Gab1 transduces PI3K-mediated erythropoietin signals to the Erk pathway and regulates erythropoietin-dependent proliferation and survival of erythroid cells.
In this study, we examined the biological functions of Gab1 in erythropoietin receptor (EPOR)-mediated signaling in vivo. Knockdown of Gab1 by the introduction of the Gab1 siRNA expression vector into F-36P human erythroleukemia (F-36P-Gab1-siRNA) cells resulted in a reduction of cell proliferation and survival in response to EPO. EPO-induced activation of Erk1/2 but not of Akt was significantly suppressed in F-36P-Gab1-siRNA cells compared with mock-transfected F-36P cells. The co-immunoprecipitation experiments revealed an EPO-enhanced association of Gab1 with the Grb2-SOS1 complex and SHP-2 in F-36P cells. A selective inhibitor of phosphatidylinositol 3-kinase (PI3K) LY294002 and short interfering RNA (siRNA) duplexes targeting the p85 regulatory subunit of PI3K (p85-siRNA) independently suppressed tyrosine phosphorylation of Gab1; its association with Grb2, SHP-2 and p85; and the activation of Erk in EPO-treated F-36P cells. LY294002 inhibited EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 in human primary EPO-sensitive erythroid cells. The co-immunoprecipitation experiments using the Jak inhibitor AG490 or siRNA duplexes targeting Jak2 and in vitro binding experiments demonstrated that Jak2 regulated Gab1-mediated Erk activation through tyrosine phosphorylation of Gab1. Taken together, these results suggest that Gab1 couples PI3K-mediated EPO signals with the Ras/Erk pathway and that Gab1 plays an important role in EPOR-mediated signal transduction involved in the proliferation and survival of erythroid cells.
Fukumoto
Tetsuya
T
Division of Hematology, Internal Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita-gun, Kagawa 761-0793, Japan. tf1163id@jt3.so-net.ne.jp
Kubota
Yoshitsugu
Y
Kitanaka
Akira
A
Yamaoka
Genji
G
Ohara-Waki
Fusako
F
Imataki
Osamu
O
Ohnishi
Hiroaki
H
Ishida
Toshihiko
T
Tanaka
Terukazu
T
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D041905:Erythroid Cells / Q000166:cytology* / Q000378:metabolism; D004921:Erythropoietin / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000378:metabolism; D020837:SOS1 Protein / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002867:Chromones; D004791:Enzyme Inhibitors; C098587:GAB1 protein, human; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D009025:Morpholines; D017467:Receptors, Erythropoietin; D020837:SOS1 Protein; D004921:Erythropoietin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11