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.
O'Brien
V
V
Beatson Institute for Cancer Research, CRC Beatson Laboratories, Garscube Estate, Glasgow G61 1BD, Scotland, United Kingdom. vobrien@beatson.gla.ac.uk
Grindlay
G J
GJ
Campo
M S
MS
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D052688:Bovine papillomavirus 1 / Q000737:chemistry*; D052697:Bovine papillomavirus 4; D018797:Cell Cycle Proteins / Q000096:biosynthesis*; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D019938:Cyclin D1 / Q000378:metabolism*; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D015536:Down-Regulation; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D009856:Oncogene Proteins, Viral; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; C436321:oncogene protein E5-E8, bovine papillomavirus type 4; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases