Key points are not available for this paper at this time.
Glycogenin is a self-glucosylating protein involved in the initiation of glycogen biosynthesis. Self-glucosylation leads to the formation of an oligosaccharide chain, which, when long enough, supports the action of glycogen synthase to elongate it and form a mature glycogen molecule. To identify possible regulators of glycogenin, the yeast two-hybrid strategy was employed. By using rabbit skeletal muscle glycogenin as a bait, cDNAs encoding three different proteins were isolated from the human skeletal muscle cDNA library. Two of the cDNAs encoded glycogenin and glycogen synthase, respectively, proteins known to be interactors. The third cDNA encoded a polypeptide of unknown function and was designated GNIP (glycogenininteractingprotein). Northern blot analysis revealed that GNIP mRNA is highly expressed in skeletal muscle. The gene for GNIP generates at least four isoforms by alternative splicing. The largest isoform GNIP1 contains, from NH2- to COOH-terminal, a RING finger, a B box, a putative coiled-coil region, and a B30.2-like motif. The previously identified protein TRIM7 (tripartitemotif containing protein 7) is also derived from the GNIP gene and is composed of the RING finger, B box, and coiled-coil regions. The GNIP2 and GNIP3 isoforms consist of the coiled-coil region and B30.2-like domain. Physical interaction between GNIP2 and glycogenin was confirmed by co-immunoprecipitation, and in addition GNIP2 was shown to stimulate glycogenin self-glucosylation 3–4-fold. GNIPs may represent a novel participant in the initiation of glycogen synthesis. Glycogenin is a self-glucosylating protein involved in the initiation of glycogen biosynthesis. Self-glucosylation leads to the formation of an oligosaccharide chain, which, when long enough, supports the action of glycogen synthase to elongate it and form a mature glycogen molecule. To identify possible regulators of glycogenin, the yeast two-hybrid strategy was employed. By using rabbit skeletal muscle glycogenin as a bait, cDNAs encoding three different proteins were isolated from the human skeletal muscle cDNA library. Two of the cDNAs encoded glycogenin and glycogen synthase, respectively, proteins known to be interactors. The third cDNA encoded a polypeptide of unknown function and was designated GNIP (glycogenininteractingprotein). Northern blot analysis revealed that GNIP mRNA is highly expressed in skeletal muscle. The gene for GNIP generates at least four isoforms by alternative splicing. The largest isoform GNIP1 contains, from NH2- to COOH-terminal, a RING finger, a B box, a putative coiled-coil region, and a B30.2-like motif. The previously identified protein TRIM7 (tripartitemotif containing protein 7) is also derived from the GNIP gene and is composed of the RING finger, B box, and coiled-coil regions. The GNIP2 and GNIP3 isoforms consist of the coiled-coil region and B30.2-like domain. Physical interaction between GNIP2 and glycogenin was confirmed by co-immunoprecipitation, and in addition GNIP2 was shown to stimulate glycogenin self-glucosylation 3–4-fold. GNIPs may represent a novel participant in the initiation of glycogen synthesis. The biosynthesis of glycogen involves two distinguishable stages. The initiation step involves the formation of a glycoprotein primer, by self-glucosylation of glycogenin to form a covalently linked oligosaccharide. Elongation involves the bulk synthesis of glycogen through the reactions catalyzed by glycogen synthase and branching enzyme (for reviews see Refs. 1Alonso M.D. Lomako J. Lomako W.M. Whelan W.J. FASEB J. 1995; 9: 1126-1137Crossref PubMed Scopus (203) Google Scholar, 2Smythe C. Cohen P. Eur. J. Biochem. 1991; 200: 625-631Crossref PubMed Scopus (149) Google Scholar, 3Roach P.J. Skurat A.V. Prog. Nucleic Acid Res. Mol. Biol. 1997; 57: 289-316Crossref PubMed Google Scholar). Humans express two isoforms of glycogenin, one widely distributed (4Viscupic E. Cao Y. Zhang W. Cheng C. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1992; 267: 25759-25763Abstract Full Text PDF PubMed Google Scholar, 5Rodrigues I.R. Fliesler S.J. Arch. Biochem. Biophys. 1988; 260: 628-637Crossref PubMed Scopus (26) Google Scholar) and the other, glycogenin-2, predominantly expressed in liver (6Mu J. Skurat A.V. Roach P.J. J. Biol. Chem. 1997; 272: 27589-27597Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). One of the most important properties of glycogenin is its ability to self-glucosylate, using UDP-glucose as a glucose donor (6Mu J. Skurat A.V. Roach P.J. J. Biol. Chem. 1997; 272: 27589-27597Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 7Pitcher J. Smythe C. Cohen P. Eur. J. Biochem. 1988; 176: 391-395Crossref PubMed Scopus (115) Google Scholar). In rabbit glycogenin-1, self-glucosylation results in the formation of a C-1-O-tyrosyl linkage between glucose and Tyr194(8Rodrigues I.R. Whelan W.J. Biochem. Biophys. Res. Commun. 1985; 132: 829-836Crossref PubMed Scopus (101) Google Scholar, 9Smythe C. Caudwell F.B. Ferguson M. Cohen P. EMBO J. 1988; 7: 2681-2686Crossref PubMed Scopus (88) Google Scholar). Self-glucosylation continues with the formation of α-1,4-glycosidic linkages, until a chain of 8–12 residues has formed. This form of glycogenin serves as a substrate for glycogen synthase (1Alonso M.D. Lomako J. Lomako W.M. Whelan W.J. FASEB J. 1995; 9: 1126-1137Crossref PubMed Scopus (203) Google Scholar, 2Smythe C. Cohen P. Eur. J. Biochem. 1991; 200: 625-631Crossref PubMed Scopus (149) Google Scholar, 3Roach P.J. Skurat A.V. Prog. Nucleic Acid Res. Mol. Biol. 1997; 57: 289-316Crossref PubMed Google Scholar). Several lines of evidence suggest an important role of protein-protein interactions for the function of glycogenin. First, glycogenin is capable of forming dimers (10Smythe C. Watt P. Cohen P. Eur. J. Biochem. 1990; 189: 199-204Crossref PubMed Scopus (50) Google Scholar, 11Cao Y. Steinrauf L.K. Roach P.J. Arch. Biochem. Biophys. 1995; 319: 293-298Crossref PubMed Scopus (38) Google Scholar, 12Lin A., Mu, J. Yang J. Roach P.J. Arch. Biochem. Biophys. 1999; 363: 163-170Crossref PubMed Scopus (32) Google Scholar), resulting in self-glucosylation via an inter-subunit mechanism (12Lin A., Mu, J. Yang J. Roach P.J. Arch. Biochem. Biophys. 1999; 363: 163-170Crossref PubMed Scopus (32) Google Scholar, 13Alonso M.D. Lomako J. Lomako W.M. Whelan W.J. J. Biol. Chem. 1995; 270: 15315-15319Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). It was proposed that interaction between subunits of glycogenin is relatively weak (12Lin A., Mu, J. Yang J. Roach P.J. Arch. Biochem. Biophys. 1999; 363: 163-170Crossref PubMed Scopus (32) Google Scholar). Second, there are protein-protein interactions between glycogenin and glycogen synthase. For example, glycogenin co-purified with glycogen synthase from rabbit skeletal muscle in a stoichiometric 1:1 complex, indicating a relatively strong interaction (7Pitcher J. Smythe C. Cohen P. Eur. J. Biochem. 1988; 176: 391-395Crossref PubMed Scopus (115) Google Scholar). Moreover, two-hybrid analysis revealed the interaction between yeast glycogenin, Glg2p, and yeast glycogen synthase, Gsy2p (14Cheng C., Mu, J. Farkas I. Huang D. Goebl M.G. Roach P.J. Mol. Cell. Biol. 1995; 15: 6632-6640Crossref PubMed Scopus (78) Google Scholar). Third, protein-protein interactions may be involved in the subcellular distribution of glycogen. For example, glycogenin expressed in mammalian cells as a green fluorescent fusion protein was partly co-localized with actin (15Baque S. Guinovart J.J. Ferrer J.C. FEBS Lett. 1997; 417: 355-359Crossref PubMed Scopus (27) Google Scholar). Binding to actin is mediated by the carboxyl-terminal part of glycogenin. Additionally, glycogenin was found in the cytoplasm and the cell nucleus (15Baque S. Guinovart J.J. Ferrer J.C. FEBS Lett. 1997; 417: 355-359Crossref PubMed Scopus (27) Google Scholar, 16Miozzo M.C. Maldonado C. Curtino J.A. Biochem. Mol. Biol. Int. 1996; 40: 173-180PubMed Google Scholar, 17Skurat A.V. Lim S.S. Roach P.J. Eur. J. Biochem. 1997; 245: 147-155Crossref PubMed Scopus (40) Google Scholar). In order to identify other proteins that interact with glycogenin, we used the yeast two-hybrid system with rabbit glycogenin-1 as bait. We identified an as yet undescribed protein, which we named glycogenin-interacting protein (GNIP), 1The abbreviations used are: GNIPglycogenin-interacting proteinRACErapid amplification of cDNA endsUTRuntranslated regionORFopen reading frame that activates glycogenin in vitro. glycogenin-interacting protein rapid amplification of cDNA ends untranslated region open reading frame The cDNA for rabbit skeletal muscle glycogenin was generated by cutting pET15b-GN (11Cao Y. Steinrauf L.K. Roach P.J. Arch. Biochem. Biophys. 1995; 319: 293-298Crossref PubMed Scopus (38) Google Scholar) with NdeI, blunting with Klenow fragment, and subsequent digestion with SalI. The cDNA was ligated into pGBDU-C2 (18James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) that was cut with EcoRI, blunted, and digested with SalI. The resulting plasmid, which contains the glycogenin coding sequence in-frame with the DNA binding domain of Gal4p, was designated pGBDU-GN. The plasmid containing a cDNA fragment for GNIP, GNIPt-h, flanked by EcoRI-NotI-SalI adapter sequences (pGAD-GNIPt-h) was isolated from the two-hybrid library and used to create several GNIPt-h expression vectors. To construct pET28-GNIPt-h, pGAD-GNIPt-h was digested with NotI and blunt-ended, and the 1.6-kb fragment encoding GNIPt-h was ligated into pET28a (Novagen), which had been previously cut with NheI and blunt-ended. To construct pFLAG-GNIPt-h, pGAD-GNIPt-h was digested with NotI, and the 1.6-kb fragment was inserted into pFLAG-CMV-2 (Sigma), which was digested with NotI. To construct the vectors expressing GNIP1, the 0.4-kb fragment encoding the 5′ end of GNIP1 was generated by BglII and NotI digestion of DNA from EST clone AI 492496 (Fig. 1). The middle fragment of GNIP1 was amplified from a human skeletal muscle library by PCR using GNIP1- and library adapter-specific primers, as described below. This PCR product was digested with NotI and EcoRI to generate a 0.5-kb fragment. Both the 0.4- and 0.5-kb fragments were ligated with BglII- and EcoRI-cut pET32a to construct pET32-GNIP-N. The 1.2-kb fragment encoding the 3′-terminal part of GNIP1 was obtained by digestion of pGAD-GNIPt-h with EcoRI and SalI. The 1.2-kb fragment was ligated into EcoRI- and SalI-cut to To construct was digested with BglII and and the fragment was inserted into and To generate the fragment of GNIP2 encoding residues from to and an at the 5′ end was generated by This product was digested with and EcoRI to the fragment. The 1.2-kb fragment encoding the 3′-terminal part of GNIP2 was obtained by digestion of pGAD-GNIPt-h with EcoRI and Both the and the 1.2-kb fragments were ligated with and to construct To construct was digested with and EcoRI, and the fragment was used to the fragment in The yeast (18James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) was with and the human skeletal muscle cDNA library in plasmid using the The were and were from to and the with in and from the were into cells and were by yeast two-hybrid and DNA The were into and in Genetics. Scholar) were human Northern blot was with 1.6-kb GNIP fragment from pGAD-GNIPt-h by NotI The was to the cDNA from human skeletal muscle was used as for Two of were to from and from Two were used to and identify the 5′ end of In the the amplification was of cDNA with of adapter of the containing and of in a of were used with an step at for by addition of the enzyme at This was by of for and for and a step for The was at for For and were that and were of the product was used as and of for for and for were of amplified was using DNA was using and PCR were identified by DNA By using the 5′ end of two-hybrid clone was 1). of the human EST in using the sequence of clone PCR as a identified one EST clone AI with sequence that the sequence of clone PCR This clone was obtained from the and by DNA the sequences from EST clone AI PCR and GNIPt-h generate a cDNA sequence for an isoform designated GNIP1 The two-hybrid GNIPt-h, the fragment of GNIP1 (Fig. 1). The cDNA for GNIP1 was in pET32a and vectors using the fragment of EST