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• W2004914600 abstract "Similar to phosphorylation, GlcNAcylation (the addition of O-GlcNAc to Ser(Thr) residues on polypeptides) is an abundant, dynamic, and inducible post-translational modification. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription. Here we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is highly GlcNAcylated in vivo. In addition, we show that upon activation of HEK293 cells, hemagglutinin-tagged CaMKIV GlcNAcylation rapidly decreases, in a manner directly opposing its phosphorylation at Thr-200. Correspondingly, there is an increase in CaMKIV interaction with O-GlcNAcase during CaMKIV activation. Furthermore, we identify at least five sites of GlcNAcylation on CaMKIV. Using site-directed mutagenesis, we determine that the GlcNAcylation sites located in the active site of CaMKIV can modulate its phosphorylation at Thr-200 and its activity toward cAMP-response element-binding transcription factor. Our results strongly indicate that the O-GlcNAc modification participates in the regulation of CaMKIV activation and function, possibly coordinating nutritional signals with the immune and nervous systems. This is the first example of an O-GlcNAc/phosphate cycle involving O-GlcNAc transferase/kinase cross-talk. Similar to phosphorylation, GlcNAcylation (the addition of O-GlcNAc to Ser(Thr) residues on polypeptides) is an abundant, dynamic, and inducible post-translational modification. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription. Here we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is highly GlcNAcylated in vivo. In addition, we show that upon activation of HEK293 cells, hemagglutinin-tagged CaMKIV GlcNAcylation rapidly decreases, in a manner directly opposing its phosphorylation at Thr-200. Correspondingly, there is an increase in CaMKIV interaction with O-GlcNAcase during CaMKIV activation. Furthermore, we identify at least five sites of GlcNAcylation on CaMKIV. Using site-directed mutagenesis, we determine that the GlcNAcylation sites located in the active site of CaMKIV can modulate its phosphorylation at Thr-200 and its activity toward cAMP-response element-binding transcription factor. Our results strongly indicate that the O-GlcNAc modification participates in the regulation of CaMKIV activation and function, possibly coordinating nutritional signals with the immune and nervous systems. This is the first example of an O-GlcNAc/phosphate cycle involving O-GlcNAc transferase/kinase cross-talk. The post-translational modification (PTM) 3The abbreviations used are: PTMpost-translational modificationGlcNAcylationaddition of O-GlcNAc to Ser(Thr) residues on polypeptidesCaMKIVcalcium/calmodulin-dependent kinase IVO-GlcNAcaseO-GlcNAc selective Β-N-acetylglucosaminidaseCREBcAMP-response element-binding transcription factorOGTUDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyltransferase (O-GlcNAc transferase)CaMcalmodulinCaMKKcalcium/calmodulin-dependent kinase kinasePUGNAcO-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate (an inhibitor of Β-N-acetylglucosaminidase)HAYPYDVPDYA influenza hemagglutinin-HA (epitope)HEK293human embryonic kidney cells 293pEF-HAplasmid vector based upon eukaryotic translation elongation factor 1 promoter with HA tagGSTglutathione S-transferasepCMVplasmid based upon cytomegaloviral promoterGal-T1 Y289Lmutant Β1,4-galactosyltransferase with larger binding pocketUDP-GalNAzuridine diphosphate-N-azidoacetylgalactosamineTAMRA5(6)-carboxytetramethylrhodamineGalNAzN-azidoacetylgalactosamineDTTdithiothreitolBEMADΒ-elimination, Michael Addition, dithiothreitolMES4-morpholineethanesulfonic acidMS/MStandem mass spectrometry. of proteins with O-GlcNAc was discovered over 2 decades ago (1Torres C.R. Hart G.W. J. Biol. Chem. 1984; 259: 3308-3317Abstract Full Text PDF PubMed Google Scholar). Unlike “traditional glycosylation,” O-GlcNAc is not elongated into more complex structures and is localized mostly in nucleocytoplasmic compartments. Similar to phosphorylation, GlcNAcylation is abundant, dynamic, and inducible and occurs on Ser and Thr residues (for review see Ref. 2Hart G.W. Housley M.P. Slawson C. Nature. 