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  • 1. ”Bittersweet Roles of O-GlcNAc in Physiology & Analytical Challenges” Gerald W. Hart, Ph.D. DeLamar Professor & Director Department of Biological Chemistry Johns Hopkins University School of Medicine 725 N. Wolfe St., Baltimore, MD 21205-2185 Email: Disclosures: Supported by NIH R01CA42486, R01DK61671; N01-HV-00240; P01HL107153, R24DK084949 and Dr. Hart is ”The Beth W. and A. Ross Myers Scholar” of the Patrick C. Walsh Prostate Cancer Research Fund. Dr. Hart receives a share of royalty received by the university on sales of the CTD 1 1 0. 6 antibody, which are managed by JHU. Warren Symp. CCFC August 2012
  • 2. O-GlcNAc is Different Than Other Glycans: Nucleocytoplasmic & Cycles Outside Plasma Membrane O-GlcNAc Discovered Early 1980s Essentials of Glycobiology Second Edition Inside Common classes of animal glycans
  • 3. Detailed Glycomic Analysis of O-GlcNAc Structural Diversity: β --Ser (Thr)- ~3000 sites mapped to date
  • 4. Properties of O-GlcNAc. O-GlcNAc is Abundant on Nuclear & Cytosolic Proteins 2-5% Glucose To Hexosamine Biosynthesis Pan >O-GlcNAc Antibody Western Blot - HeLa UDP-GlcNAc UDP (Hart et al., Nature, April 2007)  Highly Dynamic Enzymatic Modification of Ser and Thr residues by β-N-acetylglucosamine  NOT elongated to more complex structures & Localized to the cytoplasm and nucleus.  Present in all Metazoans studied, some bacteria & proteozoa, some fungi, plants & viruses.  Highly abundant PTM (>~3000 identified proteins) & Often Reciprocal (Competitive) with phosphorylation - Abundance = pancreas islets>>brain>>other tissues>liver.  Dynamically cycling on Ser/Thr residues - Time scale & stoichiometry similar to phosphate.  Cycling on proteins is Controlled by O-GlcNAc Transferase and by O-GlcNAcase
  • 5. O-GlcNAc Has Extensive Crosstalk with Phosphorylation to Serve As A Nutrient Sensor that Regulates Many Cellular Processes Ann. Rev. Biochem. (2011) UDP-GlcNAc Is a Major Node of Metabolism. “O-GlcNAc or O-linked N-acetylglucosamine or hexosamine pathway” papers in PubMed = 1709 ~3000 proteins & 2500 Sites Mapped & Counting!
  • 6. O-GlcNAc Regulates Many Cellular Processes: “O-GlcNAc or O-linked N-acetylglucosamine or hexosamine pathway” papers in PubMed = 1737> Essential Role in Lymphocyte Activation - Both B- & T-lymphocytes; NFkB & NFAT Nuclear Localization; EMBO J. (2007) 26, 4368. Regulates Protein Interactions - Prevents YY1 binding Rb; Reg. ß- Essential for Life - OGT is required at the single cell, tissue and organ level in plants and animals (Marth et al.) Polymerase II, SP1, CREB, NeuroD1, PGC-1α, PDX-1, c-myc, p53, and mSin3A, most transcription factors; Histones are O-GlcNAcylated. OGT = Polycomb gene. Regulates the Proteasome & Ubiquitination of p53 - Reduced ATPase activity 19S cap;5/19 & 9/14 of catalytic and core subunits, respectively modified. eg.eNOS;-diabetic erectile dysfunction; glycogen synthase; RNA Pol II; ER, SV-40 Large T, c-myc,p53; Cancer al.; Cole et al., Song et al. Regulates Translation - eg. Diabetes - Elevated O-GlcNAc blocks Regulates Transcription - RNA Blocks Phosphorylation - Role in Neuronal Plasticity & Synaptic Vesicle Trafficking & Axonal Branching- Vosseller et 2-5% Glucose To Hexosamine Biosynthesis catenin & E-cadherin trafficking (Andrews et al.) insulin signaling; Hyper-O-GlcNAc of transcription factors important to glucose toxicity; O-GlcNAc on NeuroD1 and PDX-1 regulates insulin transcription. 1,~3000 Proteins & 2500 Sites Mapped & Counting! p67 binding to EIF2 kinase (Gupta) Ribosome Proteins; mTOR pathway (Hart et al., Nature, April 2007) Regulates Histone Methylation MLL5 GlcNAcylation triggers cell lineage determination of HL60 through activation of its HKMT activity. Nature April 2009 - O-GlcNAc on SP1 Regulates HIV-1 latency and activation, and links viral replication to the glucose metabolism of the host cell. J. Virol. (March 2009) Neurodegeneration - OGase Maps to AD and OGT Parkinsons Loci; Most proteins linked are O-GlcNAcylated; OGlcNAc reduced on tau etc. in AD; OGlcNAc reduced on key synaptic proteins. Regulates Growth Hormone (Gibberillic Acid) in Plants) Spy and Secret Agent are both OGTs. Short-Term, Protects Cells From Stress - Protects against multiple forms of cellular stress; Protects cardiac function after trauma and ischemia. Regulates Cell Cycle & Cytokinesis Affects Rate of Transition through cellcycle; Elevated OGT causes Polyploidy by blocking cytokinesis - transient cytokinesis complex.
