Protein kinase c delta inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27(Kip1)

Distinct protein kinase C (PKC) isoforms differentially regulate cellular p roliferation in rat microvascular endothelial cells (EC), Overexpression of PKC alpha has little effect on proliferation, whereas PKC delta slows endo thelial cell proliferation and induces S-phase arrest. Analyses were perfor med on EC overexpressing PKC alpha (PKC alpha EC) or PKC delta (PKC delta E C) to determine the role of specific cell cycle regulatory proteins in the PKC delta-induced cell cycle arrest, Serum-induced stimulation of cyclins D 1, 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 fo r cyclins D1, E, and A were similar. Nuclear accumulation of cyclin D1 prot ein 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 di d not affect p27(Kip1) levels in the control vascular endothelial cell line . Immunoprecipitation-Western blotting analysis of p27(Kip1) showed serum s timulation of the vascular endothelial cell line resulted in increased amou nts of cyclin D1 bound to p27(Kip1). In the PKC delta EC line, serum did no t 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 critica l role for p27(Kip1) in the PKC delta-mediated S-phase arrest.