MINIREVIEW THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 50, pp. 34424 –34432, December 12, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

The Making of a Sweet Modification: Structure and Function of O-GlcNAc Transferase* Published, JBC Papers in Press, October 21, 2014, DOI 10.1074/jbc.R114.604405

John Janetzko‡1 and Suzanne Walker§2 From the §Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115 and the ‡Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138

Thirty years ago, Torres and Hart made the unexpected discovery that many intracellular proteins are modified with O-GlcNAc (1). Thirteen years later, the enzyme responsible for this post-translational modification was finally cloned (2, 3). This enzyme, O-GlcNAc (O-linked N-acetylglucosamine) transferase (OGT),3 differs from the glycosyltransferases of the secretory pathway in its cellular localization and biological functions. OGT is found in both the cytoplasm and nucleus, and it catalyzes transfer of a GlcNAc moiety from UDPGlcNAc to specific serine and threonine residues in its many cellular targets (Fig. 1A). Amid the large diversity of substrates, no clear glycosylation consensus sequence has been identified (3, 4). In conjunction with a partner glycosidase, O-GlcNAcase (OGA), OGT is thought to modulate cellular O-GlcNAc levels in response to signaling cues, including metabolic status and external stresses (5, 6). Analogous to protein phosphorylation (7, 8), protein O-GlcNAc modifications can affect protein local-

* This

work was supported in part by National Institutes of Health Grant R01GM094263 (to S. W.). This is the first article in the Minireview Series on the Thirtieth Anniversary of Research on O-GlcNAcylation of Nuclear and Cytoplasmic Proteins: Nutrient Regulation of Cellular Metabolism and Physiology by O-GlcNAcylation. 1 Supported by National Science and Engineering Research Council of Canada PGS-M and PGS-D3 fellowships. 2 To whom correspondence should be addressed: E-mail: suzanne_ [email protected]. 3 The abbreviations used are: OGT, O-GlcNAc transferase; TPR, tetratricopeptide repeat; OGA, O-GlcNAcase; ncOGT, nuclear and cytoplasmic OGT; mOGT, mitochondrial OGT; sOGT, smallest OGT; hOGT4.5, human OGT bearing 4.5 TPRs; 5SGlcNAc, 2-acetamido-2-deoxy-5-thio-D-glucopyranose; Int-D, intervening domain; CKII, casein kinase II; PIP3, phosphatidylinositol 3,4,5-triphosphate; GT-B, type-B glycosyltransferase.

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Structure of OGT Analysis of the primary sequence of OGT revealed a cluster of repeating motifs called tetratricopeptide repeats (TPRs) N-terminal to the catalytic domain (2, 3). TPRs are degenerate 34-amino acid motifs that typically fold into paired antiparallel ␣-helices (18). Often found in tandem arrays, TPRs are known for their role in mediating protein-protein interactions (18 – 20). Evidence implicated the TPRs of OGT in mediating interactions with protein substrates, as well as putative adaptors and proteins shown to affect its enzymatic activity (21–31). There are three physiological isoforms of OGT, differing primarily in the number of N-terminal TPRs (32–34). The longest isoform, with 13.5 TPRs, is referred to as nuclear and cytoplasmic OGT (ncOGT). A shorter form, with 9 TPRs and bearing a mitochondrial targeting sequence, is localized to the inner mitochondrial membrane (mOGT). The smallest of the three, sOGT, has only 2.5 TPRs and is perhaps the least studied. Interestingly, mOGT appears to have no in vivo catalytic function (33), but instead has been speculated to be involved, along with sOGT, in apoptosis (35, 36). The first structure of a segment of OGT was reported in 2004. This structure comprised 11.5 TPRs from the N terminus of the human protein and showed that the TPRs assemble into an elongated superhelix (Fig. 2A) (37). Several features of the TPR structure are noteworthy. In the asymmetric unit, two TPR molecules pack as a homodimer, with the interface centered on TPRs 6 and 7 (37). Previous studies had suggested that OGT exists as an oligomer in solution, and the TPRs were implicated in mediating its oligomerization (27, 38). The TPR crystal structure suggested that OGT may exist as a dimer, and consistent with this, mutating the residues at the TPR dimer interface afforded a lower order species. Because mutations that disrupted this interface did not substantially affect enzymatic activity in vitro, it remains unclear what role oligomerization serves for OGT function. The TPR structure also suggested a high degree of conformational flexibility, with one monomer bent by ⬃40° between TPRs 9 and 10 (37). Although this deformation could be due to crystal-induced unfolding, the authors suggested that the conformational flexibility might have implications for substrate recognition. Finally, phylogenetic comparisons showed strong conservation in solvent-exposed residues lining the interior of the VOLUME 289 • NUMBER 50 • DECEMBER 12, 2014

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O-GlcNAc transferase is an essential mammalian enzyme responsible for transferring a single GlcNAc moiety from UDP-GlcNAc to specific serine/threonine residues of hundreds of nuclear and cytoplasmic proteins. This modification is dynamic and has been implicated in numerous signaling pathways. An unexpected second function for O-GlcNAc transferase as a protease involved in cleaving the epigenetic regulator HCF-1 has also been reported. Recent structural and biochemical studies that provide insight into the mechanism of glycosylation and HCF-1 cleavage will be described, with outstanding questions highlighted.

ization, activity, stability, and interactions with other biomolecules. In 2011, OGT was unexpectedly implicated in a second process: the proteolytic maturation of HCF-1 (host cell factor 1), a large protein involved in several crucial chromatin-modifying complexes (9, 10). Subsequent work demonstrated that OGT uses UDP-GlcNAc as a co-substrate to promote cleavage of HCF-1, and remarkably, the cleavage reaction takes place in the same active site used for glycosylation (Figs. 1B and 4C) (11). This minireview focuses on recent structural and biochemical studies of OGT, as well as its role in HCF-1 cleavage. For comprehensive reviews and references to earlier work, see Refs. 7 and 12–17.

