members of the Rho family (198). Thus, functional RhoA is required for LPA-, serum-induced transcriptional activation by SRF and Rac1 and Cdc42 also potentiate SRF activity (198). The ability of RhoA and Rac1 to cooperate with Raf in focus formation does not depend, however, on the their activation of SRF, but rather, in the case of RhoA, on the activation of ROCK-I, a RhoA effector involved in the formation of actin stress fibers (214, 215). The pathways or effectors involved in Rho-dependent SRF activation have not yet been elucidated. 7.2. Multiple pathways to control cell cycle regulation Ras proteins play a key role in integrating mitogenic signals with cell cycle progression through G1. It can be concluded that Ras plays temporally distinct, phase-specific roles throughout the cell cycle, and particularly in the G1 phase (271). Ras-dependent inputs can be activating or inhibitory signals that reach the cell cycle regulatory machinery using different downstream pathways (figure 4). Ras is required for cell cycle progression and activation of both CDK2 and CDK4 complexes until 2 h before the G1/S transition, corresponding to the restriction point. This is done basically through a Raf/MEK/Erk-dependent induction of cyclin D1 and downregulation of p27^Kip1. However, although Ras-dependent cyclin D1 induction is only dependent on the Erk pathway, degradation of p27^Kip1 seems to require additional events. The effect of the Ras/Raf/Erk pathway on cyclin expression may produce an increase in the level of cyclin D/CDK complexes which are able to sequester p27^Kip1 canceling its inhibitory effect (264). In addition, a RhoA-associated pathway, that could involve a PI3K-dependent but PKB-independent pathway, has been proposed to cooperate in the Ras-dependent p27^Kip1 downregulation (299, 300). Since dominant negative forms of Erk inhibit p27^Kip1 degradation, the Erk pathway seems to be necessary but not sufficient for this activity. The PI3K pathway has been shown to synergize with the Raf pathway in inducing DNA synthesis and loss of contact inhibition (131, 204). Ras signaling to the cell cycle machinery also occurs via multiple pathways to induce anchorage-independent growth. Thus, activation of any of the three possible pairwise combinations between the Raf, RalGDS and PI3K pathways is needed in NIH 3T3 cells to induce anchorage-independent growth. Each individual pathway only partially relieves anchorage dependence of Rb phosphorylation, cyclin E/CDK activity and expression of cyclin A (301). In T cells, induction of the E2F transcription factors after IL-2 stimulation has been demonstrated to be dependent on the PI3K/PKB pathway (300). Also, multiple Ras downstream pathways, involving Erk and Rac, have been shown to contribute to regulate the nuclear factor of activated T cells (NFAT) (302). Figure 4. Regulation of the cell cycle by Ras. Ras can participate in cell cycle regulation sending activating (green lines) or inhibitory (red lines) signals through several downstream pathways, depending on the cell context. Activation of the G1->S progression is mediated by induction of cyclin expression and by triggering degradation of the p27^Kip1 inhibitor. Ras can also induce the CDK inhibitors p16^INK4a, p15^INK4b and p21^Cip1 producing G1 arrest. Recently, the Ras activation changes during the cell cycle have been analyzed. An elegant study demonstrated that in HeLa cells and NIH 3T3 fibroblasts, the increase of Ras-GTP loading achieved immediately after release from mitosis is much less than a second phase of Ras activation that occurred some 5 h latter, in mid-G1 (303). Interestingly, only the first phase of Ras activation was accompanied by Erk activation, whereas the latter, much stronger Ras activation occurred without significant Erk activation. The biological significance of Ras activation in mid-G1 phase, and the nature of the effectors recruited by activated Ras at that time is entirely unknown. Ras activity late in G1 phase is required for p27^Kip1 downregulation, resulting in activation of cyclin D/CDK4 and cyclin E/CDK2 and entry into S phase (271). Members of the INK4 and Cip/Kip family often work in concert in response to antimitogenic signals such as TGFb. Thus, diverse evidence has led to the hypothesis that CKIs establish an inhibitory threshold which must be surpassed in order for the cell cycle to proceed (250). As reported in the few last years, Ras uses multiple pathways to induce the cell cycle activators and to overcome the inhibitory activity of the CKIs. For cells to reenter the cell cycle, they require first to induce cyclin D so cyclin D-CDK4/6 complexes can exceed the threshold established by CKIs and allow activation of cyclin E-CDK2 complexes needed for downstream events. Once this task is accomplished, cyclin D is downregulated by proteolysis and perhaps upregulation of p16^INK4a (304). Since Ras signals early and late in the G1 phase, and, on the other hand, Ras is also able to induce p16^INK4a and p15^INK4b expression through specific sequences in their promoters (273; M. M. & A. P., submitted), it should be interesting to analyze the effect of Ras signaling late in the G1 phase. However, in the event of inappropriate cell proliferation induced by Ras oncogenic signals, the levels of several CKIs, including p16^INK4a, p15^INK4band p21^Cip1, are increased providing a way to control the G1 timing and to prevent excessive cell proliferation and subsequent tumorigenesis. Ras proteins, in summary, are helping us to understand how the cell works as a whole, integrating different signaling cascades. The growing number of Ras regulators and effectors is exponentially increasing the number of cellular processes where Ras activation plays a role, from kinase cascades to cell cycle progression, apoptosis or cell-to-cell interactions. All these cellular proteins and the interactions among them form a complex web that responds coordinately when an external signal reaches the cell. Probably, the web has different organization depending on the cell type, but Ras is frequently in the middle of it.

