O-Glycosylation of Nuclear and Cytosolic Proteins

Abstract

serine/threonine-linked β-N-acetylglucosamine O-GlcNAc transferase β-N-acetylglucosaminidase eukaryotic initiation factor 2 C-terminal domain of RNA polymerase II glutamine:fructose-6-phosphate amidotransferase Despite the long held view that protein glycosylation occurs exclusively on extracellular or lumenal polypeptides (1Furukawa K. Kobata A. Curr. Opin. Biotechnol. 1992; 3: 554-559Crossref PubMed Scopus (34) Google Scholar), it is clear that many nuclear and cytoplasmic proteins are multiply O-glycosylated at specific serine or threonine hydroxyl groups by single β-N-acetylglucosamine moieties (O-GlcNAc)1(2Hart G.W. Haltiwanger R.S. Holt G.D. Kelly W.G. Annu. Rev. Biochem. 1989; 58: 841-874Crossref PubMed Scopus (322) Google Scholar, 3Hart G.W. Annu. Rev. Biochem. 1997; 66: 315-335Crossref PubMed Scopus (450) Google Scholar, 4Haltiwanger R.S. Busby S. Grove K. Li S. Mason D. Medina L. Moloney D. Philipsberg G. Scartozzi R. Biochem. Biophys. Res. Commun. 1997; 231: 237-242Crossref PubMed Scopus (95) Google Scholar). O-GlcNAc modification is common to nearly all eukaryotes, including filamentous fungi, plants, animals, and animal parasites, as well as viruses that infect eukaryotes. Mounting evidence suggests a direct role for O-GlcNAc in cellular regulation. For example, the α-toxin of the gangrene causing bacteriaClostridium novyi is an O-GlcNAc transferase that exerts its toxic effects by the addition of O-GlcNAc to proteins in the Rho subfamily (5Selzer J. Hofmann F. Rex G. Wilm M. Mann M. Just I. Aktories K. J. Biol. Chem. 1996; 271: 25173-25177Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Thus, the disruption of normalO-GlcNAc-regulated pathways may be responsible for the pathology of some bacteria. Moreover, disruption of the gene forO-GlcNAc transferase demonstrates that O-GlcNAc modification is essential for life, even at the single cell level (6Shafi R. Iyer S.P. Ellies L.G. O'Donnell N. Marek K.W. Chui D. Hart G.W. Marth J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5735-5739Crossref PubMed Scopus (593) Google Scholar). O-GlcNAc appears to be both as abundant and as dynamic as protein phosphorylation. In several documented instances, phosphorylation and O-GlcNAc modification are reciprocal, occurring at the same or adjacent hydroxyl moieties (7Kelly W.G. Dahmus M.E. Hart G.W. J. Biol. Chem. 1993; 268: 10416-10424Abstract Full Text PDF PubMed Google Scholar, 8Chou T.-Y. Dang C.V. Hart G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4417-4421Crossref PubMed Scopus (183) Google Scholar). Furthermore, all O-GlcNAc-modified proteins identified to date also occur as phosphorylated proteins. Nevertheless, the interrelationship between Ser/Thr O-GlcNAc modification andO-phosphorylation appears to be complex. Although there are examples of mutually exclusive O-GlcNAc modification andO-phosphorylation, it is likely that all possible combinations are represented in the complex environment of a eukaryotic cell (Fig. 1). The specific addition and removal of these two differentially regulated post-translational modifications might allow for nearly infinite modulation of protein function. The immense task of coordinating cellular activities and responding to extracellular cues with both temporal and spatial accuracy is likely to require the concerted action of both of these regulatory modifications. Reports that alkali-induced β-elimination of adenovirus fiber proteins releases GlcNAcitol hinted at the existence ofO-linked GlcNAc (9Ishibashi M. Maizel Jr., J.V. Virology. 1974; 58: 345-361Crossref PubMed Scopus (58) Google Scholar). Subsequent analysis confirmed the presence of β-O-GlcNAc and suggested that the modification may be involved in adenovirus fiber assembly or stabilization (10Mullis K.G. Haltiwanger R.S. Hart G.W. Marchase R.B. Engler J.A. J. Virol. 1990; 64: 5317-5323Crossref PubMed Google Scholar). Although another study suggested the existence of O-glycosidically linked GlcNAc on extracellular proteins (11Lin T.S. Kolattukudy P.E. Eur. J. Biochem. 1980; 106: 341-351Crossref PubMed Scopus (53) Google Scholar), later structural analyses suggested that these workers were likely observing α-linked GlcNAc, a mucin-like modification common in primitive eukaryotes (12Jung E. Gooley A.A. Packer N.H. Karuso P. Williams K.L. Eur. J. Biochem. 1998; 253: 517-524Crossref PubMed Scopus (22) Google Scholar). Another early study showed that the GlcNAc-binding lectin wheat germ agglutinin blocked ATP-dependent RNA nuclear transport (13Baglia F. Maul G.G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2285-2289Crossref PubMed Scopus (46) Google Scholar). The characterization of β-O-GlcNAc in 1984 explained some of these preliminary observations and establishedO-GlcNAc as a major form of intracellular protein glycosylation (14Torres C.-R. Hart G.W. J. Biol. Chem. 1984; 259: 3308-3317Abstract Full Text PDF PubMed Google Scholar). The nuclear pore proteins were among the first structurally charac