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.
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).
Lee
Jeong Goo
JG
Doheny Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Kay
EunDuck P
EP
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*
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
Invest Ophthalmol Vis Sci. 2008 Jan;49(1):189-96
18172092
Cancer Res. 2005 Jun 1;65(11):4870-80
15930308
J Biol Chem. 2004 Apr 2;279(14):13755-63
14742443
J Biol Chem. 1982 Jun 25;257(12):7116-21
6282831
Neurochem Int. 2008 May;52(6):1266-75
18319192
Arch Ophthalmol. 1982 Jan;100(1):122-34
7055463
Cornea. 1998 Nov;17(6):669-71
9820949
Biochem Pharmacol. 2010 Mar 15;79(6):926-37
19883628
Nat Cell Biol. 1999 Aug;1(4):193-9
10559916
Cancer Biol Ther. 2006 Oct;5(10):1340-7
16969075
Nat Med. 2002 Oct;8(10):1153-60
12244302
Mol Cell Biol. 2009 Apr;29(8):2254-63
19204084
Invest Ophthalmol Vis Sci. 1996 Jul;37(8):1566-75
8675399
Mol Biol Cell. 2009 Apr;20(8):2186-95
19244340
Invest Ophthalmol Vis Sci. 2009 May;50(5):2067-76
19136710
Oncogene. 2009 Oct 15;28(41):3652-62
19648963
Invest Ophthalmol Vis Sci. 2003 Apr;44(4):1521-8
12657588
J Biol Chem. 2007 Mar 2;282(9):6444-54
17209046
Neurosci Lett. 2009 Sep 25;461(3):252-7
19539699
J Neurosci. 2009 Jan 21;29(3):775-88
19158303
J Biol Chem. 2001 Jun 22;276(25):22404-9
11304535
Mol Vis. 2000 Mar 07;6:15-23
10731515
J Biol Chem. 2004 Jul 30;279(31):32325-32
15173165
Nephrol Dial Transplant. 2007 Dec;22(12):3431-41
17893107
J Biol Chem. 2002 Aug 9;277(32):28706-13
12042314
Genes Dev. 1999 May 1;13(9):1181-9
10323868
Invest Ophthalmol Vis Sci. 2000 Mar;41(3):660-7
10711678
J Biol Chem. 2008 Nov 7;283(45):30911-8
18715870
Invest Ophthalmol Vis Sci. 2010 Feb;51(2):822-9
19797202
Invest Ophthalmol Vis Sci. 1994 Apr;35(5):2427-35
8163333
Cell Cycle. 2005 Jan;4(1):87-95
15611642
EMBO J. 2002 Jul 1;21(13):3390-401
12093740
Invest Ophthalmol Vis Sci. 1982 Feb;22(2):200-12
7035394
EMBO Rep. 2008 Aug;9(8):766-73
18566599
Eur J Biochem. 2001 Feb;268(3):487-98
11168386
J Biol Chem. 2002 Apr 26;277(17):14355-8
11889117
Invest Ophthalmol Vis Sci. 2009 Aug;50(8):3680-7
19387080
Cell Cycle. 2003 Jul-Aug;2(4):339-45
12851486
J Cell Physiol. 2005 Aug;204(2):437-44
15672448
Invest Ophthalmol Vis Sci. 2006 Jun;47(6):2358-68
16723445
J Biol Chem. 2002 Dec 6;277(49):47469-75
12359725
Invest Ophthalmol Vis Sci. 2005 Dec;46(12):4495-503
16303940
Invest Ophthalmol Vis Sci. 2006 Apr;47(4):1376-86
16565371