clone AI the fragment of PCR and the fragment GNIPt-h clone identified in PCR (Fig. has a 5′ end sequence that the sequence of In two for and are in-frame with the of the 5′ clone contains a which and is in-frame with This clone the isoform designated GNIP3 To that the coding sequence for GNIP1 is in human skeletal a PCR was In the and were used with PCR to the for amplification of DNA with The amplified were inserted into the by of One cells The resulting were by using a 0.4-kb fragment from EST clone AI 492496 as a from were and the PCR fragments were by DNA One of the the open reading frame for This clone the 5′ end of cDNA for GNIP1 by as with the EST clone AI and contains a in-frame with and the In to the 5′ end of GNIP, was a human skeletal muscle cDNA library using and The largest PCR fragments were for sequence and an 3′-terminal region that is from the sequence in sequences in the 5′ ends of are three in-frame the in the protein which was designated the sequences from the 5′ of three were found in the GNIP gene to the gene and the sequences from and The of three mRNA for GNIP2 was confirmed by of several obtained from human skeletal muscle cDNA as a of cells were by using of plasmid DNA of was used to cells in a were for and cells expressing the of were in of containing and The were isolated at for at The were in of The were with and protein in the of The were three with containing and and proteins were with The the and the were by and by of GNIP2 was in from the plasmid cells were at until the and at for were by at for The cell was with of cell containing The cells were by through a cell at of the cell at for the was and with at for the was by in with containing and with containing were and the containing and rabbit skeletal muscle glycogenin was expressed and as described previously (12Lin A., Mu, J. Yang J. Roach P.J. Arch. Biochem. Biophys. 1999; 363: 163-170Crossref PubMed Scopus (32) Google Scholar). The the of into glycogenin as described previously Y. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The was in and at for as Two were to glycogenin In of the were which was three with for The was and to In the an was to of and to and To for cDNA encoding proteins that interact with glycogenin-1, we the coding sequence for rabbit skeletal muscle glycogenin with the DNA binding domain and a human skeletal muscle library expressed from the and library were isolated three different cDNAs as by cDNA One clone a cDNA for glycogen synthase of the region Two a cDNA for glycogenin-1 with of The of the isolated cDNAs the of coding for glycogen synthase and glycogenin indicating that are expressed in yeast as with a of the fusion protein by the Two other 1.6-kb fragments encoding a polypeptide of an unknown protein and This protein was designated glycogenin-interacting protein (GNIP), and the protein sequence encoded by two-hybrid library plasmid was designated GNIPt-h 1). To the the isolated from two-hybrid were into yeast that had been with pGBDU-GN. The resulting were to express and which are of the sequence in yeast the interaction between GNIPt-h and glycogenin-1 was relatively strong with the interaction between glycogen synthase and glycogenin-1 (Fig. The glycogenin-1 inter-subunit interaction was in with (12Lin A., Mu, J. Yang J. Roach P.J. Arch. Biochem. Biophys. 1999; 363: 163-170Crossref PubMed Scopus (32) Google Scholar). GNIPt-h with human glycogenin-1 as by using and expression as To GNIP with glycogenin in mammalian we expressed protein with glycogenin in GNIPt-h was as a polypeptide of in the and the of the cells (Fig. The were with shown in protein was in the from cells with cells with and glycogenin. a glycogenin was in from cells with glycogenin and (Fig. that the interaction between glycogenin and GNIP in mammalian The sequence of the GNIPt-h an open reading frame in-frame with the domain of The was by a at the 5′ there was a putative at The sequence of the a protein of with of of the protein using the several proteins protein and several RING and proteins of unknown The region was at the of proteins and a B30.2-like domain