MINIREVIEW: Structure and Function of O-GlcNAc Transferase OH O

HO HO AcHN

O

O NH

O O O O P P O O

O

N

O HO HO AcHN

HO

OH

OH O

O

O OO O P P O O O HO

OGT HO

OH O O

HO HO AcHN

Protein Ser/Thr

NH O N

O

OH

OGT HCF-1N

HCF-1N

Protein

Ser/Thr

HCF-1C

HCF-1C

OGA Full length HCF-1

H O

Mature HCF-1

CKII peptide TPRs

C

UDP

C-Cat

TPR1

N-Cat Int-D

N

D mOGT sOGT hOGT4.5 (372) TPR1

TPR1

1

(167)

(313)

ncOGT

3

TPRs 466 H3

C-Cat (828-997)

Int-D (698-827)

N-Cat (496-697) 1028

FIGURE 2. A, a single chain from the 11.5-TPR structure. The C and N termini are labeled, as is the position of the first TPR. B, structure of hOGT4.5. The complex shown contains UDP (purple spheres) and a peptide derived from a well studied OGT substrate (orange balls and sticks), CKII (27). The intervening domain (green), two catalytic lobes of the GT-B fold (blue and red), and the TPRs (gray) are shown in ribbon representation. C, schematic representation of OGT using the same coloring as described for B. D, model of ncOGT constructed from the 11.5-TPR structure and the hOGT4.5 structure. The dark-gray TPRs are derived from the hOGT4.5 structure, whereas the light-gray TPRs are from the 11.5-TPR structure. C-Cat and N-Cat, C- and N-terminal catalytic domains, respectively.

TPRs, particularly several asparagine residues near the center of the cavity, and suggested an unexpected similarity to armadillo repeat-containing proteins, such as importin-␣ and ␤-catenin (37). DECEMBER 12, 2014 • VOLUME 289 • NUMBER 50

More recently, structures of a bacterial homolog of OGT from Xanthomonas campestris, xcOGT (39 – 41), and the catalytic domain of human OGT bearing 4.5 TPRs (hOGT4.5) (Fig. 2, B and C) were reported (4). Structures of hOGT4.5 have been JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 1. A, OGT/O-GlcNAcase (OGA)-mediated dynamic glycosylation of nucleocytoplasmic proteins. B, the cleavage of HCF-1 by OGT at one of several repeats (red squares) results in two fragments: HCF-1C and HCF-1N. OGT also glycosylates HCF-1 at several sites, mostly located in the N-terminal segment (blue hexagons).

MINIREVIEW: Structure and Function of O-GlcNAc Transferase

Mechanism of Protein Glycosylation Although it was initially proposed that OGT follows a random Bi Bi kinetic mechanism (27), structural information showed that the polypeptide substrate bound above the UDPsugar, which suggested an ordered mechanism in which UDP-GlcNAc binds first. Product inhibition studies also supported an ordered Bi Bi mechanism (4). Hence, UDP-GlcNAc

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binds first to OGT, followed by the polypeptide substrate, which makes extensive contacts with the sugar donor (Fig. 3A). Notably, kinetic studies have shown that the Km(app) values for UDP-GlcNAc can differ by up to an order of magnitude depending on the polypeptide substrate examined, which underscores the importance of substrate-substrate interactions in the mechanism (27, 53). After the ternary substrate complex forms, the acceptor side chain attacks the anomeric carbon, with loss of UDP, to form a ␤-glycosidic linkage. Finally, the glycopeptide dissociates, followed by UDP. In addition to binding the sugar donor and acceptor substrates in close proximity (in the case of OGT, UDP-GlcNAc and a polypeptide, respectively), it has long been presumed that glycosyltransferases must do two things to catalyze glycosylation: 1) activate the leaving group of the glycosyl donor toward departure and 2) remove the proton from the hydroxyl group on the glycosyl acceptor (48). GT-A superfamily glycosyltransferases use a metal ion to activate the leaving group, but OGT and other GT-B glycosyltransferases are metal ion-independent enzymes and use a different strategy (Fig. 3B) (48). Leaving group activation in OGT is achieved through several elements that stabilize the buildup of negative charge. First, the ␤-phosphate of UDP-GlcNAc is anchored by hydrogen bonds to the N terminus of an ␣-helix; these include key interactions with His-920, Thr-921, and Thr-922. Further stabilization from this helix is derived from electrostatic interactions between the negatively charged phosphate and the net helix dipole (43). Other GT-B superfamily members use a similar mode of activation (48), with some anchoring the ␣-phosphate, rather than the ␤-phosphate, of the leaving group (54, 55). Second, the side chain of a lysine residue (Lys-842) is positioned directly below the ␤-phosphate (Fig. 3C) and presumably further activates the leaving group toward departure. Lys-842 plays an essential role in the enzymatic activity of OGT (4, 40, 43, 44). Because Lys842 is chemically reactive, as evidenced by its nucleophilicity toward a covalent OGT inhibitor (45), its pKa may be suppressed due to its proximity to the aforementioned helix dipole; however, its protonation state remains to be established explicitly (45). This combination of a helix dipole and carefully positioned hydrogen bonds from the N terminus of the helix and a critical lysine to the ␤-phosphate activates UDP-GlcNAc toward nucleophilic attack. Nevertheless, glycosylation is favored over hydrolysis, raising the question of how selectivity for reaction with an acceptor hydroxyl instead of water is achieved, especially given that several ordered water molecules are visible within the active site of all structures below 2.0 Å resolution (Fig. 3C). It seems likely that the aforementioned contact between the ␣-phosphate of UDP-GlcNAc and the amide N-H of the acceptor serine (Fig. 3B) is an important element for this selectivity. This contact may serve to bring the acceptor and donor into close proximity and position the acceptor hydroxyl in a more favorable position to form a bond with the anomeric carbon (labeled C1 in Fig. 3D) compared with any water molecule (Fig. 3C) (43). It may also further activate the leaving group for departure. When substrate and product ternary complexes are examined, modest changes in several positions can be seen, with the C1, C2, and O5 atoms of the GlcNAc displaying the greatest VOLUME 289 • NUMBER 50 • DECEMBER 12, 2014

