Jeong Goo Lee1, EunDuck P Kay. 1. Doheny Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Abstract
PURPOSE: To determine the mechanism of p27 phosphorylation through common and differential pathways triggered by FGF-2 in corneal endothelial cells (CECs). METHODS: A GTP pull-down assay was performed to identify Rac1-GTP. Expression and activation of protein were analyzed by immunoblotting. Cell proliferation was measured by an MTT assay. Transfection of CECs with kinase-interacting stathmin (KIS) siRNA was performed. RESULTS: FGF-2 activated Rac1 through Akt, and Rac1 inhibitor greatly inhibited the FGF-2-stimulated cell proliferation. Rac1 inhibitor reduced p27 phosphorylation at both serine 10 (Ser10) and threonine 187 (Thr187). ERK1/2 was also involved in FGF-2-stimulated CEC proliferation and phosphorylation of p27 at Ser10 and Thr187 in parallel to phosphatidylinositol (PI) 3-kinase. In both PI 3-kinase/Rac1 and ERK1/2 pathways, Ser10 of p27 is phosphorylated by KIS, confirmed by siRNA to KIS, which subsequently hampered the FGF-2-stimulated cell proliferation, while Thr187 of p27 was phosphorylated through Cdk2 activated by Cdc25A. Cdc25A inhibitor blocked activation of Cdk2, phosphorylation of p27 at Thr187, and cell proliferation. FGF-2 induced both KIS and Cdc25A during the G1 phase; the maximum KIS expression was observed 4 hours after FGF-2 stimulation, while the maximum Cdc25A expression was observed at 12 hours. Blockade of ERK1/2 and Rac1 greatly reduced KIS and Cdc25A expression. CONCLUSIONS: Results suggest that FGF-2 uses both PI 3-kinase/Rac1 and ERK pathways for cell proliferation; two signals employ common pathways for phosphorylating p27 according to the sites (KIS for Ser10 and Cdc25A/Cdk2 for Thr187) with their characteristic kinetics (early G1 for Ser10 and late G1 for Thr187).
PURPOSE: To determine the mechanism of p27 phosphorylation through common and differential pathways triggered by FGF-2 in corneal endothelial cells (CECs). METHODS: A GTP pull-down assay was performed to identify Rac1-GTP. Expression and activation of protein were analyzed by immunoblotting. Cell proliferation was measured by an MTT assay. Transfection of CECs with kinase-interacting stathmin (KIS) siRNA was performed. RESULTS:FGF-2 activated Rac1 through Akt, and Rac1 inhibitor greatly inhibited the FGF-2-stimulated cell proliferation. Rac1 inhibitor reduced p27 phosphorylation at both serine 10 (Ser10) and threonine 187 (Thr187). ERK1/2 was also involved in FGF-2-stimulated CEC proliferation and phosphorylation of p27 at Ser10 and Thr187 in parallel to phosphatidylinositol (PI) 3-kinase. In both PI 3-kinase/Rac1 and ERK1/2 pathways, Ser10 of p27 is phosphorylated by KIS, confirmed by siRNA to KIS, which subsequently hampered the FGF-2-stimulated cell proliferation, while Thr187 of p27 was phosphorylated through Cdk2 activated by Cdc25A. Cdc25A inhibitor blocked activation of Cdk2, phosphorylation of p27 at Thr187, and cell proliferation. FGF-2 induced both KIS and Cdc25A during the G1 phase; the maximum KIS expression was observed 4 hours after FGF-2 stimulation, while the maximum Cdc25A expression was observed at 12 hours. Blockade of ERK1/2 and Rac1 greatly reduced KIS and Cdc25A expression. CONCLUSIONS: Results suggest that FGF-2 uses both PI 3-kinase/Rac1 and ERK pathways for cell proliferation; two signals employ common pathways for phosphorylating p27 according to the sites (KIS for Ser10 and Cdc25A/Cdk2 for Thr187) with their characteristic kinetics (early G1 for Ser10 and late G1 for Thr187).
Authors: Bin Huang; Paul R Krafft; Qingyi Ma; William B Rolland; Basak Caner; Tim Lekic; Anatol Manaenko; Mai Le; Jiping Tang; John H Zhang Journal: Neurobiol Dis Date: 2012-01-24 Impact factor: 5.996