Involvement of Rac in G1/S Transition via Phosphorylation of p27 at Both Thr187 and Ser10 Sites in PI 3-Kinase Pathway but not in ERK1/2 Pathway in Rabbit Corneal Endothelial Cells

Abstract

Purpose: : The small GTPase, Rac1, is a key transducer of proliferative signals in various cell types. But its significance to cell proliferation has not been studied in rabbit corneal endothelial cells (CECs). We investigated whether the ERK1/2 pathway is involved in the mitogenic pathway via degradation of p27 and whether Rac is involved in G1/S transition via PI 3-kinase, ERK1/2, or both pathways.

Methods: : GTP pull-down assays were used to identify activated Rac and Rho GTPases. Cell proliferation was measured by MTT assay. Expression of Rac, ERK1/2, Akt, KIS, Cdc25a, Lamin B, and tubulin was analyzed by immunoblotting. Pharmacological inhibitors were used to block Rac, Rho, ERK1/2, or Akt.

Results: : In CECs pretreated with specific inhibitors for ERK1/2 (U0126) or Rac (NSC23766), both inhibitors markedly inhibited cell proliferation stimulated with FGF-2, but not RhoA inhibitor (Y27632). Our previous data showed FGF-2 stimulates cell proliferation using PI 3-kinase and ERK1/2 pathways in parallel and PI 3-kinase works as upstream regulator of Rac1. We investigated cross-talk between Rac1 and ERK1/2. Rac1 and ERK1/2 inhibitor failed to block phosphorylation of ERK1/2 and activation of Rac1, respectively, suggesting the Rac1 and ERK1/2 pathways are parallel and independent in response to stimulation with FGF-2, similar to PI 3-kinase and ERK1/2. It was also defined whether Rac1 and ERK1/2 are involved in phosphorylation of p27 at Ser10 and Thr187 site, events that are prerequisite for degradation. Both Rac1 and ERK1/2 inhibitors reduced about 50% of phospho-p27 at Ser10; phosphorylation was completely blocked by a combination of inhibitors for Rac1 and ERK1/2. Each inhibitor also completely blocked phosphorylation of p27 at Thr187 site. We further defined that expression of KIS (kinase for phosphorylation of p27 at Ser10 site) and Cdc25a (upstream regulator of Cdk2 for phosphorylation of p27 at Thr187 site) was also completely blocked by each inhibitor.

Conclusions: : Rac1 and ERK1/2 are involved in cell proliferation in parallel pathways mediated by FGF-2 through phosphorylation of p27 at Ser10 by KIS and at Thr187 site through the Cdc25a pathway.

Keywords: cornea: endothelium • signal transduction • cornea: basic science