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

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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 p27Kip1 expression. Repression of p27Kip1 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 p27Kip1. Consistent with these observations, pharmacological inhibition of MEK or PI3′-kinase inhibited the effects of activated RAS on the expression of p27Kip1 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 p21Cip1 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.