C. J. M. M. S. P. J. Mol. PubMed Scopus Google Scholar). of the GNIPt-h protein sequence with the J. P. Nucleic Res. PubMed Scopus Google Scholar) that residues of a which is a of the region J. P. S. PubMed Scopus Google Scholar, C. M. 1999; PubMed Scopus Google Scholar). the of GNIPt-h, the identified a of residues with the of forming an coiled-coil The B30.2-like domain and coiled-coil region in of the protein known as of a of a RING finger, one two B and a coiled-coil domain Nucleic Res. 1991; PubMed Scopus Google Scholar, Biol. 1992; Full Text PDF PubMed Scopus Google Scholar) (Fig. we had identified the it was possible that the GNIPt-h was and that it sequence to the RING and B This was by the human EST in we found several EST and containing sequences to in the region of the GNIPt-h to the 5′ region of the region an in-frame described in we were to the of GNIP that to at least three different which we GNIP1, and The cDNA for GNIP1, the a protein with a of that contains, to an RING domain by a B domain in addition to the coiled-coil region and B30.2-like domain of the GNIPt-h clone (Fig. GNIP2 is a of GNIP1, which the RING and B GNIP3 a in GNIP1, and the that is in three isoforms (Fig. that we are the GNIP3 amplification of the 5′ end and the sequence we are that the is the one of two the third were the the product of be the as with the GNIP1 protein sequence as we the sequence of the protein TRIM7 The residues of TRIM7 are to the of GNIP1 and RING and B (Fig. The of TRIM7 is and contains residues to the EST we identified three Two of EST were obtained from and We found several in the sequence of clone and the sequence This sequence contains an encoding a polypeptide of to The for the is by an in-frame that it to a The GNIP2 is to its human The sequence of the EST is to in the part that The 5′ end of clone is different and an in-frame that a isoform of GNIP We identified a clone of in the sequences that to The clone is derived from we found the GNIP gene in a sequence of human were by of the sequences with GNIP and TRIM7 cDNA the to the and sequences The gene for GNIP contains and (Fig. The that we identified be by alternative by by using generate of and and in The cDNA for GNIP1 and and in cDNA are by the and We identified three different GNIP2 cDNAs that are by to from and (Fig. GNIP3 cDNA is composed of from to The isoform TRIM7 is derived from and The of the from to and The the for GNIP1 and TRIM7 and the region encoding the RING and B The the for is part of the for GNIP2 part of the coiled-coil region in the for The is in the cDNA for TRIM7 and the coiled-coil region and the The of was found in the cDNA for This and part of coiled-coil motif. contains the for GNIP2 and part of coiled-coil region in GNIP1 and The the B30.2-like domain and the for isoforms of sequence of The sequence the is shown with the and donor sequences shown by are the of the and the in a The sequence the is shown with the and donor sequences shown by are the of the and the which is to the fragment of cDNA for GNIP1 was to from It is yet a GNIP gene an To that the different isoforms of GNIP interact with glycogenin, we the cDNAs for GNIP1 and GNIP2 with the in the and yeast cells expressing fusion Both GNIP1 and GNIP2 were for interaction with glycogenin ability to in the of and in the yeast and an and of results that isoforms of GNIP interact with glycogenin in Glycogenin and be from cells expressing proteins We were to of and glycogenin to of with the from To the distribution of GNIP we a human Northern blot using the cDNA fragment from the two-hybrid This of in skeletal muscle and of in (Fig. The of was in and skeletal muscle of and and of and The of mRNA is with the of by the cDNA In order to the possible of the interaction between GNIP and glycogenin, we the of GNIP the self-glucosylation catalyzed by glycogenin. GNIP2 and glycogenin were as proteins expressed in E. of GNIP1 in the of protein in and protein was of glycogenin with by and revealed a polypeptide with to (Fig. In the of of into glycogenin was indicating that GNIP2 activates the was with other proteins