The mechanism of cell cycle regulation by v-Src

The tyrosine kinase oncoprotein v-Src can overcome the requirements for ser um growth factors and anchorage which restrain normal cell growth. Here we investigated the biochemical mechanisms whereby v-Src induces quiescent cel ls to enter S phase in the absence of serum mitogens. Activating a temperat ure sensitive v-Src in quiescent cells sequentially induced cyclins D1, E a nd A and also down regulated p27. We addressed whether p27 down regulation was required to activate cyclin D1/ CDK4/6 or cyclin E/CDK2 by engineering cells with inducible p27. Both S phase entry and activation of cyclin/CDKs were inhibited by over expression of p27. Using cells engineered with induc ible p16 we showed that Cyclin D/CDK4/6 activity was required for v-Src to increase expression of cyclin A but not cyclin E. To determine which downst ream kinases mediated these effects of v-Src we added pharmacological inhib itors of phosphatidylinositol 3-kinase (PI3-K), LY294002 or mitogen activat ed protein kinase kinase (MEK), U0126. PI3-K was required for v-Src to acti vate MEK and MEK was required for v-Src to increase expression of cyclins D I and E. However, the MEK inhibitor prevented p27 protein down regulation w hereas the PI3K inhibitor did not. This was because reduced PI3-K activity lead to proteolytic degradation of p27.