Molecular cloning of a rat brain cDNA, with homology to a tyrosine kinase substrate, that induces galactosylceramide expression in COS-7 cells.
A rat brain cDNA clone has been isolated, using a eukaryotic cell transient expression system in conjunction with an anti-galactosylceramide (anti-GalCer) monoclonal antibody that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). A good correlation between GalCer expression and the level of the enzyme activity of UDP-galactose:ceramide galactosyltransferase (CGT) was demonstrated. The cDNA insert encoded a polypeptide of 771 amino acids with a calculated molecular mass of 85,787 Da. The cDNA hybridized to a single mRNA of 3.1 kb in all rat organs examined, including brain, testis, and skeletal muscle. The cDNA product was determined to be a tyrosine-phosphorylated protein with a molecular mass of 110 kDa in transfected COS-7 cells and adult rat brain. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes: The transfected cells appeared to be fibroblast-like cells, whereas the parent COS-7 cells were typical epithelial-like cells. The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate but no homology to CGT. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer expression in the brain.
Ogura
K
K
Department of Tumor Immunology, Tokyo Metropolitan Institute of Medical Science, Japan.
Kohno
K
K
Tai
T
T
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D019556:COS Cells / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D056827:Endosomal Sorting Complexes Required for Transport; D005260:Female; D005699:Galactosylceramides / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics* / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014018:Tissue Distribution; D014162:Transfection
D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D005699:Galactosylceramides; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases; D000251:Adenosine Triphosphatases; C115505:Hgs protein, rat