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Gerald Hart Portrait

Gerald Hart
DeLamar Professor and Director of Biological Chemistry
Johns Hopkins University School of Medicine

JHU School of Medicine
725 N. Wolfe St. 515 WBSB
Baltimore, MD21205
Office Phone: 410-614-5993
Lab Phone: 410-614-1265
Fax: 410-614-8804
Lab Web Site

Click Here for PDF of CV

Dynamic O-GlcNAcylation of nuclear and cytosolic proteins.

In the early 1980’s, the Hart laboratory discovered a new type of protein modification (O-GlcNAc), present on proteins within the nucleus and cytoplasm of cells, in which a glucose-derived sugar (N-acetylglucosamine; simply glucose with a nitrogen and an acetyl group attached) is attached to serine or threonine side chains of proteins, exactly analogous to phosphorylation. After twenty-six years of research, it is now known that this modification (termed O-GlcNAc) is nearly as common as phosphorylation, often competes with it at the same or proximal sites on proteins, and serves to regulate cellular functions in response to nutrients and stress by cycling on and off sites on proteins exactly like phosphorylation. O-GlcNAc is required for life at the single cell level in mammals. Recent studies, have shown that O-GlcNAc plays an important role in diabetes and glucose toxicity, Alzhemier’s disease and in the functions of oncogenes and tumor suppressors important to cancer. Since the cycling of O-GlcNAc is similar to phosphate cycling, and since they have similar abundance and distribution in cells, and since they can be attached competitively to the same or proximal sites, it was postulated that O-GlcNAc and phosphate have a ‘yin-yang’ relationship in the regulation of cellular processes. Very recent studies have established that the crosstalk between GlcNAcylation and phosphorylation is extensive and results not only from competition at the same or proximal sites, but also by the cycling enzymes for each PTM regulating the other’s activities.

Recent Reviews: Science 291, 2376-2378; Nature 446, 1017-1022; Ann. Rev. Biochem. 80:825-58; Nature Reviews Cancer 11, 678-684; Cell, 143, 672-676.

Some On-Going Projects:


Cancer & O-GlcNAcylation. Protein phosphorylation-mediated regulation of signaling, growth and transcription is not only key to normal cellular regulation, but also is commonly dysregulated in cancers. Many anti-cancer drugs are specifically directed at kinases. Protein GlcNAcylation (O-GlcNAc) is nearly as abundant as Ser(Thr) phosphorylation. O-GlcNAc cycles like phosphorylation, and is often competitive with it. O-GcNAc regulates signaling, transcription, and cytoskeletal functions in response to nutrients and stress. Recent glycoproteomic studies have revealed surprisingly extensive crosstalk or interplay between GlcNAcylation and phosphorylation. This crosstalk results not only from competition for occupancy at the same or proximal sites, but also by each modification regulating the activities of the other’s enzymes. For example, O-GlcNAc Transferase is regulated by phosphorylation and kinases are regulated by GlcNAcylation. The goal of our study is to elucidate the global crosstalk between GlcNAcylation and phosphorylation at the individual site level and to understand the extent and mechanisms as to how GlcNAcylation regulates kinases.

Aim 1: Elucidate the Dynamic Crosstalk Between GlcNAcylation and Phosphorylation.

Aim 2: Regulation of Kinases by GlcNAcylation. We will continue to identify GlcNAcylated kinases and study their regulation by O-GlcNAc, focusing initially on CAMKIV, ERK5, PKCa and Src.
Aim 3: Study the Roles of O-GlcNAc in Cytokinesis.

