Cell transformation by the E5/E8 protein of bovine papillomavirus type 4. p27(Kip1), Elevated through increased protein synthesis is sequestered by cyclin D1-CDK4 complexes.

The E5/E8 hydrophobic protein of BPV-4 is, at only 42 residues, the smallest transforming protein identified to date. Transformation of NIH-3T3 cells by E5/E8 correlates with up-regulation of both cyclin A-associated kinase activity and, unusually, p27(Kip1) (p27) but does not rely on changes in cyclin E or cyclin E-CDK2 activity. Here we have examined how p27 is prevented from functioning efficiently as a CDK2 inhibitor, and we investigated the mechanisms used to achieve elevated p27 expression in E5/E8 cells. Our results show that normal subcellular targeting of p27 is not subverted in E5/E8 cells, and p27 retains its ability to inhibit both cyclin E-CDK2 and cyclin A-CDK activities upon release from heat-labile complexes. E5/E8 cells also have elevated levels of cyclins D1 and D3, and high levels of nuclear p27 are tolerated because the inhibitor is sequestered within an elevated pool of cyclin D1-CDK4 complexes, a significant portion of which retain kinase activity. In agreement with this, pRB is constitutively hyperphosphorylated in E5/E8 cells in vivo. The increased steady-state level of p27 is achieved largely through an increased rate of protein synthesis and does not rely on changes in p27 mRNA levels or protein half-life. This is the first report of enhanced p27 synthesis as the main mechanism for increasing protein levels in continuously cycling cells. Our results are consistent with a model in which E5/E8 promotes a coordinated elevation of cyclin D1-CDK4 and p27, as well as cyclin A-associated kinase activity, which act in concert to allow continued proliferation in the absence of mitogens.