NUCLEAR-ASSOCIATION OF CYCLIN D1 IN HUMAN FIBROBLASTS - TIGHT-BINDINGTO NUCLEAR-STRUCTURES AND MODULATION BY PROTEIN-KINASE INHIBITORS

The association of cyclin D1 with nuclear structures was investigated in normal human fibroblasts by using hypotonic detergent extraction pr ocedures, immunofluorescence quantitation with flow cytometry, and Wes tern blot analysis, About 20% of the total cellular levels of cyclin D 1 was found to be tightly bound to nuclear structures, being the compl ex formation resistant to DNase I treatment and to high salt extractio n. Maximal levels of the insoluble form of the protein were found in t he middle to late G1 phase of the cell cycle. Cell fractionation and i mmunoprecipitation techniques after in vivo P-32-labeling showed that both soluble and nuclear-bound forms of cyclin D1 were phosphorylated. Both fractions were reactive to an anti-phosphotyrosine antibody, whi le only the latter was detectable with an anti-phosphoserine antibody. Treatment with the protein kinase inhibitor staurosporine, which indu ces a cell cycle arrest in early G1 phase, strongly reduced cyclin D1 phosphorylation. Concomitantly, the ratio of nuclear-bound/total cycli n D1 levels was reduced by about 60%, compared with the control value, The protein kinase A specific inhibitor isoquinoline-suIfonamide (H-8 9) induced a similar reduction in the ratio, with no significant modif ication in the total amount of protein, In contrast, both calphostin C and bisindolylmaleimide, specific inhibitors of protein kinase C, con sistently increased by 30-50% the ratio of nuclear-bound/total amount of the cyclin protein. These results suggest that, during the G1 phase , formation of an insoluble complex of cyclin D1 occurs at nuclear mat rix structures and that this association is mediated by a protein kina se A-dependent pathway. (C) 1997 Academic Press.