Constitutively Active K-cyclin/cdk6 Kinase in Kaposi Sarcoma–Associated Herpesvirus–Infected Cells

• Published in: JNCI : Journal of the National Cancer Institute Vol. 97; no. 9; pp. 656 - 666
• Main Authors: Van Dross, Rukiyah, Yao, Shan, Asad, Shaheena, Westlake, Grant, Mays, Deborah J., Barquero, Laura, Duell, Stephanie, Pietenpol, Jennifer A., Browning, Philip J.
• Format: Journal Article
• Language: English
• Published: Cary, NC Oxford University Press 04-05-2005; Oxford Publishing Limited (England)
• Subjects: Binding sites; Biological and medical sciences; Blotting, Western; Cancer; CDC2-CDC28 Kinases - metabolism; Cell Cycle Proteins - metabolism; Cell Line, Tumor; Cyclin D; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases - metabolism; Cyclins - metabolism; Dermatology; Enzyme Inhibitors - metabolism; Half-Life; Herpes viruses; Herpesviridae Infections - enzymology; Herpesviridae Infections - metabolism; Herpesviridae Infections - virology; Herpesvirus 8, Human; Humans; Immunoprecipitation; Lymphoma; Medical sciences; Oncology; Phosphorylation; Proteins; Proto-Oncogene Proteins - metabolism; Recombinant Fusion Proteins - metabolism; Tumor Suppressor Proteins - metabolism; Tumors; Tumors of the skin and soft tissue. Premalignant lesions; Gammaherpesvirinae; Human; Skin disease; Enzyme; Herpesviridae; Transferases; Malignant tumor; Carcinogenesis; Virus; Kaposi sarcoma; Human herpesvirus 8; Cancerology; Kinase; Cell cycle; Cyclin K
• ISSN: 0027-8874; 1460-2105
• DOI: 10.1093/jnci/dji113

Background: Kaposi sarcoma–associated human herpesvirus (KSHV) encodes K-cyclin, a homologue of D-type cellular cyclins, which binds cyclin-dependent kinases to phosphorylate various substrates. K-cyclin/cdk phosphorylates a subset of substrates normally targeted by cyclins D, E, and A. We used cells naturally infected with KSHV to further characterize the biochemical features of K-cyclin. Methods: We used immunoprecipitation with K-cyclin antibodies to examine the association of K-cyclin with cdk2, cdk6, p21Cip1, and p27Kip1 proteins in BC3 cells. We separated populations of BC3 cells enriched in cells in G1, S, or G2/M phases by elutriation and measured K-cyclin protein and the kinase activity of K-cyclin/cdk6 complexes. The half-life of K-cyclin and cyclin D2 proteins was determined by blocking protein synthesis with cycloheximide and measuring proteins in cell lysates by western blot analysis. We fused the entire K-cyclin sequence to the carboxyl-terminal sequence of cellular cyclin D that contains the PEST degradation sequence to produce K-cyclin/D2 and transfected K-cyclin/D2 into K-cyclin–negative cells to investigate the effect of the PEST sequence on K-cyclin's stability. Results: Viral K-cyclin interacted with cyclin-dependent kinases cdk2, cdk4, and cdk6 and with the cyclin/cdk inhibitory proteins p21Cip1 and p27Kip1 in BC3 cell lysates. Unlike D-type cyclins, whose expression is cell cycle dependent, the level of K-cyclin was stable throughout the cell cycle, and the kinase associated with the K-cyclin/cdk6 complex was constitutively active. The half-life of K-cyclin (6.9 hours) was much longer than that of cellular cyclin D2 (0.6 hour) and that of K-cyclin/D2 (0.5 hour), probably because K-cyclin lacks the PEST degradation sequence present in D-type cyclins. Conclusion: The constitutive activation of K-cyclin/cdk complexes in KSHV-infected cells appears to result from the extended half-life of K-cyclin and may explain its role in Kaposi sarcoma.