Constitutively active K-cyclin/cdk6 kinase in Kaposi sarcoma-associated herpesvirus-infected cells.
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.


CONCLUSIONS
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.
Van Dross
Rukiyah
R
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City 66160, USA. rvandross@kumc.edu
Yao
Shan
S
Asad
Shaheena
S
Westlake
Grant
G
Mays
Deborah J
DJ
Barquero
Laura
L
Duell
Stephanie
S
Pietenpol
Jennifer A
JA
Browning
Philip J
PJ
D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D056741:Cyclin D; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D006207:Half-Life; D006566:Herpesviridae Infections / Q000201:enzymology / Q000378:metabolism* / Q000821:virology; D019288:Herpesvirus 8, Human; D006801:Humans; D047468:Immunoprecipitation; D008223:Lymphoma; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D056741:Cyclin D; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases