E7 Abolishes Raf-Induced Arrest via Mislocalization of p21^Cip1

Abstract

The cellular response to oncogenic Ras depends upon the presence or absence of cooperating mutations. In the absence of immortalizing oncogenes or genetic lesions, activation of the Ras/Raf pathway results in a p21^Cip1-dependent cellular arrest. The human papillomavirus oncoprotein E7 transforms primary cells in cooperation with Ras and abolishes p21^Cip1-mediated growth arrest in the presence of various antimitogenic signals. Here we have utilized a conditional Raf molecule to investigate the effects of E7 on p21^Cip1 function in the context of Raf-induced cellular arrest. E7 bypassed Raf-induced arrest and alleviated inhibition of cyclin E-CDK2 without suppressing Raf-specific synthesis of p21^Cip1 or derepressing p21^Cip1-associated CDK2 complexes. Activation of Raf led to nuclear accumulation of p21^Cip1, and we provide evidence that this effect is mediated by inhibition of Akt, a regulator of p21^Cip1 localization. Loss of Akt activity appears to be an important event in the cellular arrest associated with Raf-induction, since maintenance of Akt activity was necessary and sufficient to bypass Raf-induced arrest. In agreement, expression of E7 sustained Akt activity and reduced nuclear accumulation of p21^Cip1, resulting in decreased association between p21^Cip1 and cyclin E-CDK2. Taken together, these data suggest that E7 inhibits p21^Cip1 function in the context of Raf signaling by altering Raf-Akt antagonism and preventing the proper subcellular localization of p21^Cip1. We propose that E7 elicits a proliferative response to Raf signaling by targeting p21^Cip1 function via a novel mechanism.

We thank E. Harlow, P. Coffer, R. Roth, R. Freeman, and B. Amati for plasmids and antibodies and M. O'Reilly for tet-E7 Mv1Lu cells. For feedback and support during the course of this work and on the manuscript, we acknowledge J. Zhao, S. Butler, S. Dewhurst, S. Huang, D. Patel, H. McMurray, L. Baglia, L. DeLeu, J. Mendler, and C. Westbrook. We are particularly grateful to H. Land for cell lines, reagents, and helpful discussions. We thank P. Keng for assistance with flow cytometry.

T.F.W. was the recipient of a fellowship on NAIAID Training Grant T32AIO7362. This work was supported by NIAID grant RO1AI30798.