2007; 446: 1017-1022Crossref PubMed Scopus (1084) Google Scholar). To date, all O-GlcNAc-modified proteins are also phosphoproteins. Interestingly, many phosphorylation sites are also known GlcNAcylation sites (e.g. endothelial nitric-oxide synthase (3Du X.L. Edelstein D. Dimmeler S. Ju Q. Sui C. Brownlee M. J. Clin. Invest. 2001; 108: 1341-1348Crossref PubMed Scopus (723) Google Scholar), c-Myc (4Kamemura K. Hayes B.K. Comer F.I. Hart G.W. J. Biol. Chem. 2002; 277: 19229-19235Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), estrogen receptor-Β (5Cheng X. Hart G.W. J. Biol. Chem. 2001; 276: 10570-10575Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), and RNA polymerase II (6Comer F.I. Hart G.W. Biochemistry. 2001; 40: 7845-7852Crossref PubMed Scopus (237) Google Scholar)); however, the relationship between the two PTMs is not simply reciprocal. Several proteins can be concomitantly phosphorylated and GlcNAcylated or the adjacent phosphorylation and GlcNAcylation sites can regulate the addition of the other moiety (e.g. p53 (2Hart G.W. Housley M.P. Slawson C. Nature. 2007; 446: 1017-1022Crossref PubMed Scopus (1084) Google Scholar, 7Yang W.H. Kim J.E. Nam H.W. Ju J.W. Kim H.S. Kim Y.S. Cho J.W. Nat. Cell Biol. 2006; 8: 1074-1083Crossref PubMed Scopus (340) Google Scholar)). Recently, different groups were able to show an extensive interplay between GlcNAcylation and phosphorylation using proteomic approaches (8Khidekel N. Ficarro S.B. Peters E.C. Hsieh-Wilson L.C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13132-13137Crossref PubMed Scopus (254) Google Scholar, 9Vosseller K. Trinidad J.C. Chalkley R.J. Specht C.G. Thalhammer A. Lynn A.J. Snedecor J.O. Guan S. Medzihradszky K.F. Maltby D.A. Schoepfer R. Burlingame A.L. Mol. Cell. Proteomics. 2006; 5: 923-934Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 10Wang Z. Pandey A. Hart G.W. Mol. Cell. Proteomics. 2007; 6: 1365-1379Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 11Wang Z. Gucek M. Hart G.W. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13793-13798Crossref PubMed Scopus (262) Google Scholar). post-translational modification addition of O-GlcNAc to Ser(Thr) residues on polypeptides calcium/calmodulin-dependent kinase IV O-GlcNAc selective Β-N-acetylglucosaminidase cAMP-response element-binding transcription factor UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyltransferase (O-GlcNAc transferase) calmodulin calcium/calmodulin-dependent kinase kinase O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate (an inhibitor of Β-N-acetylglucosaminidase) YPYDVPDYA influenza hemagglutinin-HA (epitope) human embryonic kidney cells 293 plasmid vector based upon eukaryotic translation elongation factor 1 promoter with HA tag glutathione S-transferase plasmid based upon cytomegaloviral promoter mutant Β1,4-galactosyltransferase with larger binding pocket uridine diphosphate-N-azidoacetylgalactosamine 5(6)-carboxytetramethylrhodamine N-azidoacetylgalactosamine dithiothreitol Β-elimination, Michael Addition, dithiothreitol 4-morpholineethanesulfonic acid tandem mass spectrometry. Unlike phosphorylation, where more than 600 enzymes regulate the turnover of phosphate (12Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6257) Google Scholar), O-GlcNAc cycling in mammals is regulated by only one known gene encoding the catalytic subunit for O-GlcNAc transferase (13Kreppel L.K. Blomberg M.A. Hart G.W. J. Biol. Chem. 1997; 272: 9308-9315Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar, 14Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar) and one known gene encoding the catalytic subunit for removal, O-GlcNAcase (15Gao Y. Wells L. Comer F.I. Parker G.J. Hart G.W. J. Biol. Chem. 2001; 276: 9838-9845Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). However, in a process similar to that used by Ser/Thr phosphatases (16Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar), both O-GlcNAc transferase and O-GlcNAcase transiently associate with binding partners to dynamically produce holoenzymes with unique properties and specificities (17Yang X. Zhang F. Kudlow J.E. Cell. 2002; 110: 69-80Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 18Iyer S.P. Hart G.W. J. Biol. Chem. 2003; 278: 24608-24616Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 19Cheung W.D. Sakabe K. Housley M.P. Dias W.B. Hart G.W. J. Biol. Chem. 2008; 283: 33935-33941Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 20Clarke A.J. Hurtado-Guerrero R. Pathak S. Schüttelkopf A.W. Borodkin V. Shepherd S.M. Ibrahim A.F. van Aalten D.M. EMBO J. 2008; 27: 2780-2788Crossref PubMed Scopus (91) Google Scholar, 21Yang 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. Evans R.M. Nature. 