Abstract
In this study, we present evidence that PI 3-kinase is required for alpha-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 G(1) 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 alpha-thrombin-stimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect alpha-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 alpha-thrombin-stimulated ERK and PI 3-kinase activities. These data clearly demonstrate that the alpha-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 G(1).
Highlights
In this study, we present evidence that PI 3-kinase is required for ␣-thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells)
Activation of PI 3-kinase activity has been shown to be required for DNA synthesis in response to several mitogens [22,23,24]
PI 3-Kinase Is Required for ␣-Thrombin-induced DNA Synthesis—Recently, much attention has focused on the role of PI
Summary
We present evidence that PI 3-kinase is required for ␣-thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells). These data clearly demonstrate that the ␣-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. Cyclin D1-CDK4 Assay—Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml ␣-thrombin after preincubation in the absence or presence of 100 nM wortmannin, 10 M LY294002, or 15 M PD98059 for 30 min.
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