Protein kinase Cdelta 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 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.
Cardiovascular Division, Albert Einstein College of Medicine, Bronx, New York 10461, USA. ashton@aecom.yu.edu
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Ashton
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Watanabe
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Albanese
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Harrington
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Ware
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Pestell
D000818:Animals; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D016147:Genes, Tumor Suppressor; D007527:Isoenzymes / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051381:Rats; D016196:S Phase; D025521:Tumor Suppressor Proteins
C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D016895:Culture Media, Serum-Free; D007527:Isoenzymes; D008869:Microtubule-Associated Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D018844:Cyclin-Dependent Kinases