Induction of p27(Kip1) degradation and anchorage independence by Ras through the MAP kinase signaling pathway

While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenic transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When rat normal fibroblasts were cultured in suspension without substrate attachment, the cell cycle arrested in G1 phase and the cyclin-dependent kinase inhibitor p27(Kip1) protein and its mRNA accumulated. Conditional expression of oncogenic Ras induced the G1-S transition of the cell cycle and significantly shortened the half-life of p27(Kip1) protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevented the oncogenic Ras-induced degradation of p27(KiP1). These results suggest that the loss of substrate attachment induces the cell cycle arrest through the upregulation of p27(Kip1) mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27(Kip1) degradation through the activation of the MAP kinase signaling pathway. Furthermore, we have found that p27(Kip1) is phosphorylated by MAP kinase in vitro and the phosphorylated p27(Kip1) cannot bind to and inhibit cdk2.