Structure of the met protein and variation of met protein kinase activity among human tumour cell lines

Abstract

The human and mouse met protooncogenes encode proteins that have the characteristics of growth factor receptors. Thus, the 1408 amino acid human met protein (Park et al., 1987) can be divided into several putative domains, including an intracellular protein tyrosine kinase (PTK) domain, a transmembrane domain and a 926 amino acid extracellular domain that possesses a cysteine-rich region. An activa-ed form of the met gene that is present in the chemicallytransformed human cell line (MNNG-HOS) was originally detected by its ability to transform NIH3T3 mouse fibroblasts in DNA transfection experiments (Cooper et al., 1984a, b). Activation of met involves a chromosomal rearrangement in which the regions of the met gene encoding the transmembrane and extracellular domain are replaced by a portion of an unrelated gene that has been designated tpr (Park et al., 1986a; Tempest et al., 1986a). The chimaeric gene is transcribed to produce a 5.0 kb hybrid mRNA that is in turn translated to form a fusion protein. DNA sequence analysis of cDNA clones prepared from transcripts of the activated human met gene reveal that all of the met PTK domain is retained in the product of the activated gene and that the region of the fusion protein encoded by the tpr gene exhibits weak homology to laminin Bi (Chan et al., 1987). Alterations of met were also observed in lines of spontaneously transformed mouse fibroblasts where a modest (4-8 fold) amplification of the protooncogene is accompanied by dramatic (50-100 fold) increase in the level of an 8.5 kb met transcript (Cooper et al., 1986).Northern analysis of mRNAs from a series of human cell lines has revealed a complex pattern of transcription of the met protooncogene (Park et al., 1986). Many cell lines, including a human fibroblast cell line, contain a single 9.0 kb mRNA species. Other cell lines such as the CaLu-I lung tumour line contain both 9.0 kb and 7.0 kb mRNAs while the most complex pattern of transcription of the normal met gene is present in MNNG-HOS cells and in the parent HOS cell line, which both contain 9.0 kb, 7.0 kb and 6.0 kb mRNA species. Most B-cell and T-cell tumour lines do not contain detectable levels of met transcripts.Antibodies raised against synthetic peptides corresponding to the carboxyl terminus of the predicted met gene product have been used to detect proteins encoded by the activated and normal met genes (Park et al., 1986b;Tempest et al., 1986b Tempest et al. (1986b), although in this particular study of low level of phosphorylation of a 165 kD protein, which probably corresponds to the 160 kD protein detected by Park et al. (1986b), was also observed.As a first step in determining whether alterations in met can be implicated in the induction of human tumours we have used antipeptide antibodies to examine met protein kinase activity in a series of human tumour cell lines. In addition, to help understand the large quantitative and qualitative variations in met kinase activity observed in these experiments, we have used SDS-polyacrylamide gel electr...

Hepatocyte Growth Factor Is a Regulator of Monocyte-Macrophage Function

“…Proteins, separated by SDS-PAGE, were transferred to nitrocellulose sheets and probed with Abs raised against a synthetic peptide corresponding to 19 C-terminal amino acids (from Ser 1372 to Ser 1390 ) of the MET sequence (34), as described elsewhere (21,22,35). Rabbit anti-mouse Ig conjugated to HRP and the enhanced chemiluminescence procedure (Amersham) were used.…”

Proteolytic processing of the hepatocyte growth factor/scatter factor receptor by furin

“…The mature 190 kDa heterodimeric HGF/SF receptor is produced from a 170 kDa single-chain precursor by proteolytic processing and terminal glycosylation [15]. There is a tetrabasic sequence, Arg-Lys-Lys-Arg, in the extracellular domain of the receptor [12], and, like the cleavage site of the insulin receptor [16,17], this region has been considered to be a proteolytic processing site [13]. As Arg-X-Arg/Lys-Arg is a consensus sequence -ptperazmeethan\ulfonic acid/ NaOH (pH 7 0) contannng 0 5% Trtton X-100 and I mM CaCIZ for 5 h at 37°C.…”

“…It belongs to the receptor tyrosine kinase family [12] and consists of a 50 kDa a-subunit and a 145 kDa P-subunit, which are linked by a disulfide bond [13,14]. The a-subunit and the N-terminal region of the P-subunit are extracellular and the C-terminal tyrosine kinase domain of the p-subunit is cytoplasmic…”

The role of non-ras transforming genes in chemical carcinogenesis.

“…1) (28). Cleavage at the basic sequence present in the met protein and removal of the signal peptide would generate and N-terminal peptide of 282 amino acids that might become associated with the remaining membrane-bound portion of the met protein in a manner similar to that observed for the insulin and insulinlike growth factor I receptors (24,29).…”

“…1 (13,14). This chimeric gene is transcribed to produce a unique 5.0-kb hybrid tpr-met mRNA that is in turn translated to form a 60-to 65-kDa fusion protein in which the protein tyrosine kinase domain of met is fused to the amino-terminal region of the trp protein (13,14,29,33). The region of the trp protein present in the trp-met fusion protein exhibits weaker homology to several structural proteins, including laminin and lamin, indicating that the normal trp protein may also encode a structural protein (32).…”