that is an of at of a for the binding of glycogenin to GNIP2 of Glycogenin also glucose from UDP-glucose to GNIP2 had of glucose into that the of GNIP2 was to the self-glucosylation of glycogenin by GNIP2 revealed that the GNIP2 an in with in for UDP-glucose (Fig. of GNIP UDP-glucose Glycogenin was in the of GNIP2 and of in the of at for the were for by We identified a novel of that interact with glycogenin, a of the glycogen The for proteins also glycogen synthase and glycogenin, ability to glycogenin was GNIP1 to the of RING which a known as the B box, by a coiled-coil domain Biol. 1992; Full Text PDF PubMed Scopus Google Scholar, Biochem. 1996; Full Text PDF PubMed Scopus Google Scholar). to the GNIP1 contains a domain to the B30.2-like which was identified as a product of a coding sequence in the region containing the human C. 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PubMed Scopus Google Scholar), with with and with In to the 5′ end of GNIP cDNA obtained in two-hybrid we found One the cDNA for largest isoform of GNIP, cDNA three different for protein isoform of GNIP, GNIP2 (Fig. GNIP2 the region of GNIP1, the RING and the B (Fig. The cDNA generates the third isoform of GNIP, This isoform a sequence to the of By with the GNIP1 protein we identified a isoform of GNIP, which is in sequence to the of GNIP1 and contains a of are derived from one region of human to the GNIP of the human GNIP gene a gene with of the results from to as and (Fig. The of is and represent the of The of derived from the GNIP gene is confirmed by other Northern blot analysis of different in skeletal muscle and one in (Fig. of GNIP expression were found in and with other that one of the of the GNIP is expressed in skeletal muscle in S. D. E. S. S. S. EMBO J. PubMed Scopus Google Scholar). TRIM7 is distributed in evidence for expression of GNIP2 in skeletal muscle was obtained from of a EST clone from the library. This clone contains the coding sequence for GNIP2 flanked by and the the EST clone from represent a sequence of the GNIP1 and were found for other of the protein Y. M. M. Y. M. S. M. PubMed Scopus Google Scholar, S. D. E. S. S. S. EMBO J. PubMed Scopus Google Scholar). For example, the gene for generates at least three to alternative Y. M. M. Y. M. S. M. PubMed Scopus Google Scholar). of one form in cells was by It is possible that expression of of GNIP in skeletal muscle is by different and may be important for cell between isoforms of GNIP and glycogenin important It is to that GNIP may to glycogenin to glycogen is in The of of several of protein that of proteins S. D. E. S. S. S. EMBO J. PubMed Scopus Google Scholar). To identify other proteins with GNIP, we a two-hybrid of a human skeletal muscle library using GNIPt-h as bait. In we found interaction between GNIPt-h and Skurat and P. J. We that GNIP glycogenin to of the muscle the of is of is at the initiation and glycogenin is a to glycogen of the of glycogenin is In A., Cao Y. Roach P.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Y. Skurat A.V. Roach P.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), we shown that the ability of glycogenin to as substrate for glycogen synthase its of with glycogenin leads to We found that GNIP2 activates the ability of glycogenin to self-glucosylate, the with in for UDP-glucose (Fig. GNIP2 the of of indicating that GNIP2 as of of glycogenin. the of GNIP2 self-glucosylation via a in the of the glycogenin that a for of glucose to the we the that the is to a important of GNIP in skeletal muscle to the results obtained by Smythe and (10Smythe C. Watt P. Cohen P. Eur. J. Biochem. 1990; 189: 199-204Crossref PubMed Scopus (50) Google Scholar). that of to skeletal muscle of of the glycogen in the muscle resulting in glycogenin. The subsequent of glycogenin and glycogen synthase was and be for glycogen in the of The that muscle contains that be for the of of glycogenin and glycogen synthase (10Smythe C. Watt P. Cohen P. Eur. J. Biochem. 1990; 189: 199-204Crossref PubMed Scopus (50) Google Scholar), and we that GNIP be a
Skurat et al. (Wed,) studied this question.