Nuclear association of cyclin D1 in human fibroblasts: tight binding to nuclear structures and modulation by protein kinase inhibitors

Experimental Cell Research
A I ScovassiE Prosperi

Abstract

The association of cyclin D1 with nuclear structures was investigated in normal human fibroblasts by using hypotonic detergent extraction procedures, immunofluorescence quantitation with flow cytometry, and Western blot analysis. About 20% of the total cellular levels of cyclin D1 was found to be tightly bound to nuclear structures, being the complex formation resistant to DNase I treatment and to high salt extraction. Maximal levels of the insoluble form of the protein were found in the middle to late G1 phase of the cell cycle. Cell fractionation and immunoprecipitation techniques after in vivo 32P-labeling showed that both soluble and nuclear-bound forms of cyclin D1 were phosphorylated. Both fractions were reactive to an anti-phosphotyrosine antibody, while only the latter was detectable with an anti-phosphoserine antibody. Treatment with the protein kinase inhibitor staurosporine, which induces a cell cycle arrest in early G1 phase, strongly reduced cyclin D1 phosphorylation. Concomitantly, the ratio of nuclear-bound/total cyclin D1 levels was reduced by about 60%, compared with the control value. The protein kinase A specific inhibitor isoquinoline-sulfonamide (H-89) induced a similar reduction in the ratio, with no signi...Continue Reading

Citations

Jul 9, 2008·Molecular and Cellular Biology·Masahiro HitomiDennis W Stacey
Oct 1, 2009·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Pattatheyil ArunCarter Van Waes
Oct 6, 2000·American Journal of Respiratory Cell and Molecular Biology·C F Thomas, A H Limper
May 1, 2007·Translational Research : the Journal of Laboratory and Clinical Medicine·San-Cher ChenLea-Yea Chuang
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Aug 29, 2002·In Vitro Cellular & Developmental Biology. Animal·Jeannine BotosDeborah T Kochevar

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