Constitutively active Kcyclin/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 phosphory-late 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 character-ize the biochemical features of K-cyclin. Methods: We used immunoprecipitation with K-cyclin antibodies to examine the association of K-cyclin with cdk2, cdk6, p21 Cip1, and p27 Kip1 proteins in BC3 cells. We separated populations of BC3 cells enriched in cells in G 1, S, or G 2 /M phases by elu-triation and measured K-cyclin protein and the kinase ac-tivity 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 cel