  • 7. Recent Mass Spectrometric Methods: Chemico- enzymatic Enrichment combined With Electron Transfer Dissociation (ETD) Crosstalk Between GlcNAcylation & Phosphorylation is Surprisingly Extensive!
  • 8. a. Competitive occupancy at same site G P c-myc 1 58 439 OR 58 1 HLH TAD 439 HLH TAD b. Reciprocal occupancy at different sites P C/EBPβ G P P 179 180 181 184 189 1 RD TAD OR 345 179 180 181 184 189 1 RD TAD DBD G 345 DBD c. Simultaneous occupancy at different sites P IRS-1 P 307 1 PHD PTBD G P 632 635 914 G G G 1009 1036 1041 1242 PRD SRD d. Site-dependent reciprocal or simultaneous occupancy G CaMKIV 1 57/58 AD P G 189 200 PKD 473 CaMBD OR 1 57/58 AD 189 200 PKD 473 CaMBD
  • 9. Simple Question: In the absence of other stimuli, how Does a Short Term (~2.5 hours) Elevation (~3 fold) in Global O-GlcNAcylation (caused by inhibiting O-GlcNAcase) Affect Site Specific Phosphorylation?
  • 10. Exp. Design: Affect on Phosphorylation if O-GlcNAc is Globally Increased?: Okadaic Acid OGase Inhibitors Both Inhibitors Quantitative proteomic approach to delineate global interplay between phosphorylation and O-GlcNAcylation. A. Flow chart for phosphorylation detection and sitespecific quantitation. B. Protein level quantitation using iTRAQ. C. Formula used to A. IMAC at Polypeptide level calculate ROR Titanium Dioxide C. = [P-peptide] (B) [Protein] (B) [P-peptide] (A) = [P-peptide] (A) [Protein] (A) B. iTRAQ ROR (A/B) [Protein] (B) [P-peptide] (B) X [Protein] (A) 1 = ROR = Relative Occupancy Ratio Proceedings Natl. Acad. Sci. (USA) (2008) 105 ,13793–13798. Ratio [1] X Ratio [2]
  • 11. Site-Specific Phosphorylation Dynamics: Selected Examples C OA P/N Both Cycling Site – OA increases P/N decreases ATP-citrate synthase R.TASFSESR.A C Site is Not Cycling. P/N increases global O-GlcNAc about 2-3 fold in 3hours OA P/N Both Insulin receptor substrate 2 (IRS-2) R.VASPTSGLK.R *All Data is Normalized to Spiked Internal STDs - 32 phosphorylation sites
  • 12. Effects of a ~2-3-fold increase in Global O-GlcNAc for 2.5h: Summary of ~700 Phosphorylation Sites Identified and Quantified: Effects of Inhibiting O-GlcNAcase on P Site Occupancy: ~148 increased by P/N ~280 Sites Not Affected by 3h inhibitor ~280 decreased by P/N Nearly Every Phosphorylation Site That is Actively Cycling is Affected by GlcNAcylation! Zihao Wang Proceedings Natl. Acad. Sci. (USA) (2008) 105 ,13793–13798.
