Two populations of p27 use differential kinetics to phosphorylate Ser-10 and Thr-187 via phosphatidylinositol 3-Kinase in response to fibroblast growth factor-2 stimulation.
The cyclin-dependent kinase inhibitor p27 regulates cell cycle progression. We investigated whether FGF-2 uses PI 3-kinase to facilitate phosphorylation of p27 on serine 10 (Ser-10) and threonine 187 (Thr-187) and whether the two phosphorylation sites were differentially regulated. FGF-2 stimulation dramatically increased p27 phosphorylation at Ser-10 and Thr-187 using differential kinetics, and the FGF-2-induced p27 phosphorylation was completely blocked at both sites by LY294002. We determined the physical and biochemical interaction of p27 with the Cdk2-cyclin E complex in response to FGF-2 stimulation. Maximal p27 binding to Cdk2-cyclin E occurred at 12 h; the maximal level of p27 phosphorylation at Thr-187 in the ternary complex was observed at 16 h; ubiquitination of the Thr-187-phosphorylated p27 (pp27Thr-187) was observed starting at 12 h and continuing up to 24 h. However, maximum p27 phosphorylation at Ser-10 occurred in the nucleus 6 h after FGF-2 stimulation; maximal export of Ser-10-phosphorylated p27 (pp27Ser-10) occurred 8 h after FGF-2 treatment, and pp27Ser-10 was simultaneously ubiquitinated. We further investigated which of the two phosphorylated p27 was involved in G(1)/S progression. LY294002 blocked 64% of the cell proliferation stimulated by FGF-2. Use of leptomycin B to block nuclear export of pp27Ser-10 greatly decreased the FGF-2-stimulated cell proliferation (44%), suggesting that phosphorylation of p27 at Ser-10 is the major mechanism for G(1)/S transition. Our results suggest that differential kinetics are observed in p27 phosphorylation at Ser-10 and Thr-187 and that pp27Thr-187 and pp27Ser-10 may represent two populations of p27 observed in the G(1) phase of the cell cycle.
Lee
Jeong Goo
JG
Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California 90089, USA.
Kay
Eunduck P
EP
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D004728:Endothelium, Corneal / Q000166:cytology; D005123:Eye / Q000166:cytology; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism* / Q000494:pharmacology; D007399:Interphase / Q000187:drug effects; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D009687:Nuclear Proteins; D016222:Fibroblast Growth Factor 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D012694:Serine; D019869:Phosphatidylinositol 3-Kinases