title: Adaptor molecule Crk is required for sustained phosphorylation of Grb2-associated binder 1 and hepatocyte growth factor-induced cell motility of human synovial sarcoma cell lines.
abstract: Activation of the c-Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes mitogenic, motogenic, and morphogenic cellular responses. Aberrant HGF/c-Met signaling has been strongly implicated in tumor cell invasion and metastasis. Both HGF and its receptor c-Met have been shown to be overexpressed in human synovial sarcoma, which often metastasizes to the lung; however, little is known about HGF-mediated biological effects in this sarcoma. Here, we provide evidence that Crk adaptor protein is required for the sustained phosphorylation of c-Met-docking protein Grb2-associated binder 1 (Gab1) in response to HGF, leading to the enhanced cell motility of human synovial sarcoma cell lines SYO-1, HS-SY-II, and Fuji. HGF stimulation induced the sustained phosphorylation on Y307 of Gab1 where Crk was recruited. Crk knockdown by RNA interference disturbed this HGF-induced tyrosine phosphorylation of Gab1. By mutational analysis, we identified that Src homology 2 domain of Crk is indispensable for the induction of the phosphorylation on multiple Tyr-X-X-Pro motifs containing Y307 in Gab1. HGF remarkably stimulated cell motility and scattering of synovial sarcoma cell lines, consistent with the prominent activation of Rac1, extreme filopodia formation, and membrane ruffling. Importantly, the elimination of Crk in these cells induced the disorganization of actin cytoskeleton and complete abolishment of HGF-mediated Rac1 activation and cell motility. Time-lapse microscopic analysis revealed the significant attenuation in scattering of Crk knockdown cells following HGF treatment. Furthermore, the depletion of Crk remarkably inhibited the tumor formation and its invasive growth in vivo. These results suggest that the sustained phosphorylation of Gab1 through Crk in response to HGF contributes to the prominent activation of Rac1 leading to enhanced cell motility, scattering, and cell invasion, which may support the crucial role of Crk in the aggressiveness of human synovial sarcoma.
authors.affiliation: Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Watanabe
authors.forename: Masumi
authors.initials: M
authors.lastname: Tsuda
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Makino
authors.forename: Shin
authors.initials: S
authors.lastname: Ichihara
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Sawa
authors.forename: Akio
authors.initials: A
authors.lastname: Minami
authors.forename: Naoki
authors.initials: N
authors.lastname: Mochizuki
authors.forename: Kazuo
authors.initials: K
authors.lastname: Nagashima
authors.forename: Shinya
authors.initials: S
authors.lastname: Tanaka
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D005260:Female; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010766:Phosphorylation; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D013584:Sarcoma, Synovial / Q000378:metabolism / Q000473:pathology*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C485285:CRK protein, human; C098587:GAB1 protein, human; D050737:Proto-Oncogene Proteins c-crk; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met; D020830:rac1 GTP-Binding Protein