Prostate Cancer & O-GlcNAcylation. Using well-defined normal prostate (PrEC), prostate cancer non-aggressive (LNCaP), and prostate cancer aggressive (PC-3) cells, we are addressing four aims:
Aim 1) Systematically compare site-specific O-GlcNAcylation/phosphorylation of nucleocytoplasmic proteins and the enzymes controlling O-GlcNAc cycling as a function of prostate cancer phenotype.
Aim 2) Compare subcellular localiza¬tions, expression levels, & molecular associations of O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) in PrEC, LNCaP and PC3 cell lines.
Aim 3) How does heat stress affect the O-GlcNAcylation of cellular proteins and the en¬zymes of O-GlcNAc cycling in these cells, PrEC, LNCaP and PC3? Aim 4) Evaluate how specific al¬terations in O-GlcNAcylation affect growth properties, aneuploidy and expression and activities of androgen and estrogen receptors in the prostate cancer cells and their responses to stress.

Diabetes & O-GlcNAcylation. The modification of proteins by b-N-Acetylglucosamine (O-GlcNAc) has extensive crosstalk with Ser(Thr)-protein phosphorylation to regulate signaling and gene expression in response to nutrients/stress. The hexosamine biosynthetic pathway and its endpoint product, O-GlcNAc, plays important roles in glucose toxicity and insulin resistance. O-GlcNAcylation plays a direct role in insulin signaling and insulin resistance at many points in the insulin signaling pathway, particularly on insulin receptor substrate proteins (IRS). Another key sensor of the cell’s energy state is the 5’-AMP-dependent protein kinase, AMPK. AMPK isoforms are O-GlcNAcylated and many key AMPK substrates of are also regulated by O-GlcNAc. Hyperglycemia-induced and insulin stimulated O-GlcNAcylation of the transcription factor, Sp1 underlie deregulated transcription that contributes to glucose toxicity and metabolic disease. This project is elucidating molecular events leading to insulin resistance and glucose toxicity by focusing on the roles of O-GlcNAc on proteins regulating cellular metabolism in response to nutrients, including IRS-1, AMPK and Sp1.

Specific Aim 1: a. Elucidate the Regulation of Insulin Receptor Substrate (IRS) by Crosstalk Between O-GlcNAcylation and Phosphorylation. b. Systematic Analysis of the Kinetics of Insulin Signaling by Concomitantly Quantifying Changes at the Individual Site Level on key proteins in the insulin signaling pathway in O-GlcNAcylation and Phosphorylation using Novel MS Methods.

Specific Aim 2. Continue to Elucidate the Roles of the Crosstalk Between O-GlcNAc and the AMP-Dependent Protein Kinase (AMPK) in Cellular Regulation. a. Characterization of AMPK’s modification by O-GlcNAc, including site mapping. b. Analysis of site-specific and global regulation of AMPK’s activity, targeting and subcellular localization by O-GlcNAc. c. Analysis of AMPK’s regulation of O-GlcNAc transferase activity, targeting and subcellular localization.

Specific Aim 3. Elucidate the Roles of GlcNAcylation of the General Transcription Factor Sp1 in its Activity, Promoter Specificity, Localization, Molecular Associations and Turnover. a. Map and quantify site occupancy of O-GlcNAc and phosphate on Sp1 in different cell types and as a function of diabetic state. b. How does nutrient-mediated O-GlcNAcylation of Sp1 affect its activity in living cells? c. Do different glycoforms of Sp1 have distinct promoter specificities? d. Do different glycoforms of Sp1 have different molecular associations, or subcellular localizations or degradation rates?

The results of these studies are not only elucidate novel key mechanisms underlying deregulation in diabetes and the metabolic syndrome, but also will uncover novel avenues for therapeutics.

O-GlcNAcylation and Cardiovascular Disease. Diabetes is a major risk factor for cardiovascular disease, culminating in myocardial infarction, and heart failure. Prolonged hyper-O-GlcNAcylation, due to nutrient excess and hyperglycemia, is a major molecular cause of glucose toxicity and insulin resistance. Increased O-GlcNAcylation directly contributes to diabetic cardiomyopathy and to dysfunctional mitochondria, perhaps contributing to excessive production of reactive oxygen species (ROS). Even though O-GlcNAcylation clearly plays an important role in diabetic cardiovascular disease, virtually nothing is known about O-GlcNAcylation in the cardiomyocyte. This project is elucidating the roles of O-GlcNAc in diabetic cardiomyopathy and will define the “O-GlcNAcome” of the cardiomyocyte at the site-specific level.

