
<ArticleTitle>Destabilization of the <prot>retinoblastoma</prot> tumor suppressor by human papillomavirus type 16 <prot>E7</prot> is not sufficient to overcome cell cycle arrest in human keratinocytes.</ArticleTitle>
<AbstractText>The <prot>E7</prot> oncoprotein of human papillomavirus type 16 promotes cell proliferation in the presence of antiproliferative signals. Mutagenesis of <prot>E7</prot> has revealed that this activity requires three regions, conserved regions 1 and 2 and a C-terminal zinc finger. Binding to the <prot>retinoblastoma tumor repressor</prot> (<prot>Rb</prot>) through an LxCxE motif in conserved region 2 is necessary, but not sufficient, for <prot>E7</prot> to induce proliferation. We tested the hypothesis that binding to <prot>Rb</prot> is not sufficient because conserved region 1 and/or the C terminus are required for <prot>E7</prot> to functionally inactivate <prot>Rb</prot> and thus induce proliferation. One mechanism proposed for how <prot>E7</prot> inactivates <prot>Rb</prot> is by blocking <prot>Rb</prot>-<prot>E2F</prot> binding. Either conserved region 1 or the C terminus was necessary, in combination with the LxCxE motif, for <prot>E7</prot> to block <prot>Rb</prot>-<prot>E2F</prot> binding in vitro. While all full-length <prot>E7</prot> proteins with mutations outside of the LxCxE motif inhibited <prot>Rb</prot>-<prot>E2F</prot> binding, some failed to abrogate cell cycle arrest, demonstrating that blocking <prot>Rb</prot>-<prot>E2F</prot> binding is not sufficient for abrogating antiproliferative signals. Another mechanism proposed for how <prot>E7</prot> inactivates <prot>Rb</prot> is by promoting the destabilization of <prot>Rb</prot> protein. Mutations in conserved region 1 or the LxCxE motif prevented <prot>E7</prot> from reducing the half-life of <prot>Rb</prot>. Though no specific C-terminal residues of <prot>E7</prot> were essential for destabilizing <prot>Rb</prot>, a novel class of mutations that uncouple the destabilization of <prot>Rb</prot> from the deregulation of keratinocyte proliferation was discovered. Destabilization of <prot>Rb</prot> correlated with the abrogation of <prot>Rb</prot>-induced quiescence but was not sufficient for overriding DNA damage-induced cell cycle arrest or for increasing keratinocyte life span. Finally, the same regions of <prot>E7</prot> required for destabilizing <prot>Rb</prot> were required for reducing <prot>p107</prot> and <prot>p130</prot> levels. Together, these results suggest that inactivation of all three <prot>Rb</prot> family members is not sufficient to deregulate keratinocyte cell cycle control.</AbstractText>
