Cooperative Regulation of the Cell Division Cycle by the Protein Kinases RAF and AKT

Abstract

The RAS-activated RAF→MEK→extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′-kinase (PI3′-kinase)→PDK1→AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G₁ cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27^Kip1 expression. Repression of p27^Kip1 was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27^Kip1. Consistent with these observations, pharmacological inhibition of MEK or PI3′-kinase inhibited the effects of activated RAS on the expression of p27^Kip1 in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21^Cip1 from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF→MEK→ERK and PI3′K→PDK→AKT signaling pathways can cooperate to promote G₀→G₁→S-phase cell cycle progression in both normal and cancer cells.

We thank all of the members of the McMahon lab for advice, constructive criticism, and continued support, especially David Dankort and Steen Hansen. We are most grateful to Emma Lees and David Parry for the provision of materials and reagents and for critical review of the manuscript. We also thank members of the UCSF Cancer Center for the provision of reagents and materials and for ongoing advice and suggestions, especially Frank McCormick, Osamu Tetsu, David Stokoe, and Christian Brandts.

M.M. gratefully acknowledges Schering-Plough Corporation for initial funding to support this research. This work was also supported by funds from the UCSF Cancer Center and grants from the Lustgarten Foundation for Pancreas Cancer Research and the Black Foundation to M.M. A.M.M. was supported in part by funds from an NIH Institutional Training Grant (T32 CA 09270). S.G. was a recipient of a postdoctoral fellowship from the Swiss National Science Foundation and the Novartis Foundation.