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

7. PERSPECTIVE

7.1. Multiple pathways to control transcription, morphology and other cellular processes

Ras proteins act at a central point of many signal transduction pathways from extracellular signals to the cytoplasm and the nucleus. Ras activation results in two main cellular changes: reorganization of cytoskeleton and mitogenesis. The analysis of these cellular processes using different effector loop mutants of Ras (129, 131, 204) and the study of different Ras effectors has allowed the dissection of the different pathways downstream of Ras. Although the Raf-Erk pathway has been largely thought to be the main pathway for Ras response, the use of these mutants showed that at least another pathway is responsible for the morphological changes associated with the actin cytoskeleton and is also required, together with the Raf-Erk pathway, for a complete mitogenesis response (204). This pathway is dependent on the Rho family proteins and is frequently mediated by PI3K regulating polymerization of actin, stress fiber formation and focal adhesion (9, 131, 194, 294). Using different downstream pathways, Ras proteins are involved in many cellular processes including transcription, translation, control of cytoskeleton, vesicle formation, cell-cell junctions, etc. Several of these pathways converge in the regulation of transcription.

Transcription of the early-response gene c-fos is regulated by different promoter elements. Among these, the serum response element (SRE) is believed to play a central regulatory role (295) (figure 3). Induction through the SRE requires binding of two cellular proteins, the serum response factor (p67^SRF) and one from a family of proteins able to form a ternary complex with SRE and SRF, therefore termed ternary complex factors (TCF: Elk-1, SAP-1). The ets-domain transcription factor Elk-1 is a substrate for three distinct classes of MAP kinases. Elk-1 has been shown to regulate SRE activity in response to the activation of the Ras-Raf-Erk pathway (144). Furthermore, Elk-1 can also be phosphorylated by JNK (296, 297) and the p38 MAP kinases (146, 298).

Figure 3. Ras mediates control of c-fos expression through different pathways. Rac/Cdc42 and Rho proteins activate the serum response factor (SRF), whereas the JNK and Erk kinases transactivate Elk-1 (TCF). RalGDS family proteins also activate c-fos expression by a mechanism which is not clear.

In addition, SRE can also be regulated in a TCF-independent manner through members of the Rho family (198). Thus, functional RhoA is required for LPA-, serum-induced transcriptional activation by SRF and Rac1 and Cdc42 also potentiate SRF activity (198). The ability of RhoA and Rac1 to cooperate with Raf in focus formation does not depend, however, on the their activation of SRF, but rather, in the case of RhoA, on the activation of ROCK-I, a RhoA effector involved in the formation of actin stress fibers (214, 215). The pathways or effectors involved in Rho-dependent SRF activation have not yet been elucidated. 7.2. Multiple pathways to control cell cycle regulation

