Hepatocyte growth factor receptor (Met) plays an important role in the progression of multiple cancer types. The overexpression of Met in DLD-1 colon carcinoma cells with kirsten rat sarcoma oncogene homolog (KRAS) oncogene activation resulted in enhanced subcutaneous and orthotopic tumor growth rate and increased metastatic potential. To elucidate the mechanism of this effect, we stably expressed kinase-inactive Met K1110A , Src homology 2 (SH2)-binding domain-inactive Met Y1349/1356F , growth

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... factor receptor-bound protein 2 (Grb2) non-binding Met N1358H and mutant receptors with ability to selectively recruit signaling proteins Grb2, src homology domain c-terminal adaptor homolog (Shc), phospholipase c-gamma (PLCg) and p85 phosphatidyl inositol 3 kinase. As subcutaneous implants, DLD-1 cells that expressed the majority of these receptor constructs failed to recapitulate the tumor growth-enhancing effect of the wild-type Met receptor. The Grb2-and Shc-recruiting Met mutants demonstrated slight but consistent tumor-suppressive activity, whereas the expression of N1358H mutant stimulated tumor growth rate comparable with the wild-type receptor. This suggests that direct Grb2/Shc binding does not contribute to the tumor progression activity of Met receptor. The tumors expressing Grb2-and Shcrecruiting Met receptors demonstrated a marked loss in Grb2associated adaptor protein 1 (Gab1) protein levels, which was not observed in the cell lines, consistent with a post-translationally regulated process. Moreover, a moderate level of Gab1 overexpression stimulated tumor growth. The findings suggest a delicate balance for intact Y1349/1356 SH2-binding domain to mediate the tumor progression activity of the coactivated Metrat sarcoma oncogene homolog (RAS) pathways. Selectivity for specific adaptor protein involvement may be the key that determines the tissue-and cell-type specificity of Met-mediated tumorigenicity in human cancers. Abbreviations: c-Cbl, Casitas B-lineage lymphoma proto-oncogene; Crk, Sarcoma CT10 oncogene homolog; Gab1, Grb2-associated adaptor protein 1; Gab1 WT , Gab1 wild type; GFP, green fluorescent protein; HGF, hepatocyte growth factor; KRAS, Kirsten rat sarcoma oncogene homolog; mRNA, messenger RNA; MAPK, mitogen-activated protein kinase; Met, hepatocyte growth factor receptor; Met WT , wild-type Met; PCR, polymerase chain reaction; PI3K, phosphatidyl inositol 3 kinase; PLC, phospholipase c-gamma; RAS, rat sarcoma oncogene homolog; SH2, Src homology 2; Shc, Src homology domain c-terminal adaptor homolog; Shp2, Src homology-containing tyrosine phosphatase 2; ShRNA, short hairpin RNA; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; Tpr, translocated promoter region. y Drs Seiden-Long and Navab contributed equally to this work.