Protein kinase cδ inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27Kip1

Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC), Overexpression of PKC alpha has little effect on proliferation, whereas PKC delta slows endothelial cell proliferation and induces S-phase arrest. Analyses were performed on EC overexpressing PKC alpha (PKC alpha EC) or PKC delta (PKC delta EC) to determine the role of specific cell cycle regulatory proteins in the PKC delta-induced cell cycle arrest, Serum-induced stimulation of cyclins D1, E, and A-associated kinase activity was delayed by 12 h in the PKC delta EC line in association with S-phase arrest. However, the protein levels for cyclins D1, E, and A were similar. Nuclear accumulation of cyclin D1 protein in response to serum was also delayed in PKC delta EC. In the PKC delta EC line, serum induced p27(Kip1) hut not p15(Ink4a) or p21(Cip1). Serum did not affect p27(Kip1) levels in the control vascular endothelial cell line. Immunoprecipitation-Western blotting analysis of p27(Kip1) showed serum stimulation of the vascular endothelial cell line resulted in increased amounts of cyclin D1 bound to p27(Kip1). In the PKC delta EC line, serum did not increase the amount of cyclin D1 bound to p27(Kip1). Transfection of full-length p27(Kip1) antisense into the PCK delta EC line reversed the S-phase arrest and resulted in normal cell cycle progression, suggesting a critical role for p27(Kip1) in the PKC delta-mediated S-phase arrest.