Specific Aims:

Aim 1: Quantify the Site-Specific Crosstalk Between O-GlcNAcylation and Phosphorylation in the cardiomyocyte proteome and in purified cardiomyocyte mitochondria from Normal and Diabetic Rats. Using chemico-enzymatic photocleavable tag enrichment combined with electron transfer dissociation (ETD) tandem mass spectrometry, we will quantify site occupancy for both O-GlcNAc and phosphate in cardiomyocyte contractile and mitochondrial proteins from normal and diabetic rats.
Aim 2: Determine the Specific Roles of O-GlcNAcylation in normal cardiomyocyte mitochondria, and the sites of action and mechanisms of diabetes-induced dysfunction, leading to ROS production. We will specifically alter O-GlcNAcylation using methods developed during the past 20-years, and correlate alterations with specific mitochondrial function.
Aim 3: Elucidate the properties and regulation of cardiomyocyte mitochondrial isoforms of O-GlcNAc Transferase and O-GlcNAcase. Virtually nothing is known about the mitochondrial isoforms of O-GlcNAc Transferase (OGT) or O-GlcNAcase (OGA). We will elucidate their localization, activities, molecular associations and kinetic activities in mitochondria from normal and diabetic rats.
Aim 4: Evaluate the affects and roles of diabetes-induced mitochondrial dysfunction and increased O-GlcNAcylation of cardiomyocyte contractile machinery on cardiac physiology and function. We will systematically evaluate the importance of the crosstalk between O-GlcNAcylation and phosphorylation of cardiomyocyte contractile and mitochondrial proteins on the physiological functions of cardiomyocytes. These studies will open a new paradigm for understanding the regulation of cardiac functions and in diabetic cardiomyopathies. They will lead to totally unexplored avenues of possible therapeutic interventions.

Mechanisms of Glucose Toxicity Diabetes is a leading cause of blindness, cardiovascular disease, end-stage renal disease and debilitating neuropathies. Hyperglycemia underlies each of these pleiotropic maladies. However, the biochemical and physiological mechanisms for glucose toxicity remain unclear. Several biochemical mechanisms of glucose toxicity have been proposed, including increased flux through the hexosamine and polyol pathways, increased non-enzymatic, chemical glycation, and activation of protein kinase C isoforms. It has been proposed that glucose-induced production of reactive oxygen species (ROS) by the mitochondria is a unifying process affecting these disparate mechanisms of glucose toxicity. Our working hypothesis is that hyperglycemia-induced protein O-GlcNAcylation is a major fundamental mechanism leading to ROS production and to other mechanisms of glucose toxicity important to the morbidity and mortality of diabetes. We are jointly investigating mechanisms of glucose toxicity in well-defined animal model systems and in patient samples by a multi-disciplinary team comprised of both basic scientists and clinical experts with highly complimentary expertise.

Our team is working synergistically to systematically evaluate the biochemical and physiological roles of enzymatic protein O-GlcNAcylation in diabetic neuropathy, diabetic cardiomyopathy, diabetic retinopathy, in glucose-induced beta-cell destruction, and in mitochondrial ROS production.

Recent Publications

Mary Katherine Tarrant, Hee-Sool Rho, Zhi Xie, Yu Lin Jiang, Christopher Gross, Jiang Qian, Yoshitaka Ichikawa, Tatsuji Matsuoka, Natasha Zachara, Felicia Etzkorn, Gerald W. Hart, Jun-Seop Jeong, Seth Blackshaw, Heng Zhu, Philip A. Cole (2012) Multi-faceted Regulation of Protein Kinase CK2 by Phosphorylation and O-GlcNAcylation Revealed through Semisynthesis. Nature Chemical Biology, doi: 10.1038/nchembio.771.
PubMed Reference

Natasha E. Zachara, Keith Vosseller, and Gerald W. Hart (2011) Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine Current Protocols Protein Sci. UNIT 12.8.1-12.8.33.
PubMed Reference

C. Slawson and Gerald W. Hart. (2011) Alterations in O-GlcNAc Signaling: Implications for Cancer Cell Biology (2011) Nature Reviews Cancer 11, 678-684 | doi:10.1038/nrc3114.
PubMed Reference

Kaoru Sakabe and Gerald W. Hart (2011) Sweet-talking the Histone Code: O-GlcNAcylation of Histones. Cell Cycle, In Press. 

