Two Populations of p27 Use Differential Kinetics to Phosphorylate Ser10 and Thr187 via PI3-kinase in Response to FGF-2 Stimulation

Abstract

Purpose:: 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.

Methods:: 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.

Results:: 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.

Conclusions:: 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.

Keywords: cornea: endothelium • proliferation • cornea: basic science