2008; 451: 964-969Crossref PubMed Scopus (448) Google Scholar). GlcNAcylation is crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription, affecting protein-protein interactions, activity, stability, and expression (2Hart G.W. Housley M.P. Slawson C. Nature. 2007; 446: 1017-1022Crossref PubMed Scopus (1084) Google Scholar). O-GlcNAc transferase deletion in mice led to embryonic lethality (22Shafi 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 (598) Google Scholar), highlighting the importance of OGT and GlcNAcylation for cell viability. In addition, dysfunctional protein GlcNAcylation/phosphorylation seems to be important for the pathology of diabetes and Alzheimer disease (23Dias W.B. Hart G.W. Mol. Biosyst. 2007; 3: 766-772Crossref PubMed Scopus (204) Google Scholar). GlcNAcylation is involved in several signaling pathways, including regulating cellular responses to insulin (24Copeland R.J. Bullen J.W. Hart G.W. Am. J. Physiol. Endocrinol. Metab. 2008; 295: E17-28Crossref PubMed Scopus (193) Google Scholar), cell cycle control (25Slawson C. Zachara N.E. Vosseller K. Cheung W.D. Lane M.D. Hart G.W. J. Biol. Chem. 2005; 280: 32944-32956Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), stress protection (26Zachara N.E. O’Donnell N. Cheung W.D. Mercer J.J. Marth J.D. Hart G.W. J. Biol. Chem. 2004; 279: 30133-30142Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar), and calcium cycling (27Clark R.J. McDonough P.M. Swanson E. Trost S.U. Suzuki M. Fukuda M. Dillmann W.H. J. Biol. Chem. 2003; 278: 44230-44237Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Recently, several studies have shown a direct link between GlcNAcylation and Ca2+ homeostasis in cardiomyocytes (28Jones S.P. Zachara N.E. Ngoh G.A. Hill B.G. Teshima Y. Bhatnagar A. Hart G.W. Marbán E. Circulation. 2008; 117: 1172-1182Crossref PubMed Scopus (189) Google Scholar, 29Ramirez-Correa G.A. Jin W. Wang Z. Zhong X. Gao W.D. Dias W.B. Vecoli C. Hart G.W. Murphy A.M. Circ. Res. 2008; 103: 1354-1358Crossref PubMed Scopus (106) Google Scholar). Calcium (Ca2+) is a central secondary messenger involved in regulating cytoskeletal proteins, ion pumps, and activities of enzymes, including Ca2+/calmodulin-dependent protein kinases (CaMKs). Calcium/calmodulin-dependent kinase IV (CaMKIV) is a multifunctional Ser/Thr kinase found predominantly in the brain, T-cells, and testis. CaMKIV is activated in the presence of increased intracellular [Ca2+], acting as a potent mediator of Ca2+-induced gene expression. The regulation of CaMKIV activity is complex and involves a multistep process to achieve full activation (30Means A.R. Mol. Endocrinol. 2000; 14: 4-13Crossref PubMed Scopus (159) Google Scholar, 31Racioppi L. Means A.R. Trends Immunol. 2008; 29: 600-607Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). First, Ca2+/CaM binds to CaMKIV, exposing its activation loop to allow calcium/calmodulin-dependent kinase kinase (CaMKK) (32Anderson K.A. Means R.L. Huang Q.H. Kemp B.E. Goldstein E.G. Selbert M.A. Edelman A.M. Fremeau R.T. Means A.R. J. Biol. Chem. 1998; 273: 31880-31889Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) to phosphorylate Thr-200 within this activation loop, resulting in a 10–20-fold increase in total activity to generate Ca2+/CaM-independent activity (33Soderling T.R. Trends Biochem. Sci. 1999; 24: 232-236Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). CaMKIV may then also undergo an autophosphorylation event on Ser-11 and Ser-12 in its N terminus (34Chatila T. Anderson K.A. Ho N. Means A.R. J. Biol. Chem. 1996; 271: 21542-21548Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). CaMKIV has been shown to phosphorylate and regulate the activity of several proteins, such as c-AMP-response element-binding protein (CREB), oncogene 18, AP-1 (activator protein 1), MEF2 (myocyte enhancer factor 2), and O-GlcNAc transferase, among others. Interestingly, KCl-induced depolarization of neuroblastoma cells rapidly activates O-GlcNAc transferase to increase GlcNAcylation levels, suggesting a potential regulatory role for O-GlcNAc during Ca2+ signaling (35Song M. Kim H.S. Park J.M. Kim S.H. Kim I.H. Ryu S.H. Suh P.G. Cell. Signal. 