Yoshihiro Akimoto, Yuri Miura, Tosifusa Toda, Margreet A Wolfert, Lance Wells, Geert-Jan Boons, Gerald W Hart, Tamao Endo, and Hayato Kawakami (2011) Morphological Changes in Diabetic Kidney Are Associated with Increased O-GlcNAc-Modification of Cytoskeletal Proteins including a-Actinin 4 Clinical Proteomics 2011, 8:15 doi:10.1186/1559-0275-8-15.
PubMed Reference

Jokela TA, Makkonen KM, Oikari S, Koli E, Gerald W. Hart, Tammi RH, Carlberg C, Tammi MI (2011) Cellular Content of UDP-N-Acetylhexosamines Controls Hyaluronan Synthase 2 (Has2) by Transcription Factors YY1 and SP1 and Associates to O-GlcNAc Modification, J. Biol. Chem. 286, 33632-33640.
PubMed Reference

Helen Yu, Nazar Mashtalir, Salima Daou, Ian-Hammond Martel, Julie Ross, Guang-Chao Sui, Gerald W. Hart, Frank J. Rauscher III, Winship Herr, Elliot Drobetsky, Eric Milot, Yang Shi and El Bachir Affar1 (2011) The Ubiquitin Carboxyl Hydrolase BAP1 Forms a Ternary Complex with YY1 and HCF-1 and is a Critical Regulator of Gene Expression. Molecular and Cellular Biology 21:5071-85.
PubMed Reference

Wang J, Torii M, Liu H, Gerald W. Hart, Hu ZZ (2011) dbOGAP - An Integrated Bioinformatics Resource for Protein O-GlcNAcylation. BMC Bioinformatics. 2011 Apr 6;12(1):91. [Epub ahead of print]
PubMed Reference

Gerald W. Hart, Chad Slawson, Genero Ramirez-Correa, and Olof Lagerlof (2011) Crosstalk Between O-GlcNAcylation and Phosphorylation: Roles in Signaling, Transcription and Chronic Disease. Ann. Rev. Biochem. 80, 825-58.
PubMed Reference

Lance Wells, Chad Slawson, Gerald W Hart (2011), The E2F-1 Associated Retinoblastoma-Susceptibility Gene Product is Modified by O-GlcNAc Amino Acids Aug 1. [Epub ahead of print]
PubMed Reference

Natasha E. Zachara, Henrik Molina, Ker Yi Wong, Akilesh Pandey, Gerald W. Hart (2011). The Dynamic Stress-Induced “O-GlcNAcome” Highlights Functions for O-GlcNAc in Regulating DNA Damage/Repair and Other Cellular Pathways. Amino Acids Jul 31. [Epub ahead of print]
PubMed Reference

Gerald W. Hart and Ronald J. Copeland (2010) Glycomics hits the big time. Cell 143, 672-676.
PubMed Reference

Kaoru Sakabe and Gerald W. Hart (2010) O-GlcNAc Transferase Regulates Mitotic Chromatin Dynamics J. Biol. Chem. Nov 5;285(45):34460-8. Epub 2010 Aug 30.
PubMed Reference

Ping Hu, Shino Shimoji and Gerald W. Hart (2010) Site-Specific Interplay Between O-GlcNAcylation and Phosphorylation in Cellular Regulation. FEBS Lett. 18;584(12):2526-38.
PubMed Reference

Chad Slawson, Ron Copeland and Gerald W. Hart (2010) O-GlcNAc Signaling: A Metabolic Link Between Diabetes and Cancer? Trends in Biochemical Sciences 35, 547-555. (Link not available).

