Purchase this article with an account.
J. Lee, E. P. Kay; Two Populations of p27 Use Differential Kinetics to Phosphorylate Ser10 and Thr187 via PI3-kinase in Response to FGF-2 Stimulation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2700.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We investigated whether FGF-2 uses PI 3-kinase to facilitate phosphorylation of p27 at serine 10 (Ser10) and threonine 187 (Thr187) and the two phosphorylation sites were differentially regulated in corneal endothelial cells.
p27 phosphorylation and protein expression were analyzed by 2-D and immunoblotting. The association between proteins and ubiquitination of p27 were determined by co-immunoprecipitation and immunoblotting. Cdk2 kinase activity was determined by measuring the phosphorylation rate of Histone-1.
FGF-2 stimulation dramatically increased the phosphorylation of p27 at Ser10 and Thr187 using differential kinetics: the maximum phosphorylation at Ser10 and Thr187 were observed 6 and 16 h after FGF-2 stimulation, respectively. FGF-2-induced p27 phosphorylation at both sites was completely blocked by LY294002. We determined the physical and biochemical interaction of p27 with CycE/Cdk2 complex in response to FGF-2 stimulation; maximum binding of p27 to CycE/Cdk2 complex occurred at 12 h; maximum level of phosphorylation of p27 at Thr187 in the ternary complex was observed at 16 h; ubiquitination of the Thr187 phospho-p27 (pp27Thr187) was observed starting at 12 h and continuing for up to 24 h. However, the phosphorylation of p27 at Ser10 occurred in the nucleus with a maximum rate 6 h after FGF-2 stimulation. The Ser10 phospho-p27 (pp27Ser10) was ubiquitinated and was simultaneously exported to cytoplasm at a maximum rate 8 h after FGF-2 treatment. We further investigated which of the two phosphorylated p27 was involved in G1/S progression. When LY294002 was used, the inhibitor blocked 64% of cell proliferation stimulated by FGF-2; the blocking of nuclear export of pp27Ser10 by leptomycin B greatly decreased the FGF-2 stimulated cell proliferation (44%), suggesting that p27 phosphorylation of p27 at Ser10 is the major mechanism for G1/S transition.
Our results suggest that different kinetics are observed in p27 phosphorylation at Ser10 and Thr187 and that pp27Thr187 and pp27Ser10 may represent two separate populations of p27 observed during G1/S transition.
This PDF is available to Subscribers Only