E7 Abolishes Raf-Induced Arrest via Mislocalization of p21Cip1

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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 p21Cip1-dependent cellular arrest. The human papillomavirus oncoprotein E7 transforms primary cells in cooperation with Ras and abolishes p21Cip1-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 p21Cip1 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 p21Cip1 or derepressing p21Cip1-associated CDK2 complexes. Activation of Raf led to nuclear accumulation of p21Cip1, and we provide evidence that this effect is mediated by inhibition of Akt, a regulator of p21Cip1 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 p21Cip1, resulting in decreased association between p21Cip1 and cyclin E-CDK2. Taken together, these data suggest that E7 inhibits p21Cip1 function in the context of Raf signaling by altering Raf-Akt antagonism and preventing the proper subcellular localization of p21Cip1. We propose that E7 elicits a proliferative response to Raf signaling by targeting p21Cip1 function via a novel mechanism.