Quira Zeidan and Gerald W. Hart (2010) Crosstalk between GlcNAcylation and phosphorylation: implications for signal transduction and transcription. J. Cell Sci. 123, 13-22.
PubMed Reference

Kyoungsook Park, Christopher D. Saudek, and Gerald W. Hart (2010) Increased Expression of β-N-Acetylglucosamindase (O-GlcNAcase) in Erythrocytes from Prediabetic and Diabetic Individuals. Diabetes. 59(7):1845-50.
PubMed Reference

Shino Shimoji, Kyoungsook Park and Gerald W. Hart (2010) Dynamic Crosstalk Between GlcNAcylation and Phosphorylation: Roles in Signaling, Transcription and Human Disease. Current Signal Transduction Therapy, 5, 25-40. (Link not available).

Zihao Wang; Namrata Udeshi; Chad Slawson; Philip Compton; Jeffrey Shabanowitz; Donald F. Hunt; and Gerald W. Hart (2010) Extensive Crosstalk Between GlcNAcylation and Phosphorylation Regulates Cytokinesis Science Signaling 3, (104) ra2.
PubMed Reference

Zihao Wang; Namrata Udeshi; Meaghan O’Malley; Jeffrey Shabanowitz; Donald F. Hunt; and Gerald W. Hart (2010) Highly Specific Enrichment and Site-Mapping of O-GlcNAcylation Using Chemoenzymatic Tagging, Solid-Phase Photocleavage, and Electron Transfer Dissociation Mass Spectrometry. Molecular and Cellular Proteomics 9: 153-160.
PubMed Reference

Yonghong Shi, J. Tomic, Frances Wen, A. Bahlo, R. Harrison, James Dennis, R. Williams, Benjamin J Gross, Suzanne Walker, J. Zuccolo, J. P. Deans, Gerald W Hart, and David E. Spaner (2010) Aberrant O-GlcNAcylation characterizes chronic lymphocytic leukemia Leukemia 24(9):1588-98.
PubMed Reference

Quira Zeidan, Zihao Wang, Antonio De Maio and Gerald W. Hart (2010) O-GlcNAc Cycling Enzymes Associate with the Translational Machinery and Modify Many Core Ribosomal Proteins. Molecular Biology of the Cell 21, 1922-1936.
PubMed Reference

Kaoru Sakabe and Gerald W. Hart (2010) O-GlcNAc is Part of the Histone Code Proc. Natl. Acad. Sci. (USA) 107, 19915-19920.
PubMed Reference

Stephen A. Whelan, Wagner Dias, T. Lakshmanan, M. Daniel Lane, Gerald W. Hart (2009) Regulation of Insulin Receptor 1 (IRS-1)/AKT Kinase Mediated Insulin Signaling by O-linked β-N-acetylglucosamine (O-GlcNAc) in 3T3-L1 Adipocytes.J. Biol. Chem. 285, 5204-5211.
PubMed Reference

Chutikarn Butkinaree, Kyoungsook Park, and Gerald W. Hart (2009) O-Linked b-N-Acetylglucosamine: Extensive Crosstalk with Phosphorylation to Regulate Signaling and Transcription in Response to Nutrients and Stress. Biochim Biophys Acta. 2010 Feb;1800(2):96-106.
PubMed Reference

Xi Li, Henrik Molina, Haiyan Huang, You-you Zhang, Mei Liu, Shu-wen Qian, Chad Slawson, Wagner B. Dias, Akhilesh Pandy, Gerald W. Hart, M. Daniel Lane, and Qi-Qun Tang (2009) O-Linked N-Acetylglucosamine Modification on C/EBPb: Role During Adipocyte Differentiation. J. Biological Chemistry 284,19248–19254.
PubMed Reference

