Protein Kinase Cδ Inhibition of S-Phase Transition in Capillary Endothelial Cells Involves the Cyclin-dependent Kinase Inhibitor p27Kip1

Anthony W Ashton,Genichi Watanabe,Chris Albanese,Elizabeth O Harrington,J Anthony Ware,Richard G Pestell

doi:10.1074/jbc.274.30.20805

Anthony W Ashton, Genichi Watanabe + Show 4 more

Open Access

https://doi.org/10.1074/jbc.274.30.20805

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Abstract

Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC). Overexpression of PKCalpha has little effect on proliferation, whereas PKCdelta slows endothelial cell proliferation and induces S-phase arrest. Analyses were performed on EC overexpressing PKCalpha (PKCalphaEC) or PKCdelta (PKCdeltaEC) to determine the role of specific cell cycle regulatory proteins in the PKCdelta-induced cell cycle arrest. Serum-induced stimulation of cyclins D1, E, and A-associated kinase activity was delayed by 12 h in the PKCdeltaEC 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 PKCdeltaEC. In the PKCdeltaEC line, serum induced p27(Kip1) but not p16(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 PKCdeltaEC line, serum did not increase the amount of cyclin D1 bound to p27(Kip1). Transfection of full-length p27(Kip1) antisense into the PCKdeltaEC line reversed the S-phase arrest and resulted in normal cell cycle progression, suggesting a critical role for p27(Kip1) in the PKCdelta-mediated S-phase arrest.

Highlights

Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC)
To determine whether the proliferative defect observed in the PKC␦EC stable line was associated with alterations in cyclin-associated kinase activity, the cell lines PKC␦EC, PKC␣EC, and V-EC were analyzed
The current studies extend our previous findings that PKC␦ delays S-phase progression in rat microvascular endothelial cells [1]

Summary

Introduction

Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC). Serum-induced stimulation of cyclins D1, E, and A-associated kinase activity was delayed by 12 h in the PKC␦EC line in association with S-phase arrest. In recent studies we showed that overexpression of PKC␦, but not PKC␣, in EC inhibited cellular proliferation through an arrest in S-phase [1] These findings were consistent with several other studies in which the loss of PKC␦ expression was associated with increased cellular proliferation or transformation (20 –22). We report here that PKC␦ overexpression (PKC␦EC) delays serum-induced expression of kinase activity associated with cyclins D1, E, and A. P27Kip1-antisense reduced p27Kip levels and relieved the cell cycle defect induced by PKC␦, strongly suggesting that increased expression of p27Kip was responsible for the prolongation of S-phase in PKC␦EC PKC␦EC contained higher nuclear levels of the cyclindependent kinase inhibitor p27Kip than vector controls. p27Kip1-antisense reduced p27Kip levels and relieved the cell cycle defect induced by PKC␦, strongly suggesting that increased expression of p27Kip was responsible for the prolongation of S-phase in PKC␦EC

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