Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity.
O-GlcNAc transferase (OGT) modifies nuclear pore proteins and transcription factors. In Arabidopsis, the OGT homolog participates in the gibberellin signaling pathway. We and others have proposed that mammalian OGT is the terminal step in a glucose-sensitive signal transduction pathway that becomes disregulated in insulin resistance. To facilitate mutational analysis of OGT in the absence of competing endogenous activity, we expressed the 103-kDa human OGT in Escherichia coli. Kinetic parameters for the purified recombinant enzyme (K(m) = 1.2 microM for Nup 62; K(m) = 0.5 microM for UDP-GlcNAc) are nearly identical to purified mammalian OGT. Deletions in the highly conserved C terminus result in a complete loss of activity. The N-terminal tetratricopeptide repeat domain is required for optimal recognition of substrates. Removal of the first three tetratricopeptide repeats greatly reduces the O-GlcNAc addition to macromolecular substrates. However, this altered enzyme retains full activity against appropriate synthetic peptides. Autoglycosylation of OGT is augmented when the first six tetratricopeptide repeats are removed showing that these repeats are not required for catalysis. Given its proposed role in modulating insulin action, OGT may modify kinases involved in this signaling cascade. Among the many kinases tested, OGT glycosylates glycogen synthase kinase-3 and casein kinase II, two enzymes critical in the regulation of glycogen synthesis.
Lubas
W A
WA
Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hanover
J A
JA
D047390:Casein Kinase II; D006006:Glycogen Synthase / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013379:Substrate Specificity; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism*
D011994:Recombinant Proteins; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; D006006:Glycogen Synthase; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases