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. 1997 Oct 14;94(21):11445-50.
doi: 10.1073/pnas.94.21.11445.

Activating mutations for the met tyrosine kinase receptor in human cancer

M Jeffers  1 , L Schmidt, N Nakaigawa, C P Webb, G Weirich, T Kishida, B Zbar, G F Vande Woude
Affiliations

Activating mutations for the met tyrosine kinase receptor in human cancer

M Jeffers et al. Proc Natl Acad Sci U S A. .

Abstract

Recently, mutations in the Met tyrosine kinase receptor have been identified in both hereditary and sporadic forms of papillary renal carcinoma. We have introduced the corresponding mutations into the met cDNA and examined the effect of each mutation in biochemical and biological assays. We find that the Met mutants exhibit increased levels of tyrosine phosphorylation and enhanced kinase activity toward an exogenous substrate when compared with wild-type Met. Moreover, NIH 3T3 cells expressing mutant Met molecules form foci in vitro and are tumorigenic in nude mice. Enzymatic and biological differences were evident among the various mutants examined, and the somatic mutations were generally more active than those of germ-line origin. A strong correlation between the enzymatic and biological activity of the mutants was observed, indicating that tumorigenesis by Met is quantitatively related to its level of activation. These results demonstrate that the Met mutants originally identified in human papillary renal carcinoma are oncogenic and thus are likely to play a determinant role in this disease, and these results raise the possibility that activating Met mutations also may contribute to other human malignancies.

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Figures

Figure 1
Figure 1
Expression, autophosphorylation, and exogenous kinase activity of wild-type and mutant Met in NIH 3T3 cells. Samples labeled control and wild type are from cells stably transfected with empty vector or vector expressing wild-type Met, respectively. All other samples are from cells stably transfected with vectors expressing the indicated Met mutant. Cells were cultured in DMEM/10% CS before harvest. (A, Top) Fifty micrograms of cell lysate/sample were resolved on a 7.5% gel and examined by Western analysis using anti-Met antibody. (A, Second from Top) The filter was stripped and reprobed with anti-phosphotyrosine antibody. (A, Third from Top) Four hundred micrograms of cell lysate/sample were immunoprecipitated with anti-Met antibody, resolved on an 8% gel, and analyzed by Western analysis using anti-phosphotyrosine antibody. (A, Bottom) The filter was stripped and reprobed with anti-Met antibody. Molecular mass markers are indicated on the left. (B) Two hundred micrograms of cell lysate/samples were immunoprecipitated with anti-Met antibody and assessed for kinase activity toward an exogenous substrate (gastrin) using a tyrosine kinase assay kit. Results are reported as fold increase relative to cells transfected with empty vector. Samples were performed in triplicate, and SDs were ≤ 5% of the mean.
Figure 3
Figure 3
Morphology of and Met expression in explants derived from tumors induced by NIH 3T3 cells expressing wild-type or mutant Met. Explants prepared from tumors induced by cells expressing the indicated construct were photographed at a magnification of ×100. (Insets) Fifty micrograms of cell lysate/sample were resolved on a 7.5% gel and examined by Western analysis using anti-Met antibody. P, samples from a pool of cells, transfected with the indicated construct, that were used as the inoculum for tumor induction. T, samples from explants prepared from tumors induced by cells transfected with the indicated construct.
Figure 2
Figure 2
Focus induction by wild-type and mutant Met in NIH 3T3 cells. Cells transfected with the indicated constructs were assessed for focus-forming ability. A representative field of view is shown for each sample. See Table 1 for additional information on focus-forming activity.
Figure 4
Figure 4
An alignment of a portion of the COOH-terminal lobe of a cytoplasmic tyrosine kinase (c-src) and representative receptor tyrosine kinases (all other samples) (49, 53). The locations and relative activities of seven of the Met mutations analyzed are shown above affected residues. Conserved amino acids are shown in black. Residues, which, when mutated, activate the Kit and Ret receptors, are shown in broken black boxes. See text for additional details. See note added in proof.
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