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solved with UDP, UDP-GlcNAc, the hydrolysis-resistant analog UDP-2-acetamido-2-deoxy-5-thio-D-glucopyranose (UDP-5SGlcNAc) (42), and several different peptide and glycopeptide substrates bound. Hence, binary as well as ternary substrate-like and product complexes are available (4, 11, 43– 45). A structure containing a bisubstrate analog has also been reported (46). Taken together, these structures offer perhaps the most complete structural picture yet obtained for any glycosyltransferase. By combining the TPR structure with the catalytic domain structure, a model for human ncOGT, containing all 13.5 TPRs, can be created (Fig. 2D). In this model the TPRs form two complete superhelical turns and extend upward from the active site. The structures of OGT have revealed many important features. As expected, the catalytic domain of OGT possesses a GT-B fold, reminiscent of that found in MurG (CAZy glycosyltransferase family 28), GtfB (CAZy glycosyltransferase family 1), and many other glycosyltransferases (47–52). GT-B glycosyltransferases possess a catalytic domain comprising two lobes that each adopt a Rossmann-like fold. OGT differs from other GT-B glycosyltransferases in several key respects, however. The N-terminal catalytic domain bears two additional helices that are crucial components of the active site (4). Moreover, it possesses an ⬃120-amino acid insertion (termed the intervening domain (Int-D)) between its two catalytic lobes (4). This insertion forms a domain that packs exclusively against the C-terminal catalytic domain. Although its structure is now known (Fig. 2B), its function remains mysterious. The peptide substrate binds over the UDP-GlcNAc, and the peptide-binding site spans a groove formed between the two catalytic lobes and the TPR domain (Fig. 2B). The extended conformation of the peptide substrate likely explains the preference for proline and ␤-branched amino acids near the site of glycosylation (4). There is an interaction between the ␣-phosphate of the nucleotide sugar and the amide N-H of the acceptor serine/threonine that is proposed to play a role in peptide binding to OGT (4) and may further serve to orient the substrate for reaction (Fig. 3A) (43). Although several OGT residues contact the backbone of the peptide substrate, few make specific contacts with the peptide side chains, consistent with the lack of a clear consensus sequence (4). Peptide binding is accompanied by widening of the cleft between the catalytic domain and the TPRs, and molecular dynamics simulations have suggested that the TPRs are able to pivot about a hinge region between TPRs 12 and 13, giving rise to open and closed states for the active site (4). Such molecular motion would be crucial for accommodating larger substrates, such as loops in folded proteins. Moreover, the flexibility is consistent with that seen in the TPR crystal structure and with the previous suggestion that the TPRs mediate substrate binding (37).

MINIREVIEW: Structure and Function of O-GlcNAc Transferase

B

B +

H

CKII peptide

HO HO HO AcHN + + +

UDP-5SGlcNAc

O

HO

HO HO AcHN

R

+

AcHN + +

O

+

O

HO HO

O O P O OUMP +

D

C

R

O

BH

HO

H

O O P O OUMP

+ + +

+ +

O

O

R

O O P O OUMP +

CKII

W2

gCKII

D554

W1 O5

H498

S5

C2

UDP

C1 UDP

T921 K842

UDP-5SGlcNAc UDP-5 GlcNAc

E

D554 O Ser HO

Proposal I:

HO HO H 3C N O

H

O

O

O H

O

O

O O O P P O O HO

Ser NH

O

O

H

W2 H O

N

O

OH

HO

Proposal II:

HO HO H 3C

O

H O

W1 H O H O NH

N

O O H O P O P O O O O HO

O

N

O

OH

FIGURE 3. A, UDP-5SGlcNAc (magenta spheres), a UDP-GlcNAc mimic, makes extensive interactions with a peptide substrate (yellow sticks and surface). A hydrogen bond is shown as a black dotted line. B, a generic GT-B glycosyltransferase mechanism. A Brønsted base (B) and one or more groups to stabilize the accumulation of negative charge in the leaving group (␦⫹) are thought to be required. A possible transition state, bearing oxocarbenium character, is shown. C, view of the OGT active site in a ternary product complex of hOGT4.5, glycosylated CKII (gCKII; yellow sticks), and UDP (cyan sticks). Key residues (purple sticks), two ordered water molecules (W1 and W2; red spheres), and hydrogen bonds (black dotted lines) are also shown. D, overlay of a product complex containing a glycosylated CKII peptide and UDP (both yellow sticks) and a substrate complex containing CKII and UDP-5SGlcNAc (both cyan sticks). Several positions on the sugars are labeled. E, two main proposals for the operative proton transport mechanism in OGT.

displacement (Fig. 3D). A net upward rotation of the sugar moves the anomeric carbon away from the ␤-phosphate of UDP and into bonding distance with the acceptor hydroxyl. The inferred reaction trajectory is consistent with an electrophilic migration-type mechanism (43). Another notable change is the rotation of the C2 acetamide, which contacts His-498 and rotates away from C1 along the reaction coordinate (Fig. 3D). The importance of the C2 acetamide was further demonstrated by the fact that OGT can transfer UDP-GalNAc, but not UDPglucose or UDP-2-keto-Glc, a UDP-GlcNAc analog in which the C2 N-H is replaced with CH2 (43). In addition to providing information about leaving group stabilization and reaction trajectory, the OGT ternary structures DECEMBER 12, 2014 • VOLUME 289 • NUMBER 50

revealed a surprise: there is no OGT side chain that can serve as a catalytic base in the immediate vicinity of the reactants (43, 44). Two different side chains, His-498 and His-558, had previously been suggested as candidates for the catalytic base, but both were ruled out after the examination of ternary structures containing substrate analogs implicated them in other roles (43, 44). Two alternatives for how proton transport is achieved have been suggested (Fig. 3E). In one, the ␣-phosphate of UDP is proposed to act as a base to deprotonate the acceptor hydroxyl (Fig. 3E, Proposal I) (44). In the other, a chain of water molecules leading to an aspartate side chain (Asp-554) is proposed to facilitate transport of the proton liberated during the reaction from the active site (Fig. 3E, Proposal II) (43). To test the first JOURNAL OF BIOLOGICAL CHEMISTRY

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gCKII

MINIREVIEW: Structure and Function of O-GlcNAc Transferase

HCF-1 Is Cleaved by OGT In 2011, OGT was implicated as the cellular factor responsible for the maturation of an essential transcriptional co-activator, HCF-1 (9, 10). HCF-1 was first identified in the late 1980s as a component from HeLa cell lysate required for the stable formation of the herpes simplex VP16-induced complex and the expression of immediate-early genes during herpes virus infection (60 – 65). HCF-1 has since been implicated as a critical element in cell cycle regulation, including both G1-to-S and M phase progression (66 –73). Biochemically, the first report that described the purification of HCF-1 (then called C1), by Kristie and Sharpe (74), suggested its existence as a set of heterogeneous, but related, polypeptide fragments; later work revealed that these polypeptide fragments were derived from a large precursor protein by some proteolytic maturation process (75). Although the protease responsible for this cleavage remained elusive, pulse-chase studies suggested that cleavage took place largely in the nucleus, but also, albeit to a lesser extent, in the cytoplasm (76). Cleavage appeared to take place at one or more of six 26-amino acid repeating elements, which constituted an atypically large protease recognition motif (75, 76). Based on N-terminal peptide sequencing, cleavage was reported to occur between Glu-10 and Thr-11 of a given repeat (76, 77). One striking feature of this cleavage was the fact that even though it was shown to be required for proper HCF-1 function (78), the two halves of the protein remain noncovalently associated post4

Numbering in Ref. 59 is according to mOGT. As a result, Asp-554 is referred to as Asp-438.