  • 13. Kinases Are Regulated by O-GlcNAcylation.
  • 14. CTD110.6 (1:5000) – O-GlcNAcylation of Yeast Kinase (human) Chip Print Array  OGT + UDP-GlcNAc  Immunoblot + Fluor 2ndary Ab No OGT Printed in Identical Pair Spots + OGT Guanghui Han (collaboration Heng Zhu’s Lab.)
  • 15. Some O-GlcNAcylated Kinases I.D. so far: ERK-5 MAP2K3 p38 MAP3K14 MAPK1 MAP2K6 MAP3K6 CDK2 CDK5 CCRK Polo-like K1 AURKB PCTAIRE K1 pim-1 oncogene src PKCζ PKCalpha AKT PIK3C3 TANK-binding K1 casein kinase 2* PRKD2 C20orf97 CAMKK2 beta CAMK IV CAMK1G CAMKII Ser/thr Ser/thr Ser/thr Ser/thr Ser/thr K16 K24 K25 K17B K33 S6K1 PKLR PDK3 PTK9 PAK4 BRD3 APEG1 SNARK STE20-like kinase AP2 associated K1 Heat Shock 27 p8 PACE-1 SFRS K1 RIO K2 RIO K3 LATS1 MATK TRIB2 NEK8 FGR CDK9 FLJ23356 ACUR2B RIPK2 PNCK ALS2CR2 CAMK2A STK16 BMPRIB PRKC1 CSNK1G2 PRKX CDC2 STK4 CSNK2A1 CAMKK1 CSNK1E HIPK1 ITK PRK2 URK1 MAPK8 TESK2 CAMKK2 KSR2 ARK5 CAMKK2 TNK2 MAPK14 NUAK1 MAPK10 MYO3A STK17A PKN3 RP6-213H19.1 Dias et al. Biochemical and Biophysical Research Communications (2012) 46 O-GlcNAcylated Kinases in Synapse -”Disproportionately O-GlcNAcylated” MCP Papers in Press. Published on May 29, 2012 (Burlingame) – i.d. 1750 O-GlcNAc sites in synapse.
  • 16. O-GlcNAc at the Active Site Inhibits CAMKIV: *Song et al. Cellular Signaling (2007) O-GlcNAc transferase is activated by CaMKIV-dependent phosphorylation under potassium chloride-induced depolarization in NG-108-15 cells
  • 17. Relative Sizes of pT200 and og189 on CAMKIV Surface models of N-acetylglucosamine (left foreground) and inorganic phosphate (right foreground), along with a cartoon model of the kinase domain from human wild-type CaMKIV (center background) modeled from an X-ray crystal structure of human CaMKIγ. The amino acid residues colored in green and red are those that are modified by GlcNAcylation and phosphorylation, respectively.
  • 18. O-GlcNAc is a Negatively Modulates - Preventing Activation of CAMKIV CaMKIV Activates OGT PT OGT 137 Inactive OGA ATP Pocket GS GS 189 137 CaMKIV (Basal State) GS A GS Stimulation 356 Ca2+ increase GS Ca2+ CaM GS *Song et al. Cellular Signaling (2007) O-GlcNAc transferase is activated by CaMKIV-dependent phosphorylation under potassium chloride-induced depolarization = O-GlcNAc-Phosphate Reg. Cycle. * Activation loop CaMKIV GS T57/S58 Activation is a 2 Step Process: 1 . Removal of O- GlcNAc 2. Activation by Phosphorylation & Ca/CM 189 GS 356 B T57/S58 CaMKK 137 200 PT GS CaMKIV GS C GS Active 356 (Stimulated State) T57/S58 Dias et al. (2009) J. Biol. Chem. 284, 21327–21337 J. Cell Sci. 1 23, 13-22.
