Characterization of the functional domains of galactosylceramide expression factor 1 in MDCK cells

We previously reported that GalCer expression factor 1 (GEF-1), a rat homol ogue of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), induced GalCer expression, morphological changes, and cell growth inhibiti on in COS-7 cells. In this study, we describe the characterization of GEF-1 in MDCK cells. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed G alCer-deirived sulfatide expression as well as dramatic morphological chang es, but not cell growth suppression. The enzyme activity and the mRNA level of UDP-galactose:ceramide galactosyltransferase (CGT) increased significan tly in MDCK/GEF-1 cells compared with control cells. GEF-1 molecule is comp osed of four domains; a zinc-finger (Z), a proline-rich (P), a coiled-coil (C), and a proline/glutamine-rich (Q) domain. MDCK cells transfected with v arious GEF-1 deletion mutants were examined for morphology and for glycolip id expression. MDCK cells transfected with Z-domain deletion mutant (MDCK/P CQ) and those with both Z- and P-domains deletion mutant (MDCK/CQ) were sim ilar to those with a wild-type GEF-1 (MDCK/ZPCQ) in shape, exhibiting fibro blast-like cells, whereas those with the other deletion mutants showed no m orphological changes, exhibiting typical epithelial-like cells. On the othe r hand, MDCK/ZPCQ, MDCK/PCQ, MDCK/CQ, and MDCK/Q cells expressed sulfatide, whereas those with the other deletion mutants that did not include the Q-d omain showed neither GalCer nor sulfatide expression. Thus, the correlation between fibroblast-like cells in shape and the glycolipid expression was g ood in these deletion mutants except MDCK/Q cells, which showed epithelial- like cells, but expressed sulfatide. The glycolipid expression paralleled C GT mRNA levels. Taking these results together, it is suggested that only th e Q-domain may be essential for the role of GEF-1 in inducing CGT mRNA, whe reas the Q-domain together with the C-domain may be required for the induct ion of morphological changes in MDCK cells.