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processing (76, 79, 80). Although several mechanisms for cleavage were considered (9, 10, 81), the finding that HCF-1 interacts stably with and is modified by OGT (9, 67, 82, 83) led to the suggestion that OGT may somehow promote cleavage. This raised several questions. Was there a second OGT active site responsible for this new transformation? What was the mechanism of the transformation? Why would nature connect OGT activity with the cleavage of HCF-1? The most recent studies into HCF-1 proteolysis exploited a combination of structural and biochemical tools to confirm that OGT indeed catalyzes cleavage, and they have provided additional insights into this intriguing process (11). A cleavage substrate devoid of Ser/Thr glycosylation sites was developed, and reconstitution of the cleavage reaction established that UDP-GlcNAc is required for HCF-1 cleavage, whereas UDP or UDP-5SGlcNAc inhibits cleavage (11). Inactivation of OGT, through either a K842A mutation or modification with a covalent OGT inhibitor (45), also blocked cleavage (11). The finding that UDP-5SGlcNAc, which is resistant to glycosylation and hydrolysis (42, 43), is unable to promote cleavage, even though it binds identically to UDP-GlcNAc (43), suggested that the nucleotide sugar must serve a functional role rather than simply a structural one. Mass spectrometry established that cleavage in fact occurs between Cys-9 and Glu-10, rather than between Glu-10 and Thr-11 (Fig. 4A) (11), and results in formation of a C-terminal cleavage product whose N terminus has cyclized to form a pyroglutamate residue (11). The formation of this product would require activation of the side chain carboxylate of the glutamate. Although there are several possible mechanisms by which this could occur, structural studies have established that the cleavage region of the HCF-1 proteolytic repeat binds in the active site in a manner almost identical to that of the casein kinase II (CKII) glycosylation substrate and with the glutamate side chain (Glu-10) in the same position as the reactive serine of CKII (Fig. 4A) (11). Indeed, converting the glutamate residue to serine transforms a cleavage substrate into a glycosylation substrate (11). These findings implied that cleavage occurs in the same active site of OGT as glycosylation and suggested that the glutamate carboxyl group might react with the UDP-GlcNAc substrate to form a glutamyl-GlcNAc intermediate (Fig. 4B). Although enzymatic glycosylation on glutamate has not, to our knowledge, been reported, there is precedent for the spontaneous hydrolysis of internal pyroglutamates that form by other mechanisms (84 – 86). Additional studies will be required to elucidate details of the catalytic mechanism of HCF-1 cleavage by OGT. There are outstanding questions about the possible roles of the N-terminal pyroglutamate in the biological function of HCF-1 and whether some of the effects of OGT that have been attributed to its glycosylation function are in fact due to its role in HCF-1 cleavage. However, as has been suggested (9, 11), there is a unifying theme: both glycosylation and cleavage depend on UDP-GlcNAc concentration, and cleavage may thus be another mechanism for linking cell cycle regulation to the metabolic status of the cell, as for glycosylation. VOLUME 289 • NUMBER 50 • DECEMBER 12, 2014

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hypothesis, both diastereomeric phosphodithioate analogs of UDP-GlcNAc were prepared, and it was found that although both had a similar binding affinity for OGT, only one of them functioned as a donor substrate (44). Although this finding suggests that the ␣-phosphate is involved in the catalytic mechanism, results involving substrate mimics must be examined cautiously. Replacing the acceptor serine with an aminoalanine may, depending on the protonation state of the amine, bring the amino group in closer proximity to the ␣-phosphate than it would be in the native substrate. Furthermore, oxygen-to-sulfur substitution on the phosphate would likely affect the previously mentioned hydrogen bond to the backbone of the acceptor amide (Fig. 3, C and D). Given that the importance of this interaction has not been rigorously examined, its energetic contribution to binding should be quantified. The proposal that the proton is shuttled out of the active site by a Grotthuss-like (56, 57) mechanism invokes the active-site ordered water molecules in the mechanism in lieu of implicating a catalytic base within OGT. In a transition state involving an oxocarbenium ion-like species, as computational studies have suggested for OGT (58), a general base may not be needed to deprotonate the acceptor hydroxyl for the reaction to occur. In this case, the ordered water molecules leading to Asp-554 may simply provide a pathway for proton extrusion from the active site. It has been shown that mutation of Asp-554 4 to alanine abolishes catalytic activity (59), but mutation to asparagine does not (44). Further study is required to resolve the issue of how the proton on the acceptor hydroxyl is removed.

MINIREVIEW: Structure and Function of O-GlcNAc Transferase

The TPR Domain Plays a Role in Substrate Recognition Although the TPR domain of OGT has been implicated in protein-protein interactions (see above) and is presumed to play a key role in substrate selection, there is little information on how protein substrates or adaptor/enhancer proteins are recognized by this domain. Structural studies involving the HCF-1 proteolytic repeat bound to OGT provided the first information about how the TPRs of OGT recognize at least some of its substrates. In addition to the portion of an HCF-1 repeat where cleavage takes place, there is a C-terminal threonine-rich region that was implicated in binding to the TPRs of OGT. Recent crystal structures revealed how the threoninerich region binds to OGT (Fig. 4, C and D) (11). The structures show an elaborate network of hydrogen bonds that envelop the threonine-rich C terminus of the HCF-1 repeat within the TPRs of OGT (Fig. 4C) (11). Within this region, HCF-1 is bound in an extended conformation, with the highly conserved ladder of OGT asparagine residues (see above) engaging the peptide, mostly through interactions with the amide backbone. By engaging the peptide backbone, this asparagine network positions the side chains, which, in this case, are largely threonines, to make additional hydrogen bonds with polar residues, mostly aspartates, that line the interior of the TPR DECEMBER 12, 2014 • VOLUME 289 • NUMBER 50