2008; 20: 94-104Crossref PubMed Scopus (53) Google Scholar). Here we show that CaMKIV is highly GlcNAcylated in vivo. We also show that upon ionomycin treatment, CaMKIV GlcNAcylation decreases, whereas its interaction with O-GlcNAcase increases in a manner directly opposing the phosphorylation of Thr-200 on CaMKIV. Furthermore, we identify at least five sites of GlcNAcylation on CaMKIV. Using site-directed mutagenesis, we have determined that the GlcNAcylation sites located in the active cleft of CaMKIV modulate the phosphorylation of CaMKIV at Thr-200 and its activity toward CREB. Our results strongly indicate that the O-GlcNAc modification participates in the regulation of CaMKIV activation and function. HEK293A cells were grown in Dulbecco’s modified Eagle’s medium (25 mm glucose; Mediatech) containing 10% (v/v) fetal bovine serum (Gemini Bio-Products) and penicillin/streptomycin (Mediatech). Jurkat human T-lymphocytes were maintained in RPMI 1640 medium, 10 mm sodium pyruvate, nonessential amino acids, penicillin/streptomycin, and 10% fetal bovine serum. Cells were stimulated with 1 μm ionomycin for the indicated times. Cerebella were dissected from rat brains (Pel-Freez) and homogenized in 25 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 1 μm PUGNAc with protease and phosphatase inhibitors. The homogenate was clarified by centrifugation and filtration. HEK293A cells were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The human CaMKIV cDNA was obtained from the ATCC and subcloned into pEF-HA (36Cheung W.D. Hart G.W. J. Biol. Chem. 2008; 283: 13009-13020Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) to create HA-CaMKIV. CaMKIV point mutants T57A/S58A, S137A, S189A, S344A/S345A, S356A, T200A, and T200E were created using the QuikChange site-directed mutagenesis system (Stratagene). The mutations were confirmed by DNA sequencing by the Synthesis & Sequencing Facility at Johns Hopkins University. The GST-CREB prokaryotic expression plasmid was created by subcloning CREB from pCMV-mycCREB (37Riccio A. Alvania R.S. Lonze B.E. Ramanan N. Kim T. Huang Y. Dawson T.M. Snyder S.H. Ginty D.D. Mol. Cell. 2006; 21: 283-294Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) kindly provided by Dr. Ginty (Johns Hopkins University) into pGEX-4T2 (GE Healthcare). Recombinant GST-CREB was expressed and purified from Escherichia coli BL21 cells according to the manufacturer’s instructions. Cells were washed with phosphate-buffered saline and collected into lysis buffer (0.5% Nonidet P-40 (Sigma) in 25 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 1 μm PUGNAc with protease and phosphatase inhibitors). Cell lysates were sonicated and centrifuged to remove debris. Immunoprecipitations were performed with the indicated antibodies and captured with GammaBind G-Sepharose (GE Healthcare). The immunoprecipitates were then washed with lysis buffer and submitted for enzymatic assays or eluted in Laemmli buffer for immunoblot analysis. Samples were separated on Criterion precast SDS-polyacrylamide gels (Bio-Rad), and the gels were subsequently electroblotted to nitrocellulose (Bio-Rad). The membranes were blocked in Tris-buffered saline with 0.1% (v/v) Tween 20 with either 3% (w/v) bovine serum albumin or 3% (w/v) nonfat dry milk. The blocked membranes were then incubated overnight at 4 °C with primary antibodies against O-GlcNAc (CTD110.6), O-GlcNAcase, HA (HA.11; Covance), CaMKIV (Cell Signaling), or pT200 CaMKIV (Santa Cruz Biotechnology). CTD110.6 O-GlcNAc immunoblots were performed using bovine serum