Destabilization of the retinoblastoma tumor suppressor by human papillomavirus type 16 E7 is not sufficient to overcome cell cycle arrest in human keratinocytes

The E7 oncoprotein of human papillomavirus type 16 promotes cell proliferat ion in the presence of anti-proliferative signals. Mutagenesis of E7 has re vealed that this activity requires three regions, conserved regions 1 and 2 and a C-terminal zinc finger. Binding to the retinoblastoma tumor represso r (Rb) through an LxCxE motif in conserved region 2 is necessary, but not s ufficient, for E7 to induce proliferation. We tested the hypothesis that bi nding to Rb is not sufficient because conserved region 1 and/or the C termi nus are required for E7 to functionally inactivate Rb and thus induce proli feration. One mechanism proposed for how E7 inactivates Rb is by blocking R b-E2F binding. Either conserved region 1 or the C terminus was necessary, i n combination with the LxCxE motif, for E7 to block Rb-E2F binding in vitro . While all full-length E7 proteins with mutations outside of the LxCxE mot if inhibited Rb-E2F binding, some failed to abrogate cell cycle arrest, dem onstrating that blocking Rb-E2F binding is not sufficient for abrogating an tiproliferative signals. Another mechanism proposed for how E7 inactivates Rb is by promoting the destabilization of Rb protein. Mutations in conserve d region 1 or the LxCxE motif prevented E7 from reducing the half-life of R b, Though no specific C-terminal residues of E7 were essential for destabil izing Rb, a novel class of mutations that uncouple the destabilization of R b from the deregulation of keratinocyte proliferation was discovered. Desta bilization of Rb correlated with the abrogation of Rb-induced quiescence bu t was not sufficient for overriding DNA damage-induced cell cycle arrest or for increasing keratinocyte life span. Finally, the same regions of E7 req uired for destabilizing Rb were required for reducing p107 and p130 levels. Together, these results suggest that inactivation of all three Rb family m embers is not sufficient to deregulate keratinocyte cell cycle control.