Nuclear association of cyclin D1 in human fibroblasts: tight binding to 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 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 significant modification in the total amount of protein. In contrast, both calphostin C and bisindolylmaleimide, specific inhibitors of protein kinase C, consistently 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 matrix structures and that this association is mediated by a protein kinase A-dependent pathway.
Scovassi
A I
AI
Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R., Pavia, Italy.
Stivala
L A
LA
Rossi
L
L
Bianchi
L
L
Prosperi
E
E
D002455:Cell Division; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D019938:Cyclin D1 / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology*; D008168:Lung; D008301:Maleimides / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors; D019311:Staurosporine / Q000494:pharmacology*; D013449:Sulfonamides
D004791:Enzyme Inhibitors; D007211:Indoles; D007546:Isoquinolines; D008301:Maleimides; D009281:Naphthalenes; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; C061465:calphostin complex; D019938:Cyclin D1; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C088060:bisindolylmaleimide