Grb2-associated binder-1 plays a central role in the hepatocyte growth factor enhancement of hepatoma growth inhibition by K vitamin analog compound 5.
Compound 5 (Cpd 5), a K vitamin analog, has been shown to inhibit Hep3B human hepatoma cell growth in cultures and rat hepatoma growth in vivo through prolonged epidermal growth factor receptor (EGFR)-extracellular response kinase (ERK) phosphorylation, and hepatocyte growth factor (HGF) synergizes with Cpd 5 to enhance the inhibition of Hep3B cell and rat hepatoma growth. To explore the mechanisms mediating the HGF/Cpd 5 synergy, we examined the possible involvement of the Grb2-associated binder-1 (Gab1) docking protein because it interacts with both EGFR and HGF receptor c-Met pathways. We found that HGF enhanced Cpd 5-induced c-Met phosphorylation at Tyr-1349, a binding site for Gab1, resulting in increased c-Met binding to Gab1, and induced strong and prolonged Gab1 tyrosine phosphorylation. Prolonged Gab1 phosphorylation by HGF/Cpd 5 in turn enhanced the ability of Gab1 to bind to protein tyrosine phosphatase SHP2 and enhanced the activation of its downstream mitogen-activated protein kinase pathway. In contrast, this same HGF/Cpd 5 treatment inhibited Gab1 binding to phosphatidylinositol 3-kinase (PI3K), leading to the inactivation of the PI3K-Akt pathway. The inhibition of Akt phosphorylation by HGF/Cpd 5 further activated the Raf-MEK-ERK signaling cascade via an Akt-Raf1 interaction, leading to strong and prolonged ERK phosphorylation. The transfection of Hep3B cells with mutated Gab1 (Gab1 Y627F), which had lost its ability to bind SHP2, antagonized HGF/Cpd 5-induced ERK phosphorylation, whereas the transfection of Hep3B cells with mutated Gab1 3YF, which lost its ability to bind PI3K, further enhanced HGF/Cpd 5-induced ERK phosphorylation and cell growth inhibition.


CONCLUSIONS
Gab1 plays a central role in regulating HGF/Cpd 5 synergy in their actions on Hep3B cell growth inhibition.
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Ziqiu
Z
Wang
Lisheng
L
Ge
Meifang
M
Wang
Brian I
BI
Carr
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000503:physiopathology; D045744:Cell Line, Tumor / Q000187:drug effects; D004195:Disease Models, Animal; D004357:Drug Synergism; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D015398:Signal Transduction; D014812:Vitamin K / Q000031:analogs & derivatives* / Q000494:pharmacology
C446520:2-(2-hydroxyethylsulfanyl)-3-methyl-1,4-naphthoquinone; D048868:Adaptor Proteins, Signal Transducing; C098587:GAB1 protein, human; C490896:Gab1 protein, rat; D010750:Phosphoproteins; D014812:Vitamin K; D017228:Hepatocyte Growth Factor