Phosphatidylinositol 3-kinase activity regulates α-thrombin-stimulated G₁ progression by its effect on cyclin D1 expression and cyclin-dependent kinase 4 activity

In this study, we present evidence that PI 3-kinase is required for α- thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells). Previous results from our laboratory demonstrate that the mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) pathway controls transit through G₁ phase of the cell cycle by regulating the induction of cyclin D1 mRNA levels and cyclin dependent kinase 4 (CDK4)-cyclin D1 activity. In IIC9 cells, PI 3-kinase activation also is an important controller of the expression of cyclin D1 protein and CDK4-cyclin D1 activity. Pretreatment of IIC9 cells with the selective PI 3-kinase inhibitor, LY294002 blocks the α-thrombin-stimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect α-thrombin- induced cyclin D1 steady state message levels, indicating that PI 3-kinase acts independent of the ERK pathway. Interestingly, expression of a dominant- negative Ras significantly decreased both α-thrombin-stimulated ERK and PI 3-kinase activities. These data clearly demonstrate that the α-thrombin- induced Ras activation coordinately regulates ERK and PI 3-kinase activities, both of which are required for expression of cyclin D1 protein and progression through G₁.