albumin as the blocking agent. Streptavidin-conjugated horseradish peroxidase (Pierce) was used to probe for biotin labeling. The blots were then washed, incubated with the appropriate secondary antibody, developed using ECL (GE Healthcare), and exposed to Hyperfilm ECL (GE Healthcare). NIH Image or ImageJ software were used for densitometric analysis of immunoblots, and all measurements were normalized against HA loading controls. Immunoprecipitated HA-CaMKIV was washed with lysis buffer, washed once with water, and submitted to the following treatments. Β-Elimination was performed overnight at 4 °C using the GlycoProfile Β-elimination kit (Sigma) according to the manufacturer’s instructions. γ-Phosphatase (New England Biolabs) treatment was performed according to the manufacturer’s instructions. Β-GlcNAcase (Sigma) treatment was performed in 20 mm MES, pH 5.6, 50 mm NaCl for 2 h at 30 °C, followed by 12 h at 4 °C. All reactions were stopped and eluted with Laemmli buffer for immunoblot analysis. HA-CaMKIV was immunoprecipitated, washed with lysis buffer, and washed twice with reaction buffer containing 20 mm HEPES, pH 7.9, 50 mm NaCl, 1 μm PUGNAc, and 5 mm MnCl2 with protease and phosphatase inhibitors. Next, 2 μl of Gal-T1 Y289L (Invitrogen) and 2 μl of 0.5 mm UDP-GalNAz (Invitrogen) were added to a reaction volume of 20 μl. The reaction was performed overnight at 4 °C. The beads were washed twice with reaction buffer to remove excess UDP-GalNAz. The samples were then reacted with biotin alkyne (Invitrogen) or tetramethyl-6-carboxyrhodamine (TAMRA) alkyne (Invitrogen) according to the manufacturer’s instructions. Galactosyltransferase labeling with UDP-[3H]galactose was performed as described previously (38Slawson C. Lakshmanan T. Knapp S. Hart G.W. Mol. Biol. Cell. 2008; 19: 4130-4140Crossref PubMed Scopus (128) Google Scholar). The enzymatic reactions were eluted with Laemmli buffer for immunoblot analysis or gel-purified for site-mapping analysis. In-gel fluorescence was imaged and measured using a Typhoon Variable Mode Imager (GE Healthcare). Immunoprecipitated HA-CaMKIV was labeled with GalNAz and reacted with biotin alkyne as described above (supplemental Fig. S1). The sample was gel-purified, trypsin-digested, and enriched for O-GlcNAc-containing peptides over streptavidin-agarose (Invitrogen). Biotin-containing peptides were eluted from the streptavidin-agarose by Β-elimination followed by Michael addition (BEMAD) (39Wells L. Vosseller K. Cole R.N. Cronshaw J.M. Matunis M.J. Hart G.W. Mol. Cell. Proteomics. 2002; 1: 791-804Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), to replace the GlcNAc-GalNAz-biotin moiety with DTT (Fig. 2A), and analyzed by an LTQ mass spectrometer (Thermo Finnigan) coupled with a nano-two-dimensional liquid chromatography pump (Eksigent Technologies). The mass spectrometer was programmed to record a full precursor scan, followed by fragmentation and MS/MS scans of the top eight most intense ions. The data were analyzed using Mascot (Matrix Science) against the full human protein data base with DTT-modified Ser/Thr as variable modifications. The automated comparative modeling program SWISS-MODEL was used for homology modeling (40Arnold K. Bordoli L. Kopp J. Schwede T. Bioinformatics. 2006; 22: 195-201Crossref PubMed Scopus (6039) Google Scholar). Using the first approach automated mode, SWISS-MODEL selected CaMKIG from the RCSB Protein Data Bank as best template by sequence similarity. Using the x-ray crystal structure of CaMKIG (Protein Data Bank code 2JAM) 4J. E. Debreczeni, A. Bullock, T. Keates, F. H. Niesen, E. Salah, L. Shrestha, C. Smee, F. Sobott, A. C. W. Pike, G. Bunkoczi, F. von Delft, A. Turnbull, J. Weigelt, C. H. Arrowsmith, A. Edwards, M. Sundstrom, and S. Knapp, unpublished data. as template, SWISS-MODEL created a predicted model of the kinase domain of CaMKIV. The resulting Protein Data Bank code coordinates are found in supplemental Fig. S3. Figures and other representations of the predicted CaMKIV kinase domain structure were created using MacPyMol (DeLano Scientific). Sequence alignments, percent identity, and percent similarity scores were