cavity. This likely provides sequence selectivity, as large or hydrophobic residues would be disfavored. This structure confirms that there is indeed an analogy between the substrate-binding mode of OGT and that of some other proteins that recognize multiple related ligands (see above) (37). For example, ␤-catenin, which binds to various cadherins (87, 88); PEX5, which recognizes a peroxisomal targeting signal (PTS1) (89); karyopherin (importin)-␣, which recognizes a nuclear localization sequence (88); and the histocompatibility protein HLA-DR1, which recognizes pathogen-derived peptides (90) all employ asparagine-mediated peptide backbone contacts as a crucial component of their ligand binding. There are two major reasons for this: backbone-binding interactions provide sequence-independent binding energy and predictably splay the side chains of the ligand (Fig. 4C), which in turn allows for sequence-selective recognition at defined sites along the polypeptide chain. In the context of OGT, this might imply that a consensus sequence for adaptor proteins or for substrates might in fact exist and remains to be discovered. As such, the interaction between HCF-1 and OGT has illuminated one mechanism for substrate engagement, but whether this generalizes to other substrates remains to be seen. JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 4. A, overlay of the ternary complexes HCF-1䡠UDP-5SGlcNAc䡠hOGT4.5 and OGT䡠UDP-5SGlcNAc䡠CKII. HCF-1 (yellow sticks) resides above UDP-5SGlcNAc (gray sticks). The CKII (tan sticks) and HCF-1 peptides overlay almost perfectly. B, overall cleavage reaction catalyzed by OGT. A proposed glutamyl-GlcNAc intermediate is shown. C, the C terminus of the HCF-1 peptide (yellow sticks) makes extensive contacts within the TPRs of OGT (gray ribbon). Glu-13 and Thr-24 (indicating the relative positions of the N and C termini, respectively) are indicated in boldface italics. Hydrogen bonds are shown as black dotted lines. Important OGT residues (gray sticks) are labeled according to ncOGT numbering. Contacts from two lysine residues (gray sticks) were omitted for clarity. D, overall structure of the ternary complex containing HCF-1 (yellow balls and sticks), UDP-5SGlcNAc (magenta spheres), and hOGT4.5 (gray ribbon).

MINIREVIEW: Structure and Function of O-GlcNAc Transferase

REFERENCES 1. Torres, C. R., and Hart, G. W. (1984) Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J. Biol. Chem. 259, 3308 –3317 2. Kreppel, L. K., Blomberg, M. A., and Hart, G. W. (1997) Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats. J. Biol. Chem. 272, 9308 –9315 3. Lubas, W. A., Frank, D. W., Krause, M., and Hanover, J. A. (1997) OLinked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats. J. Biol. Chem. 272, 9316 –9324 4. Lazarus, M. B., Nam, Y., Jiang, J., Sliz, P., and Walker, S. (2011) Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature 469, 564 –567 5. Love, D. C., and Hanover, J. A. (2005) The hexosamine signaling pathway: deciphering the “O-GlcNAc code.” Sci. STKE 2005, re13 6. Kazemi, Z., Chang, H., Haserodt, S., McKen, C., and Zachara, N. E. (2010) O-Linked ␤-N-acetylglucosamine (O-GlcNAc) regulates stress-induced heat shock protein expression in a GSK-3␤-dependent manner. J. Biol.

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Chem. 285, 39096 –39107 7. Hart, G. W., Housley, M. P., and Slawson, C. (2007) Cycling of O-linked ␤-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446, 1017–1022 8. Butkinaree, C., Park, K., and Hart, G. W. (2010) O-Linked ␤-N-acetylglucosamine (O-GlcNAc): extensive crosstalk with phosphorylation to regulate signaling and transcription in response to nutrients and stress. Biochim. Biophys. Acta 1800, 96 –106 9. Capotosti, F., Guernier, S., Lammers, F., Waridel, P., Cai, Y., Jin, J., Conaway, J. W., Conaway, R. C., and Herr, W. (2011) O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1. Cell 144, 376 –388 10. Daou, S., Mashtalir, N., Hammond-Martel, I., Pak, H., Yu, H., Sui, G., Vogel, J. L., Kristie, T. M., and Affar el, B. (2011) Crosstalk between O-GlcNAcylation and proteolytic cleavage regulates the host cell factor-1 maturation pathway. Proc. Natl. Acad. Sci. U.S.A. 108, 2747–2752 11. Lazarus, M. B., Jiang, J., Kapuria, V., Bhuiyan, T., Janetzko, J., Zandberg, W. F., Vocadlo, D. J., Herr, W., and Walker, S. (2013) HCF-1 is cleaved in the active site of O-GlcNAc transferase. Science 342, 1235–1239 12. Vocadlo, D. J. (2012) O-GlcNAc processing enzymes: catalytic mechanisms, substrate specificity, and enzyme regulation. Curr. Opin. Chem. Biol. 16, 488 – 497 13. Martinez-Fleites, C., He, Y., and Davies, G. J. (2010) Structural analyses of enzymes involved in the O-GlcNAc modification. Biochim. Biophys. Acta 1800, 122–133 14. Gloster, T. M., and Vocadlo, D. J. (2010) Mechanism, structure, and inhibition of O-GlcNAc processing enzymes. Curr. Signal Transduct. Ther. 5, 74 –91 15. Hurtado-Guerrero, R., Dorfmueller, H. C., and van Aalten, D. M. (2008) Molecular mechanisms of O-GlcNAcylation. Curr. Opin. Struct. Biol. 18, 551–557 16. Harwood, K. R., and Hanover, J. A. (2014) Nutrient-driven O-GlcNAc cycling–think globally but act locally. J. Cell Sci. 127, 1857–1867 17. Hart, G. W., Slawson, C., Ramirez-Correa, G., and Lagerlof, O. (2011) Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. Annu. Rev. Biochem. 80, 825– 858 18. Allan, R. K., and Ratajczak, T. (2011) Versatile TPR domains accommodate different modes of target protein recognition and function. Cell Stress Chaperones 16, 353–367 19. Das, A. K., Cohen P. W., and Barford, D. (1998) The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPRmediated protein-protein interactions. EMBO J. 17, 1192–1199 20. Blatch, G. L., and Lässle, M. (1999) The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. BioEssays 21, 932–939 21. März, P., Stetefeld, J., Bendfeldt, K., Nitsch, C., Reinstein, J., Shoeman, R. L., Dimitriades-Schmutz, B., Schwager, M., Leiser, D., Ozcan, S., Otten, U., and Ozbek, S. (2006) Ataxin-10 interacts with O-linked ␤-N-acetylglucosamine transferase in the brain. J. Biol. Chem. 281, 20263–20270 22. Riu, I. H., Shin, I. S., and Do, S. I. (2008) Sp1 modulates ncOGT activity to alter target recognition and enhanced thermotolerance in E. coli. Biochem. Biophys. Res. Commun. 372, 203–209 23. Cheung, W. D., Sakabe, K., Housley, M. P., Dias, W. B., and Hart, G. W. (2008) O-Linked ␤-N-acetylglucosaminyltransferase substrate specificity is regulated by myosin phosphatase targeting and other interacting proteins. J. Biol. Chem. 283, 33935–33941 24. Iyer, S. P., and Hart, G. W. (2003) Roles of the tetratricopeptide repeat domain in O-GlcNAc transferase targeting and protein substrate specificity. J. Biol. Chem. 278, 24608 –24616 25. Iyer, S. P., Akimoto, Y., and Hart, G. W. (2003) Identification and cloning of a novel family of coiled-coil domain proteins that interact with O-GlcNAc transferase. J. Biol. Chem. 278, 5399 –5409 26. Yang, X., Zhang, F., and Kudlow, J. E. (2002) Recruitment of O-GlcNAc transferase to promoters by corepressor mSin3A: coupling protein O-GlcNAcylation to transcriptional repression. Cell 110, 69 – 80 27. Kreppel, L. K., and Hart, G. W. (1999) Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats. J. Biol. Chem. 274, 32015–32022 28. Zhang, Q., Liu, X., Gao, W., Li, P., Hou, J., Li, J., and Wong, J. (2014)