  • 19. DYNAMIC CROSSTALK BETWEEN TWO ESSENTIAL NUTRIENT-SENSITIVE ENZYMES: O-GlcNAc Transferase (OGT) and AMPK-activated protein kinase (AMPK) HIGHLIGHTS 1) AMPK regulates nutrient-sensitive nuclear localization of OGT in myotubes, affecting global O-GlcNAcylation of nuclear protein and K9 acetylation on histone 3. 2) AMPK phosphorylates Thr-444 on OGT, a residue that regulates nuclear localization of OGT in response to AMPK activation in myotubes 3) AMPK is extensively and dynamically O-GlcNAcylated (alpha 1 and 2 subunits & gamma subunits) 4) Acute global inhibition of O-GlcNAc cycling blunts activation of AMPK 3000 John Bullen et al. submitted
  • 20. Intein Chemical Ligation to Produce P344 & G347 CK2 Isoforms Tarrant et al. Nature Chemical Biology 2012
  • 21. Using a Large Protein Array As Substrates, The Different Modified Forms of CK2 Have Different Substrate Specificities. S-Adenyosyl homocystein lyase Good Substrate For O-GlcNAc Isoform. Nucleosomal Assembly Protein 1 Good Substrate For Phospho Isoform. Pin1 Assoc. Also Regulates Tarrant et al. Nature Chemical Biology 2012
  • 22. CK2 Specificity Is, In Part, Controlled by Its Modifications: Tarrant et al. Nature Chemical Biology 2012
  • 23. Extensive Crosstalk Between GlcNAcylation And Phosphorylation Regulates Cytokinesis
  • 24. A.OGT DNA B. OGT DNA Over-expression of OGT causes polyploidy. O-GlcNAc transferase is associated with the spindle and midbody F. GFP Sup OGT TP Sup TP G. Tubulin O-GlcNAc Tubulin O-GlcNAc OGT DNA Purified spindles demonstrate a ring of O-GlcNAc modified proteins and localization with tubulin near the spindle poles. GFP Sup OGT TP Sup O-GlcNAc modified proteins are enriched at the midbody and at the nascent nuclear envelope GFP H. Sup TP 250 kDa 250 kDa 160 kDa 105 kDa 75 kDa 75 kDa 50 kDa 50 kDa TP 105 kDa 75 kDa Sup 160 kDa 105 kDa TP 250 kDa 160 kDa OGT 50 kDa Chad Slawson IB: O-GlcNAc O-GlcNAc is enriched in the mitotic taxol pellet and OGT over-expression elevates protein GlcNAcylation. IB: MPM-2 Mitotic phosphorylation is disrupted after OGT over-expression. TP=taxol pellet Coomassie Blue OGT over-expression alters protein expression in the taxol pellet samples. Molec. Biol. Cell 1 9, 41 30- 41 4
  • 25. ETD is a Breakthrough for O-GlcNAc: CAD Synthetic C-Myc O-GlcNAc peptide ETD MS performed by Hunt et al. Univ. Virginia
  • 26. The O-GlcNAc Ion Suppression Problem: Ionization of O-GlcNAc peptide is suppressed in MALDI and ESI by naked peptides Solutions: 1. Top-down MS 1. 2. High mass accurancy instrument Need large amount pure protein. Probably not effective for complex samples 2. Enrich O-GlcNAc peptides 1. 2. Direct enrichment of O-GlcNAc peptides not effective Enzymatic/chemical derivatization available
  • 27. Method to Quantify O-GlcNAc Site Occupancy: Photocleavable Biotin Alkyne – 1 Step Synthesis from Commercial Compounds: O O C O NO2 O C O N O C N H + H H S N CH NH HN O H3C O O N C NH2 O O H2 C C Propylargylamine; (Aldrich) NHS-ester reaction with primary amine; (Ambergen) Olejnik et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7590-7594. DMF, RT O C H2 C C CH CH NH NO2 O H H S NH O H3C O HN CH C N O O C N H O Molecular and Cellular Proteomics (2010) 9: 1 53- 1 60. N C O Alkyne for Click Chemistry
