title: Characterization of the functional domains of galactosylceramide expression factor 1 in MDCK cells.
abstract: We previously reported that GalCer expression factor 1 (GEF-1), a rat homologue of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), induced GalCer expression, morphological changes, and cell growth inhibition 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 GalCer-derived sulfatide expression as well as dramatic morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of UDP-galactose:ceramide galactosyltransferase (CGT) increased significantly in MDCK/GEF-1 cells compared with control cells. GEF-1 molecule is composed 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 various GEF-1 deletion mutants were examined for morphology and for glycolipid expression. MDCK cells transfected with Z-domain deletion mutant (MDCK/PCQ) and those with both Z- and P-domains deletion mutant (MDCK/CQ) were similar to those with a wild-type GEF-1 (MDCK/ZPCQ) in shape, exhibiting fibroblast-like cells, 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 expressed sulfatide, whereas those with the other deletion mutants that did not include the Q-domain showed neither GalCer nor sulfatide expression. Thus, the correlation between fibroblast-like cells in shape and the glycolipid expression was good in these deletion mutants except MDCK/Q cells, which showed epithelial-like cells, but expressed sulfatide. The glycolipid expression paralleled CGT mRNA levels. Taking these results together, it is suggested that only the Q-domain may be essential for the role of GEF-1 in inducing CGT mRNA, whereas the Q-domain together with the C-domain may be required for the induction of morphological changes in MDCK cells.
authors.affiliation: Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Ogura
authors.forename: T
authors.initials: T
authors.lastname: Tai
mesh_terms: D000251:Adenosine Triphosphatases; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D017931:DNA Primers; D018076:DNA, Complementary; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005698:Galactosylceramidase / Q000235:genetics / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000502:physiology; D007150:Immunohistochemistry; D009024:Morphogenesis / Q000502:physiology; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D017228:Hepatocyte Growth Factor; D011505:Protein-Tyrosine Kinases; D005698:Galactosylceramidase; D000251:Adenosine Triphosphatases; C115505:Hgs protein, rat