Influence of Ras and Rho on p21~(Waf1/cip1) Protein Stability

摘要

Ras activates signaling pathways that lead to cell cycle progression, however, high intensity Ras signaling may lead to cell cycle arrest via the induction of the cyclin dependent kinase inhibitor p21~(Waf1/cip1) (p21). Ras activates transcription from the p21 promoter, however, little is known about the effect of Ras on p21 protein stability. p21 is degraded by ubiquitin-mediated proteolysis via the 26S proteasome. Pulse-chase experiments in NIH3T3 cells transiently transfected with a plasmid encoding p21 confirmed that proteasomal inhibition by Lactacystin significantly increased the stability of the p21 protein. Cotransfection with oncogenic D12 H-Ras also reduced p21 turnover. This was prevented by treatment with the MEK inhibitor UO126. Cyclin Dl cotransfection significantly stabilized p21, raising the possibility that Ras stabilizes p21 protein indirectly via induction of Cyclin Dl expression.Rho has been implicated in the downregulation of Ras-induced p21, thereby overcoming the cell cycle arrest that results from high intensity Ras signaling. The suppression of p21 is due in part to effects on transcription, however, Rho may also regulate p21 at the post-translational level. Pulse-chase experiments indicated that inhibition of Rho function by treatment of NIH3T3 with the Geranylgeranyltransferase inhibitor GGTI-298 reduced p21 turnover. Likewise, ribosylation of endogenous Rho by cotransfection with C3 ADP-ribosyltransferase also stabilized p21. This could be reversed by cotransfection with a C3-insensitive (V/I) mutant of RhoA. Inhibition of Rho function resulted in disruption of the actin cytoskeleton. Treatment with Cytochalasin D or Latrunculin B reproduced the effects of RhoA inhibition on p21 stability. These results suggest that maintenance of the actin cytoskeleton by Rho is required for regulation of p21 turnover. Further characterization of p21 degradation will provide insight into how Ras and Rho regulate p21 protein stability.