C-MET SIGNALING IN AN HGF SF-INSENSITIVE VARIANT MDCK CELL-LINE WITH CONSTITUTIVE MOTILE/INVASIVE BEHAVIOR/

The Met protein is a receptor tyrosine kinase for hepatocyte growth fa ctor/scatter factor (HGF/SF), a multifunctional growth factor with mit ogenic, motogenic and morphogenic properties, A morphologically altere d variant of the MDCK cell line, MDCK-1, spontaneously exhibits a numb er of features associated with a partial HGF/SF-Met induced phenotype (less adhesive colonies in culture, enhanced invasion and motility, na scent tubule formation), but paradoxically does not respond to HGF/SF treatment. Although the overall cell surface expression and distributi on of Met were found to be similar in parental MDCK cells and the MDCK -1 cell line, p145(met) autophosphorylation (+/- HGF/SF) was significa ntly reduced in MDCK-1 cells in vitro and in vivo when compared with p arental MDCK cells, In contrast, EGF induced cell proliferation and EG F receptor autophosphorylation to similar levels in both cell lines, T he basal levels of protein tyrosine phosphorylation were higher in MDC K-1 cells when compared with parental MDCK cells, including that of tw o prominent proteins with molecular masses of similar to 185 kDa and 2 20 kDa, Moreover, both p185 and p220 are present and tyrosine phosphor ylated in Met immunoprecipitates from MDCK-1 cells (+/- HGF/SF), but n ot parental MDCK cells, In addition,Met immunocomplexes from MDCK-1 ce lls exhibited an approximately 3-fold increased tyrosine kinase activi ty in vitro when compared with MDCK cells, correlating with the higher basal levels of total phosphotyrosine. Treatment of MDCK-1 cells with the tyrosine kinase inhibitor herbimycin A reverted the cell phenotyp e to a more MDCK-like morphology in culture, with a concomitant reduct ion in the tyrosine phosphorylation predominantly of p220. Taken toget her these data suggest that aberrations in Met activity and associated signalling render MDCK-1 cells insensitive to HGF/SF, and may also me diate alterations in MDCK-1 cell behaviour.