The role of Ras and its downstream targets on cell cycle control

The oncogene Ras can both stimulate and inhibit cell proliferation depending on the cellular context in which it is activated. Ras is believed to mediate its effects through activation of downstream effector proteins. The best characterised of these is the serine/threonine kinase, Raf. Through conditional activation of Ras or Raf we have shown that depending on the intensity of signal activation, these proteins are able to both stimulate and inhibit proliferation in murine fibroblasts. Strong signals are inhibitory to growth which is shown to be dependent upon an induction of the cell cycle inhibitor p2lCip-1 which prevents upregulated G1 cyclins from forming active complexes. Weaker signals can induce DNA synthesis in low mitogen conditions since they can upregulate cyclin D1 expression, yet are unable to stimulate p2lCip-1 expression. Thus we demonstrate that Raf controls opposing cellular responses through differential activation of cell cycle regulatory proteins. Ras however demonstrates a greater propensity to stimulate growth than Raf which correlates with an ability to downregulate Raf induction of p2lCip-1. The Ras effectors PI 3-kinase and Rif are unable to reproduce this effect and furthermore, RhoA, which has been proposed to downregulate p2lCip-1, is also unable to attenuate induction of p2lCip-1 by Raf. Indeed we show that RhoA and PI 3-kinase cooperate with Raf to induce p2lCip-1. Furthermore Raf and PI 3-kinase co-operate to induce DNA synthesis in the presence of high levels of p2lCip-1. In addition we investigate the ability of activated Rac, which has been implicated as a downstream target of Ras, to control the cell cycle. It is demonstrated that RacV12 is able to stimulate anchorage-independent growth and upregulate cell cycle regulatory proteins, yet is unable to stimulate mitogen-independent growth. Thus cell growth control by Ras depends both on signal strength and a panel of downstream targets.