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Conclusions The past few years have resulted in an enormous amount of progress in the structure and mechanism of OGT. Although these insights stand to benefit the understanding of other GT-B superfamily members, from a structural and biochemical perspective, there are still several unresolved questions regarding OGT. What is the most likely mechanism for shuttling a proton from the active site? What role does oligomerization play in OGT function? What is the function of the intervening domain? OGT has been shown to bind to phosphatidylinositol 3,4,5-triphosphate (PIP3) (91, 92), and mutagenesis suggested that Lys-981 and Lys-982, both residing in the C-terminal catalytic domain, were responsible for PIP3 binding. However, it has been reported that the K981A/K982A double mutant binds PIP3 as well as other PIPs comparably to wild-type ncOGT (4). Furthermore, several combinations of lysine residues in the C-terminal catalytic domain or Int-D were mutated, but none of these mutations seemed to abrogate PIP binding. Due to the ambiguity of these findings and given the highly basic nature of the Int-D, which might suggest a role in mediating interactions with anionic macromolecules or membrane interfaces, further investigations into the function of this domain and the location of the PIP-binding site are certainly warranted. How does OGT interact with larger polypeptides, such as folded proteins? Numerous proteins have been shown to interact with OGT directly or indirectly, although little, if anything, is known about the physical picture of these interactions. Of the characterized interactions with OGT, some are mediated by the TPRs (see above), whereas others seem to interact elsewhere (23, 28). Furthermore, TPRs in different systems have been shown to interact with proteins in several different ways (18). HCF-1 provided the first picture of a substrate engaging the TPRs of OGT, but it remains to be seen whether other substrates, adaptors, or enhancers interact with OGT analogously. Several recent publications have exploited protein microarrays as a tool that allows for precise systematic control of individual parameters, which is likely to be crucial in examining the role of possible adaptors/enhancers (93–95). More biochemical and structural data on how OGT physically interacts with different proteins would add greatly to understanding its function.

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37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

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48.

49.

50.

51.

52.

53.

54.

55.

56.

57. 58.

59.

60.

61.

62.

63.

64.

65.

66. 67.

transferases: families and functional modules. Curr. Opin. Struct. Biol. 11, 593– 600 Lairson, L. L., Henrissat, B., Davies, G. J., and Withers, S. G. (2008) Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 77, 521–555 Wrabl, J. O., and Grishin, N. V. (2001) Homology between O-linked GlcNAc transferases and proteins of the glycogen phosphorylase superfamily. J. Mol. Biol. 314, 365–374 Coutinho, P. M., Deleury, E., Davies, G. J., and Henrissat, B. (2003) An evolving hierarchical family classification for glycosyltransferases. J. Mol. Biol. 328, 307–317 Ha, S., Walker, D., Shi, Y., and Walker, S. (2000) The 1.9 Å crystal structure of Escherichia coli MurG, a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis. Protein Sci. 9, 1045–1052 Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M., and Henrissat, B. (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490 –D495 Shen, D. L., Gloster, T. M., Yuzwa, S. A., and Vocadlo, D. J. (2012) Insights into O-linked N-acetylglucosamine ([0 –9]O-GlcNAc) processing and dynamics through kinetic analysis of O-GlcNAc transferase and O-GlcNAcase activity on protein substrates. J. Biol. Chem. 287, 15395–15408 Hu, Y., Chen, L., Ha, S., Gross, B., Falcone, B., Walker, D., Mokhtarzadeh, M., and Walker, S. (2003) Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases. Proc. Natl. Acad. Sci. U.S.A. 100, 845– 849 Brown, K., Vial, S. C., Dedi, N., Westcott, J., Scally, S., Bugg, T. D., Charlton, P. A., and Cheetham, G. M. (2013) Crystal structure of the Pseudomonas aeruginosa MurG:UDP-GlcNAc substrate complex. Protein Pept. Lett. 20, 1002–1008 Grotthuss, C. J. T. (1806) Sur la décomposition de l’eau et des corps q’uelle tient en dissolution à l’aide de l’électricité galvanique. Ann. Chim. Phys. 58, 54 –74 Cukierman, S. (2006) Et tu, Grotthuss! and other unfinished stories. Biochim. Biophys. Acta 1757, 876 – 885 Tvarosˇka, I., Kozmon, S., Wimmerová, M., and Kocˇa, J. (2012) Substrateassisted catalytic mechanism of O-GlcNAc transferase discovered by quantum mechanics/molecular mechanics investigation. J. Am. Chem. Soc. 134, 15563–15571 Lazarus, B. D., Roos, M. D., and Hanover, J. A. (2005) Mutational analysis of the catalytic domain of O-linked N-acetylglucosaminyltransferase. J. Biol. Chem. 280, 35537–35544 Gerster, T., and Roeder, R. G. (1988) A herpesvirus trans-activating protein interacts with transcription factor OTF-1 and other cellular proteins. Proc. Natl. Acad. Sci. U.S.A. 85, 6347– 6351 Kristie, T. M., and Sharp, P. A. (1990) Interactions of the Oct-1 POU subdomains with specific DNA sequences and with the HSV ␣-transactivator protein. Genes Dev. 4, 2383–2396 Katan, M., Haigh, A., Verrijzer, C. P., van der Vliet, P. C., and O’Hare, P. (1990) Characterization of a cellular factor which interacts functionally with Oct-1 in the assembly of a multicomponent transcription complex. Nucleic Acids Res. 18, 6871– 6880 Stern, S., and Herr, W. (1991) The herpes simplex virus trans-activator VP16 recognizes the Oct-1 homeo domain: evidence for a homeo domain recognition subdomain. Genes Dev. 5, 2555–2566 Xiao, P., and Capone, J. P. (1990) A cellular factor binds to the herpessimplex virus type-1 transactivator Vmw65 and is required for Vmw65dependent protein-DNA complex assembly with Oct-1. Mol. Cell. Biol. 10, 4974 – 4977 apRhys, C. M., Ciufo, D. M., O’Neill, E. A., Kelly, T. J., and Hayward, G. S. (1989) Overlapping octamer and TAATGARAT motifs in the VF65-response elements in herpes simplex virus immediate-early promoters represent independent binding sites for cellular nuclear factor III. J. Virol. 63, 2798 –2812 Luciano, R. L., and Wilson, A. C. (2003) HCF-1 functions as a coactivator for the zinc finger protein Krox20. J. Biol. Chem. 278, 51116 –51124 Wysocka, J., Myers, M. P., Laherty, C. D., Eisenman, R. N., and Herr, W. (2003) Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone

JOURNAL OF BIOLOGICAL CHEMISTRY

34431

Downloaded from http://www.jbc.org/ at NYU School of Medicine Library on February 9, 2015

33.