  • 28. Photocleavable Biotin Tag for O-GlcNAc Site Mapping by ETD: OH OH O HO HO NH O O HO O NHAC O O-GlcNAc-peptides N O C H3C O NH HN O C S O Attach to GalNAz-GlcNAc by Click Chemistry CH H N C O C N H O N N NO2 H N H2 NH C O O Affinity Purify - Streptavidin Release by U.V cleavage 365 nm UV OH H3C O C NH HN NO2 O H H S C N O O C N H O O N C O + CO2 + HO HO NH O Molecular and Cellular Proteomics (2010) 9: 1 53- 1 60. N Adds Pos. Charge Recently: I.D. 274 O-GlcNAcylated proteins & Map 458 O-GlcNAc sites in Brain (Feng et al PNAS in Press 2012) H2 NH2 C OH O N N O HO O O NHAC
  • 29. Combinatorial Proteomic Approach to Identify Mitotic GlcNAcylation and Phosphorylation Sites Method GFP The chemoenzymatic approach for O-GlcNAc enrichment OGT Click-Chemistry SILAC labeling Purify MB, digest w/ trypsin/Lys-C UDP-GalNAz GalT1, O/N U.V.Cleavage C18 trap X-biotin-alkyne Cycloaddition Protein level quantification SCX Naked peptides Better tag technology CID: Diagnostic Fragment Ions FT OH FT Avidin chromatography UV-cleavage Quantification Orbitrap-CID Glyco-peptides Site-mapping FT-ETD TiO2 chromatography p-peptides NH elution O O HO O O NHAC N ID and quantification NH2 H2 C N N Orbitrap-CID Spindle Midbody: Quantified: >700 Proteins; >320 PhosphoSites by CID Orbitrap. >~450 OH O HO HO 1. 2. 3. 4. 5. Easy release from avidin beads (w/o harsh condition) Avoiding biotin fragmentation issue Reduced precursor ion m/z Adds Positive Chg for ETD; Small tryptics. Diagnostic Fragment Ions: CID=peptide, not site. GlcNAc Mass Pair:Peptides; High Res.Orbitrap CID): ~150 O-GlcNAc Sites by FT-ETD Science Signaling 2010
  • 30. Elevating OGT 2-Fold Dramatically Affects the CDK1 Circuit: Aurora Kinase Polo Kinase circuits Also Affected in a Similar Manner Science Signaling 2010
  • 31. DIBO-Alkyne based click chemistry for the enrichment of O-GlcNAc peptides Reactive group to –N3 Handle group (biotin) Linker arm (Cleavable) DIBO-Alkyne (Reagent 1) • • • • Copper Free No TBTA Fewer steps Reagent more stable and cheaper Dr. Junfeng Ma
  • 32. Conclusions: ♥ O-GlcNAc is a Major Regulatory Post-Translational Modification in all multicellular eukaryotes - Plants & Animals & Viruses (some bacteria). ♥ O-GlcNAc is Required for Life at All Levels in Mammals and Plants. ♥ Crosstalk or Interplay Between O-GlcNAcylation & Phosphorylation is Extensive and Involved in Many Cellular Processes. ♥ O-GlcNAc is Important to Transcription:Regulates Pol II & is Part of the Histone Code where Some Sites are at Contact Regions with the DNA of the Nucleosome. ♥ O-GlcNAc - “Metabolic Sensor” That Modulates Signaling & Transcription in Response to Cellular Status. ♥ Many Toxic Affects of Hyperglycemia Likely Result From Dysregulation of the Balance Between O-GlcNAc and Phosphorylation = Glucose Toxicity. ♥ O-GlcNAcylation is Elevated in Nearly All Cancers Examined and it Likely Plays a Role in Molecular Processes Leading to Cancer. Disclosures: Supported by NIH R01CA42486, R01DK61671; N01-HV-00240; P01HL107153, R24DK084949 and Dr. Hart is ”The Beth W. and A. Ross Myers Scholar” of the Patrick C. Walsh Prostate Cancer Research Fund. Dr. Hart receives a share of royalty received by the university on sales of the CTD 1 1 0. 6 antibody, which are managed by JHU.
  • 33. Acknowledgements Hart Lab Hunt Lab Donald F. Hunt Namrata D. Udeshi Univ. Virginia Genaro A. Ramirez-Correa, Weidong Gao, and Anne M. Murphy Department of Pediatrics/Division of Cardiology Rick Huganair For tools to study O- GlcNAc (eg. antibodies, plasmids, protocols): email: gwhart@jhmi. edu Brian Lewis, NIH