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Abstract
Kaposi sarcoma-associated herpes virus (KSHV) encodes a D-like cyclin (K-cyclin) that is thought to contribute to the viral oncogenicity. K-cyclin activates cellular cyclin-dependent kinases (CDK) 4 and 6, generating enzymes with a substrate selectivity deviant from CDK4 and CDK6 activated by D-type cyclins, suggesting different biochemical and biological functions. Here we report the identification of the actin- and calmodulin-binding protein caldesmon (CALD1) as a novel K-cyclin.CDK substrate, which is not phosphorylated by D.CDK. CALD1 plays a central role in the regulation of microfilament organization, consequently controlling cell shape, adhesion, cytokinesis and motility. K-cyclin.CDK6 specifically phosphorylates four Ser/Thr sites in the human CALD1 carboxyl terminus, abolishing CALD1 binding to its effector protein, actin, and its regulator protein, calmodulin. CALD1 is hyperphosphorylated in cells following K-cyclin expression and in KSHV-transformed lymphoma cells. Moreover, expression of exogenous K-cyclin results in microfilament loss and changes in cell morphology; both effects are reliant on CDK catalysis and can be reversed by the expression of a phosphorylation defective CALD1. Together, these data strongly suggest that K-cyclin expression modulates the activity of caldesmon and through this the microfilament functions in cells. These results establish a novel link between KSHV infection and the regulation of the actin cytoskeleton.
Highlights
Most cellular responses are controlled through protein phosphorylation and the protein kinases catalyzing this process represent the largest single family of enzymes (1)
Cyclin D activates both CDK4 and CDK6 to phosphorylate retinoblastoma tumor suppressor protein (Rb) to a similar extent, but cyclin D11⁄7CDK4 complexes have a higher specific kinase activity when produced in insect cells, and this kinase complex was used throughout the kinase substrate tracking and elucidation (KESTREL) screen
No incorporation of phosphate into Rb Ct was observed with CDK6 alone, indicating that a kinase activity resembling K-cyclin1⁄7CDK6 is not generated in insect cells in the absence of K-cyclin expression
Summary
Most cellular responses are controlled through protein phosphorylation and the protein kinases catalyzing this process represent the largest single family of enzymes (1). We report the identification of the actin- and calmodulin-binding protein caldesmon (CALD1) as a novel K-cyclin1⁄7CDK substrate, which is not phosphorylated by D1⁄7CDK. In addition to Rb, K-cyclin1⁄7CDK complexes can phosphorylate proteins that are not substrates for those CDKs when activated by cellular cyclin D.
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