Differential modulation of G1 cyclins and the Cdk inhibitor p27kip1 by platelet-derived growth factor and plasma factors in density-arrested fibroblasts.
Stimulation of quiescent Balb/c 3T3 fibroblasts into S phase requires the synergistic action of platelet-derived growth factor (PDGF) and progression factors found in platelet-poor plasma (PPP). Traverse of the G1/S phase boundary and the initiation of DNA replication require functional cyclin E-cyclin-dependent kinase (Cdk) 2 and cyclin A-Cdk2 complexes; however, the mechanisms by which PDGF and PPP regulate Cdk2 activation are not known. Density-arrested fibroblasts contain low levels of cyclins E and A, and high levels of the Cdk inhibitor p27kip1. Exposure of PDGF, which stimulates cell cycle entry but not progression through G1, induces the formation of cyclin D1-Cdk4 complexes that bind p27kip1 and titrate the pool of Kip1 available to inhibit Cdk2. In addition, PDGF stimulates a moderate transient reduction in the abundance of p27kip1 protein. However, limited expression of cyclin E and cyclin A is observed after PDGF treatment, and in the absence of PPP, p27 levels are sufficient to bind and inactivate existing cyclin-Cdk complexes. Although plasma does not significantly increase the proportion of Kip1 bound to cyclin D1-Cdk4, stimulation of PDGF-treated cells with plasma does overcome the threshold inhibition of p27kip1 by further increasing the expression of cyclins E and A and decreasing the amount of Kip1 over a prolonged time period. Our results indicate that the distinct mitogenic activities of PDGF and PPP differentially influence the activation of cyclin E- and cyclin A-associated kinases that ultimately regulate entry into S phase.
Winston
J
J
Department of Cell Biology Vanderbilt University, Nashville, Tennessee 37232, USA.
Dong
F
F
Pledger
W J
WJ
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells; D000818:Animals; D000077214:Becaplermin; D001769:Blood; D042846:CDC2-CDC28 Kinases; D002417:Cattle; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002772:Cholera Toxin / Q000494:pharmacology; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016213:Cyclins / Q000096:biosynthesis*; D003513:Cycloheximide / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005347:Fibroblasts; D016193:G1 Phase; D015870:Gene Expression; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D003470:Culture Media; D016213:Cyclins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D000077214:Becaplermin; D002772:Cholera Toxin; D003513:Cycloheximide; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; C495902:Cdk4 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D015056:1-Methyl-3-isobutylxanthine