Ras proteins play a key role in integrating mitogenic signals with cell cycle progression through G1. It can be concluded that Ras plays temporally distinct, phase-specific roles throughout the cell cycle, and particularly in the G1 phase (271). Ras-dependent inputs can be activating or inhibitory signals that reach the cell cycle regulatory machinery using different downstream pathways (figure 4). Ras is required for cell cycle progression and activation of both CDK2 and CDK4 complexes until 2 h before the G1/S transition, corresponding to the restriction point. This is done basically through a Raf/MEK/Erk-dependent induction of cyclin D1 and downregulation of p27^Kip1. However, although Ras-dependent cyclin D1 induction is only dependent on the Erk pathway, degradation of p27^Kip1 seems to require additional events. The effect of the Ras/Raf/Erk pathway on cyclin expression may produce an increase in the level of cyclin D/CDK complexes which are able to sequester p27^Kip1 canceling its inhibitory effect (264). In addition, a RhoA-associated pathway, that could involve a PI3K-dependent but PKB-independent pathway, has been proposed to cooperate in the Ras-dependent p27^Kip1 downregulation (299, 300). Since dominant negative forms of Erk inhibit p27^Kip1 degradation, the Erk pathway seems to be necessary but not sufficient for this activity. The PI3K pathway has been shown to synergize with the Raf pathway in inducing DNA synthesis and loss of contact inhibition (131, 204). Ras signaling to the cell cycle machinery also occurs via multiple pathways to induce anchorage-independent growth. Thus, activation of any of the three possible pairwise combinations between the Raf, RalGDS and PI3K pathways is needed in NIH 3T3 cells to induce anchorage-independent growth. Each individual pathway only partially relieves anchorage dependence of Rb phosphorylation, cyclin E/CDK activity and expression of cyclin A (301). In T cells, induction of the E2F transcription factors after IL-2 stimulation has been demonstrated to be dependent on the PI3K/PKB pathway (300). Also, multiple Ras downstream pathways, involving Erk and Rac, have been shown to contribute to regulate the nuclear factor of activated T cells (NFAT) (302).

Figure 4. Regulation of the cell cycle by Ras. Ras can participate in cell cycle regulation sending activating (green lines) or inhibitory (red lines) signals through several downstream pathways, depending on the cell context. Activation of the G1->S progression is mediated by induction of cyclin expression and by triggering degradation of the p27^Kip1 inhibitor. Ras can also induce the CDK inhibitors p16^INK4a, p15^INK4b and p21^Cip1 producing G1 arrest. Recently, the Ras activation changes during the cell cycle have been analyzed. An elegant study demonstrated that in HeLa cells and NIH 3T3 fibroblasts, the increase of Ras-GTP loading achieved immediately after release from mitosis is much less than a second phase of Ras activation that occurred some 5 h latter, in mid-G1 (303). Interestingly, only the first phase of Ras activation was accompanied by Erk activation, whereas the latter, much stronger Ras activation occurred without significant Erk activation. The biological significance of Ras activation in mid-G1 phase, and the nature of the effectors recruited by activated Ras at that time is entirely unknown. Ras activity late in G1 phase is required for p27^Kip1 downregulation, resulting in activation of cyclin D/CDK4 and cyclin E/CDK2 and entry into S phase (271). Members of the INK4 and Cip/Kip family often work in concert in response to antimitogenic signals such as TGFb. Thus, diverse evidence has led to the hypothesis that CKIs establish an inhibitory threshold which must be surpassed in order for the cell cycle to proceed (250). As reported in the few last years, Ras uses multiple pathways to induce the cell cycle activators and to overcome the inhibitory activity of the CKIs. For cells to reenter the cell cycle, they require first to induce cyclin D so cyclin D-CDK4/6 complexes can exceed the threshold established by CKIs and allow activation of cyclin E-CDK2 complexes needed for downstream events. Once this task is accomplished, cyclin D is downregulated by proteolysis and perhaps upregulation of p16^INK4a (304). Since Ras signals early and late in the G1 phase, and, on the other hand, Ras is also able to induce p16^INK4a and p15^INK4b expression through specific sequences in their promoters (273; M. M. & A. P., submitted), it should be interesting to analyze the effect of Ras signaling late in the G1 phase. However, in the event of inappropriate cell proliferation induced by Ras oncogenic signals, the levels of several CKIs, including p16^INK4a, p15^INK4band p21^Cip1, are increased providing a way to control the G1 timing and to prevent excessive cell proliferation and subsequent tumorigenesis. Ras proteins, in summary, are helping us to understand how the cell works as a whole, integrating different signaling cascades. The growing number of Ras regulators and effectors is exponentially increasing the number of cellular processes where Ras activation plays a role, from kinase cascades to cell cycle progression, apoptosis or cell-to-cell interactions. All these cellular proteins and the interactions among them form a complex web that responds coordinately when an external signal reaches the cell. Probably, the web has different organization depending on the cell type, but Ras is frequently in the middle of it.