Abstract
Platelet-derived growth factor (PDGF)-induced Ras activation is required for G1 progression in Chinese hamster embryo fibroblasts (IIC9 cells). Ras stimulates both extracellular signal-related kinase (ERK) activation and RhoA activation in response to PDGF stimulation. Inhibition of either of these Ras-stimulated pathways results in growth arrest. We have shown previously that Ras-stimulated ERK activation is essential for the induction and continued G1 expression of cyclin D1. In this study we examine the role of Ras-induced RhoA activity in G1 progression. Unstimulated IIC9 cells expressed high levels of the G1 cyclin-dependent kinase inhibitor p27(KIP1). Stimulation with PDGF resulted in a dramatic decrease in p27(KIP1) protein expression. This decrease was attributed to increased p27(KIP1) protein degradation. Overexpression of dominant-negative forms of Ras or RhoA completely blocked PDGF-induced p27(KIP1) degradation, but only dominant-negative Ras inhibited cyclin D1 protein expression. C3 transferase also inhibited PDGF-induced p27(KIP1) degradation, thus further implicating RhoA in p27(KIP1) regulation. Overexpression of dominant-negative ERK resulted in inhibition of PDGF-induced cyclin D1 expression but had no effect on PDGF-induced p27(KIP1) degradation. These data suggest that Ras coordinates the independent regulation of cyclin D1 and p27(KIP1) expression by the respective activation of ERK and RhoA and that these pathways converge to determine the activation state of complexes of cyclin D1 and cyclin-dependent kinase in response to mitogen.
Highlights
Progression through the G1 phase of the mammalian cell cycle is mediated in part through the early induction of D-type cyclins by mitogenic stimulation [1,2,3]
We have shown previously that Platelet-derived growth factor (PDGF) is a potent mitogen for IIC9 cells, and addition of PDGF to quiescent IIC9 cells resulted in up-regulation of cyclin D1 protein expression and D-type cyclin-dependent kinase activity [21, 22]
Ras but Not extracellular signal-related kinase (ERK) Activity Is Required for PDGF-induced Loss of p27KIP1—We have shown previously that PDGF-induced G1 progression requires the sustained activation of ERK in a MAP kinase/ERK kinase 1 (MEK1)-dependent manner [21]
Summary
Progression through the G1 phase of the mammalian cell cycle is mediated in part through the early induction of D-type cyclins by mitogenic stimulation [1,2,3]. In this study we demonstrate that Ras coordinates G1 progression through two independent pathways: ERK regulation of cyclin D1 expression and RhoA regulation of p27KIP1 degradation to ensure the proper activation state of cyclin D11⁄7CDK complexes following mitogenic stimulation. The addition of PDGF to IIC9 cells overexpressing dnRasϪ did not affect p27KIP1 protein levels, demonstrating the requirement of Ras activation for PDGFinduced p27KIP1 degradation.
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