O-GlcNAcylation of key nuclear and cytoskeletal proteins: reciprocity with O-phosphorylation and putative roles in protein multimerization

O-Linked N-acetylglucosamine, O-GlcNAc, was discovered (Torres and Hart, 1984) as part of our studies using highlypurified glycosyltransferases to investigate saccharide structures on lymphocytes and tumor cells (Powell et al, 1987; Whiteheart and Hart, 1987; Passaniti and Hart, 1988; Reichner et al., 1988; Whiteheart et al, 1989). Our glycosyltransferase studies are a direct outgrowth of the pioneering work of Professor Hill and his colleagues, which developed the methods for the purification of glycosyltransferases (Paulson et al., 1977; Beyer et al., 1980, 1981; Sadler et al, 1979, 1981, 1982). In particular, it was the purification (Trayer and Hill, 1971; Barkers al, 1972) and ready availability of bovine milk galactosyltransferase that allowed us to employ the enzyme as a highly specific and sensitive probe for O-GlcNAc on nuclear and cytoplasmic proteins (for methods, see Roquemore et al, 1994b). Using this enzymatic probe and other methods, we and others (for review, Hart et al, 1989, 1995a-c) have now documented that O-GlcNAc is ubiquitous and abundant on nuclear and cytoskeletal proteins of virtually all eukaryotes, including protozoans (Ortega-Barria et al, 1990; Dieckmann-Schuppert etal, 1993, 1994; Stanley etal, 1995) and fungi (Machida and Jigami, 1994). In addition, O-GlcNAc is highly dynamic, with turnover rates much higher than the protein backbones to which it is attached (Kearse and Hart, 1991; Chou et al, 1992; Roquemore et al, 19%). Virtually all O-GlcNAc proteins examined to date are also phosphoproteins, and in some instances Ser(Thr)-O-GlcNAc and Ser(Thr)-O-phosphate appear to reciprocally occupy the same hydroxyl groups (Kelly et al, 1993; Chou et al, 1995a,b). The dynamic characteristics of O-GlcNAc, and the importance of O-GlcNAcylated proteins in cellular regulation and in cytoskeletal structure, have led us to hypothesize that O-GlcNAc is a regulatory modification that not only regulates protein phosphorylation, but also is involved in modulating protein multimerization (Hart et al, 1995b,c). O-GlcNAcylation is most abundant in the nucleus