Abstract
Purpose: :
The cyclin–dependent kinase inhibitor p27 regulates cell cycle progression; its activity is controlled by its concentration and degradation through phosphorylation. We investigated whether FGF–2 facilitates phosphorylation of p27 at serine 10 site (Ser10) and threonine 187 site (Thr187) by PI 3–kinase in corneal endothelial cells, leading to a complete removal of p27. Two distinct enzymes are known to involve in phosphorylation of p27: kinase interacting stathmin for Ser10 and Cdk2 for Thr187.
Methods: :
p27 phosphorylation and protein expression were analyzed by immunoblot analysis. The association between proteins was determined by co–immunoprecipitation and immunoblot analysis. Cdk2 kinase activity was determined by measuring the phosphorylation rate of Histone–1. LY294002 was used to inhibit PI 3–kinase.
Results: :
The effect of FGF–2 treatment on the expression of p27 and Cdk2 was unremarkable; but FGF–2 stimulation dramatically increased the phosphorylation of p27 at Ser10 and Thr187. The maximum phosphorylation at Ser10 was observed 4 h after FGF–2 stimulation, whereas maximum phosphorylation at Thr187 required a 16–h exposure of cells to FGF–2. FGF–2–induced p27 phosphorylation was completely blocked by LY294002. We further determined the cascade events of phosphorylation of p27 at Thr187 using protein complex immunoprecipitated with anti–Cdk2 antibody. FGF–2 facilitated interaction between p27 and Cdk2 in a time–dependent manner (maximum association at 12 h); maximum Cdk2 activity was observed 16 h after FGF–2 stimulation; and maximum phosphorylation of p27 at Thr187 required a 16–h exposure of cells to FGF–2. All these actions of FGF–2 are inhibited by LY2940002. On the other hand, phosphorylation of p27 at Ser10 was not observed in the protein complex containing Cdk2 and phosphorylated p27.
Conclusions: :
Our results suggest that different kinetics are observed in phosphorylation of Ser10 and Thr187 of p27 in response to FGF–2 stimulation and that association of p27 and Cdk2 and subsequent activation of Cdk2 are prerequisite to the phosphorylation event of p27 at Thr187 by Cdk2.
Keywords: cornea: basic science • cornea: endothelium • proliferation