Characterization of the functionaldomains of galactosylceramide expression factor 1 in MDCK cells.

We previously reported that GalCer expression factor1 (GEF-1), a rat homologue of hepatocyte growth factor–regulatedtyrosine kinase substrate (Hrs), induced GalCer expression, morphologicalchanges, and cell growth inhibition in COS-7 cells. In this study,we describe the characterization of GEF-1 in MDCK cells. Overexpressionof GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatideexpression as well as dramatic morphological changes, but not cellgrowth suppression. The enzyme activity and the mRNA level of UDP-galactose:ceramide galactosyltransferase(CGT) increased significantly in MDCK/GEF-1 cells comparedwith control cells. GEF-1 molecule is composed of four domains;a zinc-finger (Z), a proline-rich (P), a coiled-coil (C), and aproline/glutamine-rich (Q) domain. MDCK cells transfectedwith various GEF-1 deletion mutants were examined for morphology andfor glycolipid expression. MDCK cells transfected with Z-domaindeletion mutant (MDCK/PCQ) and those with both Z- and P-domainsdeletion mutant (MDCK/CQ) were similar to those with awild-type GEF-1 (MDCK/ZPCQ) in shape, exhibiting fibroblast-likecells, whereas those with the other deletion mutants showed no morphological changes,exhibiting typical epithelial-like cells. On the other hand, MDCK/ZPCQ,MDCK/PCQ, MDCK/CQ, and MDCK/Q cells expressedsulfatide, whereas those with the other deletion mutants that didnot include the Q-domain showed neither GalCer nor sulfatide expression.Thus, the correlation between fibroblast-like cells in shape andthe glycolipid expression was good in these deletion mutants exceptMDCK/Q cells, which showed epithelial-like cells, but expressedsulfatide. The glycolipid expression paralleled CGT mRNA levels.Taking these results together, it is suggested that only the Q-domainmay be essential for the role of GEF-1 in inducing CGT mRNA, whereasthe Q-domain together with the C-domain may be required for theinduction of morphological changes in MDCK cells. [ABSTRACT FROM PUBLISHER]

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