Differential regulation of the ten-eleven translocation (TET) family of dioxygenases by O-linked ␤-N-acetylglucosamine transferase (OGT). J. Biol. Chem. 289, 5986 –5996 Chen, Q., Chen, Y., Bian, C., Fujiki, R., and Yu, X. (2013) TET2 promotes histone O-GlcNAcylation during gene transcription. Nature 493, 561–564 Lazarus, B. D., Love, D. C., and Hanover, J. A. (2006) Recombinant O-GlcNAc transferase isoforms: identification of O-GlcNAcase, yes tyrosine kinase, and tau as isoform-specific substrates. Glycobiology 16, 415– 421 Ruan, H. B., Han, X., Li, M. D., Singh, J. P., Qian, K., Azarhoush, S., Zhao, L., Bennett, A. M., Samuel, V. T., Wu, J., Yates, J. R., 3rd, and Yang, X. (2012) O-GlcNAc transferase/host cell factor C1 complex regulates gluconeogenesis by modulating PGC-1␣ stability. Cell Metab. 16, 226 –237 Hanover, J. A., Yu, S., Lubas, W. B., Shin, S. H., Ragano-Caracciola, M., Kochran, J., and Love, D. C. (2003) Mitochondrial and nucleocytoplasmic isoforms of O-linked GlcNAc transferase encoded by a single mammalian gene. Arch. Biochem. Biophys. 409, 287–297 Love, D. C., Kochan, J., Cathey, R. L., Shin, S. H., and Hanover, J. A. (2003) Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase. J. Cell Sci. 116, 647– 654 Nolte, D., and Müller, U. (2002) Human O-GlcNAc transferase (OGT): genomic structure, analysis of splice variants, fine mapping in Xq13.1. Mamm. Genome 13, 62– 64 Fletcher, B. S., Dragstedt, C., Notterpek, L., and Nolan, G. P. (2002) Functional cloning of SPIN-2, a nuclear anti-apoptotic protein with roles in cell cycle progression. Leukemia 16, 1507–1518 Shin, S. H., Love, D. C., and Hanover, J. A. (2011) Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis. Amino Acids 40, 885– 893 Jínek, M., Rehwinkel, J., Lazarus, B. D., Izaurralde, E., Hanover, J. A., and Conti, E. (2004) The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin ␣. Nat. Struct. Mol. Biol. 11, 1001–1007 Haltiwanger, R. S., Blomberg, M. A., and Hart, G. W. (1992) Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide ␤-N-acetylglucosaminyltransferase. J. Biol. Chem. 267, 9005–9013 Clarke, A. J., Hurtado-Guerrero, R., Pathak, S., Schüttelkopf, A. W., Borodkin, V., Shepherd, S. M., Ibrahim, A. F., and van Aalten, D. M. (2008) Structural insights into mechanism and specificity of O-GlcNAc transferase. EMBO J. 27, 2780 –2788 Martinez-Fleites, C., Macauley, M. S., He, Y., Shen, D. L., Vocadlo, D. J., and Davies, G. J. (2008) Structure of an O-GlcNAc transferase homolog provides insight into intracellular glycosylation. Nat. Struct. Mol. Biol. 15, 764 –765 Dorfmueller, H. C., Borodkin, V. S., Blair, D. E., Pathak, S., Navratilova, I., and van Aalten, D. M. (2011) Substrate and product analogues as human O-GlcNAc transferase inhibitors. Amino Acids 40, 781–792 Gloster, T. M., Zandberg, W. F., Heinonen, J. E., Shen, D. L., Deng, L., and Vocadlo, D. J. (2011) Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells. Nat. Chem. Biol. 7, 174 –181 Lazarus, M. B., Jiang, J., Gloster, T. M., Zandberg, W. F., Whitworth, G. E., Vocadlo, D. J., and Walker, S. (2012) Structural snapshots of the reaction coordinate for O-GlcNAc transferase. Nat. Chem. Biol. 8, 966 –968 Schimpl, M., Zheng, X., Borodkin, V. S., Blair, D. E., Ferenbach, A. T., Schüttelkopf, A. W., Navratilova, I., Aristotelous, T., Albarbarawi, O., Robinson, D. A., Macnaughtan, M. A., and van Aalten, D. M. (2012) O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis. Nat. Chem. Biol. 8, 969 –974 Jiang, J., Lazarus, M. B., Pasquina, L., Sliz, P., and Walker, S. (2012) A neutral diphosphate mimic crosslinks the active site of human O-GlcNAc transferase. Nat. Chem. Biol. 8, 72–77 Borodkin, V. S., Schimpl, M., Gundogdu, M., Rafie, K., Dorfmueller, H. C., Robinson, D. A., and van Aalten, D. M. (2014) Bisubstrate UDP-peptide conjugates as human O-GlcNAc transferase inhibitors. Biochem. J. 457, 497–502 Bourne, Y., and Henrissat, B. (2001) Glycoside hydrolases and glycosyl-

MINIREVIEW: Structure and Function of O-GlcNAc Transferase

68.

69.

70. 71.

72.

74.

75.

76. 77.

78.

79.

80.

81.

82.

