CAK-independent activation of CDK6 by a viral cyclin.
In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16(INK4a). Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16(INK4a) sensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6(T177A) together with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK.
Kaldis
P
P
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520-8114, USA. Kaldis@ncifcrf.gov
Ojala
P M
PM
Tong
L
L
Mäkelä
T P
TP
Solomon
M J
MJ
D017209:Apoptosis / Q000187:drug effects*; D051361:Cyclin-Dependent Kinase 6; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism / Q000494:pharmacology; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D019288:Herpesvirus 8, Human; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000494:pharmacology*; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D019941:Cyclin-Dependent Kinase Inhibitor p16; D016213:Cyclins; D014764:Viral Proteins; D017346:Protein Serine-Threonine Kinases; C495911:CDK6 protein, human; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase