title: 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.
abstract: 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.
authors.affiliation: Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California 90089, USA.
authors.forename: Jeong Goo
authors.initials: JG
authors.lastname: Lee
authors.forename: Eunduck P
authors.initials: EP
authors.lastname: Kay
mesh_terms: 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
chemical_list: D009687:Nuclear Proteins; D016222:Fibroblast Growth Factor 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D012694:Serine; D019869:Phosphatidylinositol 3-Kinases