generated using Clone Manager (Sci-Ed software) and BLAST2 sequences using the BLOSUM62 scoring matrix. Cells expressing various forms of HA-CaMKIV were lysed in buffer containing 20 mm Tris-HCl, pH 7.5, 0.5 mm DTT, 0.1% Tween 20, 5 mm MgCl2, 1 mm CaCl2, 1 μm CaM, and 1 μm PUGNAc with protease and phosphatase inhibitors. Lysates were sonicated and centrifuged to remove debris. Clarified lysates were incubated with 10 μl of Kinase-Bind γ-phosphate-linked high substitution ATP resin (Innova Biosciences) for 2 h at 4 °C. The binding reactions were washed with lysis buffer and eluted with Laemmli buffer for immunoblot analysis. HA-CaMKIV and its mutants were immunoprecipitated, washed with lysis buffer, washed once with lysis buffer containing 500 mm LiCl, washed twice with lysis buffer, and then washed twice with reaction buffer containing 20 mm Tris-HCl, pH 7.4, 0.1% Nonidet P-40, 8 mm MgCl2, 1 mm CaCl2, 1 μm CaM, and 1 μm PUGNAc with protease and phosphatase inhibitors. Recombinant GST-CREB (2 μg), 2 μCi of [32P]ATP, and 2 μm ATP were added to each reaction and incubated for 20 min at 30 °C. The reactions were stopped by addition of Laemmli buffer and subjected to autoradiography and immunoblot analysis. For experiments without ionomycin treatment, samples were incubated for 90 min at 30 °C, and the length of exposure to film was significantly longer than ionomycin-stimulated samples. Immunoblots shown are representative of at least three independent experiments. All experiments, excluding site-mapping analysis, were performed a minimum of three times. Error bars in all charts represent the mean ± S.E. The paired two-tailed Student’s t test was used to determine the statistical significance of any differences between the experimental samples and the control or 0-h samples. p values less than 0.05 were deemed statistically significant at the 95% confidence level. p values are indicated in each figure legend. To determine whether CaMKIV was GlcNAcylated, we used several approaches and controls to detect the O-GlcNAc modification. First, we transfected HEK293 cells with an HA-tagged form of CaMKIV, immunoprecipitated using anti-HA tag antibodies, and performed galactosyltransferase reactions using UDP-[3H]galactose. In this reaction, the radioactive galactose moiety is specifically transferred to terminal GlcNAc residues. Overexpressed HA-CaMKIV was labeled with [3H]galactose indicating the presence of terminal GlcNAc (Fig. 1A). Recently, a chemoenzymatic approach to detect O-GlcNAc was described using a mutant galactosyltransferase GalT1 Y289L and an azide derivative of UDP-GalNAc (UDP-GalNAz) as donor substrate (41Khidekel N. Arndt S. Lamarre-Vincent N. Lippert A. Poulin-Kerstien K.G. Ramakrishnan B. Qasba P.K. Hsieh-Wilson L.C. J. Am. Chem. Soc. 2003; 125: 16162-16163Crossref PubMed Scopus (217) Google Scholar). The resulting azide-labeled proteins were chemically tagged with TAMRA alkyne for detection by in-gel fluorescence. HA-CaMKIV was strongly labeled with TAMRA (Fig. 1B). We were also able to detect GlcNAcylation of HA-CaMKIV using the O-GlcNAc-specific antibody CTD110.6 (42Comer F.I. Vosseller K. Wells L. Accavitti M.A. Hart G.W. Anal. Biochem. 2001; 293: 169-177Crossref PubMed Scopus (234) Google Scholar) (Fig. 1C). As a specificity control, we performed a reductive Β-elimination reaction under mild alkaline conditions to remove O-linked but not N-linked sugars. As expected, Β-elimination treatment completely abolished the O-GlcNAc signal on CaMKIV (Fig. 1C), showing the high specificity of the CTD110.6 antibody for O-GlcNAc. Under these mild Β-elimination conditions, very little protein degradation was observed. To further confirm that CaMKIV is GlcNAcylated, we treated HA-CaMKIV with Β-GlcNAcase and γ-phosphatase. Β-GlcNAcase treatment was sufficient to remove most of the O-GlcNAc from CaMKIV without changing its protein levels, whereas γ-phosphatase treatment completely abolished the slower migrating species, indicating that phosphorylation is responsible for altering CaMKIV mobility on an SDS-polyacrylamide gel (Fig. 1D). In this experiment, the phosphorylated species of HA-CaMKIV does not appear to