34432 JOURNAL OF BIOLOGICAL CHEMISTRY

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

Exploring the O-GlcNAc proteome: direct identification of OGlcNAc-modified proteins from the brain. Proc. Natl. Acad. Sci. U.S.A. 101, 13132–13137 Mazars, R., Gonzalez-de-Peredo, A., Cayrol, C., Lavigne, A. C., Vogel, J. L., Ortega, N., Lacroix, C., Gautier, V., Huet, G., Ray, A., Monsarrat, B., Kristie, T. M., and Girard, J. P. (2010) The THAP-zinc finger protein THAP1 associates with coactivator HCF-1 and O-GlcNAc transferase: a link between DYT6 and DYT3 dystonias. J. Biol. Chem. 285, 13364 –13371 Khan, S. A., Sekulski, J. M., and Erickson, B. W. (1986) Peptide models of protein metastable binding sites: competitive kinetics of isomerization and hydrolysis. Biochemistry 25, 5165–5171 Erickson, B. W., and Khan, S. A. (1983) Synthetic lactam and thiolactone models of protein metastable binding sites. Ann. N.Y. Acad. Sci. 421, 167–177 Khan, S. A., and Erickson, B. W. (1982) An equilibrium model of the metastable binding sites of ␣2-macroglobulin and complement proteins C3 and C4. J. Biol. Chem. 257, 11864 –11867 Huber, A. H., and Weis, W. I. (2001) The structure of the ␤-catenin/Ecadherin complex and the molecular basis of diverse ligand recognition by ␤-catenin. Cell 105, 391– 402 Conti, E., Uy, M., Leighton, L., Blobel, G., and Kuriyan, J. (1998) Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin ␣. Cell 94, 193–204 Gatto, G. J., Jr., Geisbrecht, B. V., Gould, S. J., and Berg, J. M. (2000) Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat. Struct. Biol. 7, 1091–1095 Stern, L. J., Brown, J. H., Jardetzky, T. S., Gorga, J. C., Urban, R. G., Strominger, J. L., and Wiley, D. C. (1994) Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368, 215–221 Yang, X., Ongusaha, P. P., Miles, P. D., Havstad, J. C., Zhang, F., So, W. V., Kudlow, J. E., Michell, R. H., Olefsky, J. M., Field, S. J., and Evans, R. M. (2008) Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance. Nature 451, 964 –969 Kebede, M., Ferdaoussi, M., Mancini, A., Alquier, T., Kulkarni, R. N., Walker, M. D., and Poitout, V. (2012) Glucose activates free fatty acid receptor 1 gene transcription via phosphatidylinositol-3-kinase-dependent O-GlcNAcylation of pancreas-duodenum homeobox-1. Proc. Natl. Acad. Sci. U.S.A. 109, 2376 –2381 Ortiz-Meoz, R. F., Merbl, Y., Kirschner, M. W., and Walker, S. (2014) Microarray discovery of new OGT substrates: the medulloblastoma oncogene OTX2 is O-GlcNAcylated. J. Am. Chem. Soc. 136, 4845– 4848 Deng, R. P., He, X., Guo, S. J., Liu, W. F., Tao, Y., and Tao, S. C. (2014) Global identification of O-GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome. Proteomics 14, 1020 –1030 Dias, W. B., Cheung, W. D., and Hart, G. W. (2012) O-GlcNAcylation of kinases. Biochem. Biophys. Res. Commun. 422, 224 –228

VOLUME 289 • NUMBER 50 • DECEMBER 12, 2014

Downloaded from http://www.jbc.org/ at NYU School of Medicine Library on February 9, 2015

73.

H3-K4 methyltransferase are tethered together selectively by the cell-proliferation factor HCF-1. Genes Dev. 17, 896 –911 Julien, E., and Herr, W. (2004) A switch in mitotic histone H4 lysine 20 methylation status is linked to M phase defects upon loss of HCF-1. Mol. Cell 14, 713–725 Tyagi, S., Chabes, A. L., Wysocka, J., and Herr, W. (2007) E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases. Mol. Cell 27, 107–119 Mangone, M., Myers, M. P., and Herr, W. (2010) Role of the HCF-1 basic region in sustaining cell proliferation. PLoS ONE 5, e9020 Zhou, P., Wang, Z., Yuan, X., Zhou, C., Liu, L., Wan, X., Zhang, F., Ding, X., Wang, C., Xiong, S., Wang, Z., Yuan, J., Li, Q., and Zhang, Y. (2013) Mixed lineage leukemia 5 (MLL5) protein regulates cell cycle progression and E2F1-responsive gene expression via association with host cell factor-1 (HCF-1). J. Biol. Chem. 288, 17532–17543 Michaud, J., Praz, V., James Faresse, N., JnBaptiste, C. K., Tyagi, S., Schütz, F., and Herr, W. (2013) HCFC1 is a common component of active human CpG-island promoters and coincides with ZNF143, THAP11, YY1, and GABP transcription factor occupancy. Genome Res. 23, 907–916 Vogel, J. L., and Kristie, T. M. (2000) The novel coactivator C1 (HCF) coordinates multiprotein enhancer formation and mediates transcription activation by GABP. EMBO J. 19, 683– 690 Kristie, T. M., and Sharp, P. A. (1993) Purification of the cellular C1 factor required for the stable recognition of the Oct-1 homeodomain by the herpes simplex virus ␣-trans-induction factor (VP16). J. Biol. Chem. 268, 6525– 6534 Wilson, A. C., LaMarco, K., Peterson, M. G., and Herr, W. (1993) The VP16 accessory protein Hcf is a family of polypeptides processed from a large precursor protein. Cell 74, 115–125 Wilson, A. C., Peterson, M. G., and Herr, W. (1995) The HCF repeat is an unusual proteolytic cleavage signal. Genes Dev. 9, 2445–2458 Kristie, T. M., Pomerantz, J. L., Twomey, T. C., Parent, S. A., and Sharp, P. A. (1995) The cellular C1 factor of the herpes-simplex virus enhancer complex is a family of polypeptides. J. Biol. Chem. 270, 4387– 4394 Julien, E., and Herr, W. (2003) Proteolytic processing is necessary to separate and ensure proper cell growth and cytokinesis functions of HCF-1. EMBO J. 22, 2360 –2369 Wilson, A. C., Boutros, M., Johnson, K. M., and Herr, W. (2000) HCF-1 amino- and carboxy-terminal subunit association through two separate sets of interaction modules: involvement of fibronectin type 3 repeats. Mol. Cell. Biol. 20, 6721– 6730 Park, J., Lammers, F., Herr, W., and Song, J. J. (2012) HCF-1 self-association via an interdigitated Fn3 structure facilitates transcriptional regulatory complex formation. Proc. Natl. Acad. Sci. U.S.A. 109, 17430 –17435 Vogel, J. L., and Kristie, T. M. (2000) Autocatalytic proteolysis of the transcription factor-coactivator C1 (HCF): a potential role for proteolytic regulation of coactivator function. Proc. Natl. Acad. Sci. U.S.A. 97, 9425–9430 Khidekel, N., Ficarro, S. B., Peters, E. C., and Hsieh-Wilson, L. C. (2004)

Minireviews: The Making of a Sweet Modification: Structure and Function of O-GlcNAc Transferase John Janetzko and Suzanne Walker

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J. Biol. Chem. 2014, 289:34424-34432. doi: 10.1074/jbc.R114.604405 originally published online October 21, 2014