Michael P. Housley, Joseph T. Rodgers, Pere Puigserver, Gerald W. Hart (2009) A Pgc-1α:O-GlcNAc Transferase Complex Regulates FoxO Transcription Factor Activity in Response to Glucose. J. Biol. Chem. 284, 5148–5157.
PubMed Reference

Wagner B. Dias, Win D. Cheung, Zihao Wang, and Gerald W. Hart (2009) Regulation of Calcium/Calmodulin-dependent Kinase IV by O-GlcNAc Modification. J. Biol. Chem. 284, 21327–21337.
PubMed Reference

Genaro A. Ramirez-Correa, Wenhai Jin, Zihao Wang, Xin Zhong, Wei Dong Gao, Wagner B. Dias, Cecilia Vecoli, Gerald W. Hart and Anne M. Murphy (2008) O-linked GlcNAc Modification of Cardiac Myofilament Proteins: A Novel Regulator of Myocardial Contractile Function Circulation Research 103:1354-1358.
PubMed Reference

Win D. Cheung, Kaoru Sakabe, Michael P. Housley, Wagner B. Dias, and Gerald W. Hart (2008) O-GlcNAc transferase substrate specificity is regulated by MYPT1 and other interacting proteins. J. Biological Chemistry 283, 33935–33941.
PubMed Reference

Zihao Wang, Kyoungsook Park, Frank Comer1, Linda C. Hsieh-Wilson, Christopher D. Saudek, and Gerald W. Hart (2008) Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes Mellitus. Diabetes 58, 309-317.
PubMed Reference

Zihao Wang, Marjan Gucek, and Gerald W. Hart (2008) Crosstalk between GlcNAcylation and Phosphorylation: Site-Specific Phosphorylation Dynamics in Response to Globally Elevated O-GlcNAc. Proceedings Natl. Acad. Sci. (USA) 105 ,13793–13798.
PubMed Reference

Win D. Cheung and Gerald W. Hart (2008) AMP-activated Protein Kinase and p38 MAPK Activate O-GlcNAcylation of Neuronal Proteins during Glucose Deprivation J. Biol. Chem., 283: 13009 – 13020.
PubMed Reference

Ronald Copeland, John Bullen and Gerald W. Hart (2008) A Critical View of O-GlcNAc Involvement in Insulin Resistance and Diabetes? Am J Physiol Endocrinol Metab 295:17-28.
PubMed Reference

Zihao Wang and Gerald W. Hart (2008) Glycomic Approaches to Study GlcNAcylation: Protein Identification, Site-mapping, and Site-specific O-GlcNAc Quantitation. Clinical Proteomics, Vol. 4, No. 1. (1 June 2008), pp. 5-13. (Link not available).

Chad Slawson, T. Lakshmanan, Spencer Knapp, and Gerald W. Hart (2008) A Mitotic GlcNAcylation/Phosphorylation Signaling Complex Alters the Post-Translational State of the Cytoskeletal Protein Vimentin. Molec. Biol. Cell (Cover Article; Written up in INCYTES) 19, 4130-414.
PubMed Reference

Chutikarn Butkinaree, Win D. Cheung, Kyoungsook Park, Sung J. Park, Megan Barber, and Gerald W. Hart (2008) Cleavage Of ß-N-Acetylglucosaminidase (O-GlcNAcase) By Caspase-3 During Apoptosis J. Biol. Chem..2008; 283: 23557-23566.
PubMed Reference

Stephen A. Whelan, T. Lakshmanan, M. Daniel Lane, Gerald W. Hart (2008) Regulation of The O-GlcNAc Transferase By Insulin Signaling. J. Biol. Chemistry 283, 21411–21417.
PubMed Reference

Wagner B. Dias and Gerald W. Hart (2007) O-GlcNAc modification in diabetes and Alzheimer’s disease. Molecular Biosystems 3, 766-772.
PubMed Reference

Gerald W. Hart, Michael P. Housley and Chad Slawson (2007) Cycling of O-Linked b-N-Acetylglucosamine on Nucleocytoplasmic Proteins. Nature 446, 1017-1022.
PubMed Reference

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