include phosphorylation at Thr-200 (data not shown), consistent with the cells being in a basal unstimulated state. Next, we investigated the presence of O-GlcNAc on endogenously expressed CaMKIV. Toward this end, we immunoprecipitated CaMKIV from rat cerebella and Jurkat human T-lymphocytes, because it had been shown previously that CaMKIV is highly expressed in these tissues (43Ohmstede C.A. Jensen K.F. Sahyoun N.E. J. Biol. Chem. 1989; 264: 5866-5875Abstract Full Text PDF PubMed Google Scholar, 44Frangakis M.V. Chatila T. Wood E.R. Sahyoun N. J. Biol. Chem. 1991; 266: 17592-17596Abstract Full Text PDF PubMed Google Scholar). CaMKIV was found to be GlcNAcylated in both rat cerebella (Fig. 1E) and Jurkat cells (Fig. 1F). The regulation of CaMKIV activity is complex and involves binding to Ca2+/CaM, auto-phosphorylation on multiple Ser residues at its N terminus, and phosphorylation in its activation loop on Thr-200 by the upstream kinase CaMKK (32Anderson K.A. Means R.L. Huang Q.H. Kemp B.E. Goldstein E.G. Selbert M.A. Edelman A.M. Fremeau R.T. Means A.R. J. Biol. Chem. 1998; 273: 31880-31889Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 33Soderling T.R. Trends Biochem. Sci. 1999; 24: 232-236Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). The phosphorylation at Thr-200 by CaMKK increases the CaMKIV activity about 10-fold by decreasing the Km value for its substrates (34Chatila T. Anderson K.A. Ho N. Means A.R. J. Biol. Chem. 1996; 271: 21542-21548Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The phosphorylation of Thr-200 of CaMKIV has been shown to be fast and transient in response to ionomycin (45Anderson K.A. Noeldner P.K. Reece K. Wadzinski B.E. Means A.R. J. Biol. Chem. 2004; 279: 31708-31716Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Consistent with previous reports, we observed increased CaMKIV Thr-200 phosphorylation upon ionomycin stimulation, peaking at 2 min and decreasing after 5 min (Fig. 2, A and B). Interestingly, CaMKIV GlcNAcylation rapidly decreased at 2 min and returned to basal levels after 10 min, in a manner that directly opposed its phosphorylation at Thr-200 (Fig. 2, A and B). Based upon this observation, we sought to determine whether CaMKIV interacts with O-GlcNAcase during activation. Indeed, CaMKIV interacted with O-GlcNAcase, and this interaction increased with ionomycin stimulation (Fig. 2, C and D), indicating that O-GlcNAcase is recruited to CaMKIV to facilitate the removal of O-GlcNAc from CaMKIV during its activation. To determine whether phosphorylation at Thr-200 affects the GlcNAcylation of CaMKIV, we used site-directed mutagenesis to create Ala and Glu substitutions at Thr-200, to prevent and mimic constitutive phosphorylation at Thr-200, respectively. Indeed, the GlcNAcylation levels of the T200A mutant was increased 2-fold over wild-type CaMKIV, whereas the T200E mutant displayed less O-GlcNAc (Fig. 2, E and F), supporting a reciprocal relationship between the GlcNAcylation of CaMKIV and its phosphorylation at Thr-200. To explain how GlcNAcylation of CaMKIV could affect its phosphorylation at Thr-200, we identified the sites of GlcNAcylation on CaMKIV. Mapping O-GlcNAc sites has been one of the major obstacles in studying protein GlcNAcylation, mostly because O-GlcNAc is extremely labile during mass spectrometry analysis. Here we modified the newly developed chemoenzymatic labeling protocol, combining it with Β-elimination followed by Michael addition with DTT (BEMAD) for O-GlcNAc site-mapping (Fig. 3A) (10Wang Z. Pandey A. Hart G.W. Mol. Cell. Proteomics. 2007; 6: 1365-1379Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 39Wells L. Vosseller K. Cole R.N. Cronshaw J.M. Matunis M.J. Hart G.W. Mol. Cell. Proteomics. 2002; 1: 791-804Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Immunoprecipitated HA-CaMKIV was labeled with GalNAz and reacted with a biotin alkyne to covalently attach a biotin tag to the Glc" @default.
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• W2004914600 title "Regulation of Calcium/Calmodulin-dependent Kinase IV by O-GlcNAc Modification" @default.
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