title: PI 3-kinase/Rac1 and ERK1/2 regulate FGF-2-mediated cell proliferation through phosphorylation of p27 at Ser10 by KIS and at Thr187 by Cdc25A/Cdk2.
abstract: OBJECTIVE
  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.affiliation: Doheny Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Jeong Goo
authors.initials: JG
authors.lastname: Lee
authors.forename: EunDuck P
authors.initials: EP
authors.lastname: Kay
mesh_terms: D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D050777:Stathmin / Q000235:genetics; D013912:Threonine / Q000378:metabolism; D014162:Transfection; D020687:cdc25 Phosphatases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D034741:RNA, Small Interfering; D050777:Stathmin; D016222:Fibroblast Growth Factor 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D012694:Serine; D019869:Phosphatidylinositol 3-Kinases; D051357:Cyclin-Dependent Kinase 2; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020687:cdc25 Phosphatases; D020830:rac1 GTP-Binding Protein
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