PHOSPHORYLATION OF GAP JUNCTION PROTEINS

Summary

Principal Investigator: Paul D Lampe
Abstract: DESCRIPTION (provided by applicant): Gap junctions are specialized matched membrane domains that contain channels that allow exchange of small molecules including ions, metabolites, and second messengers (e.g., Ca2+ and IP3) between neighboring cells. These channels are necessary for proper development and genetic linkage analyses have implicated connexins in at least 14 human diseases. The gap junction protein connexin43 (Cx43) is regulated via phosphorylation and its interactions with other proteins. This proposal focuses on the role that these two regulatory processes play and interplay in vivo to affect tissue development and function. We will examine the role Cx43 regulation plays during fundamental biological processes such as in the heart during ischemia, tachycardia and preconditioning, in skin during wound repair, and in the eye during development. We propose to (1) determine the consequences of specific Cx43 phosphorylation events on Cx43 function. (2) characterize changes in Cx43 phosphorylation and function in skin and heart in response to conditions such as wounding and hypoxia/ischemia., and (3) investigate the in vivo role of Cx43 phosphorylation in skin, heart, ovary, and eye at different developmental stages using "knock-in" mice expressing phosphorylation site mutants of Cx43. Understanding the linkage of changes in Cx43 phosphorylation to the exquisite control of fundamental biological events is in itself important but given that drugs to affect Cx43-related cardiac function and Cx43 anti-sense gels to speed wound healing are being tested in humans, we need to fully define these medically important biological events to better understand their implications and opportunities for patient treatment. PUBLIC HEALTH RELEVANCE: We propose to investigate the linkage of changes in the phosphorylation of the gap junction protein connexin43 and gap junctional communication to the exquisite control of cellular proliferation and migration during development, cardiac stress, and wound healing. Since Cx43 anti-sense treatments for wound healing and drugs to affect Cx43-related cardiac function are currently being tested in humans, our results will help us to better understand current drug implications for patient treatment and may lead to better alternatives.
Funding Period: 1997-05-01 - 2014-12-31
more information: NIH RePORT

Top Publications

  1. pmc Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation
    Jose F Ek-Vitorin
    Department of Physiology, University of Arizona, Tucson 85724, USA
    Circ Res 98:1498-505. 2006
  2. pmc Connexin43 phosphorylation in brain, cardiac, endothelial and epithelial tissues
    Lucrecia Marquez-Rosado
    Fred Hutchinson Cancer Research Center, Seattle, WA, USA
    Biochim Biophys Acta 1818:1985-92. 2012
  3. pmc Evaluating the role of connexin43 in congenital heart disease: Screening for mutations in patients with outflow tract anomalies and the analysis of knock-in mouse models
    Guo ying Huang
    Children s Hospital of Fudan University, Shanghai, China
    J Cardiovasc Dis Res 2:206-12. 2011
  4. pmc Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability
    Clarence A Dunn
    Molecular Diagnostics Program, Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
    J Biol Chem 287:2600-7. 2012
  5. pmc CASK (LIN2) interacts with Cx43 in wounded skin and their coexpression affects cell migration
    Lucrecia Marquez-Rosado
    Molecular Diagnostics Program, Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA
    J Cell Sci 125:695-702. 2012
  6. pmc MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation
    Scott R Johnstone
    Robert M Berne Cardiovascular Research Center, Charlottesville, VA 22908, USA
    Circ Res 111:201-11. 2012
  7. pmc Changes in connexin43 expression and localization during pancreatic cancer progression
    Joell L Solan
    Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, M5C800, Box 19024, Seattle, WA 98109, USA
    J Membr Biol 245:255-62. 2012
  8. pmc Phosphorylation of serine residues in the C-terminal cytoplasmic tail of connexin43 regulates proliferation of ovarian granulosa cells
    Paul W Dyce
    Department of Physiology and Pharmacology, The University of Western Ontario and Children s Health Research Institute, London, ON N6C 2V5, Canada
    J Membr Biol 245:291-301. 2012
  9. pmc Phosphorylation of connexin43 on S279/282 may contribute to laminopathy-associated conduction defects
    Steven C Chen
    Fred Hutchinson Cancer Research Center FHCRC, Public Health Sciences Division, 1100 Fairview Ave N, Seattle, WA 98109, USA
    Exp Cell Res 319:888-96. 2013
  10. pmc Connexins and steroidogenesis in mouse Leydig cells
    Dan Li
    Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON N6A 5C1, Canada
    Can J Physiol Pharmacol 91:157-64. 2013

Research Grants

  1. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013

Detail Information

Publications28

  1. pmc Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation
    Jose F Ek-Vitorin
    Department of Physiology, University of Arizona, Tucson 85724, USA
    Circ Res 98:1498-505. 2006
    ..Our data suggest that the selectivity of gap junction channels at intercalated disks is increased early in ischemia...
  2. pmc Connexin43 phosphorylation in brain, cardiac, endothelial and epithelial tissues
    Lucrecia Marquez-Rosado
    Fred Hutchinson Cancer Research Center, Seattle, WA, USA
    Biochim Biophys Acta 1818:1985-92. 2012
    ..This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics...
  3. pmc Evaluating the role of connexin43 in congenital heart disease: Screening for mutations in patients with outflow tract anomalies and the analysis of knock-in mouse models
    Guo ying Huang
    Children s Hospital of Fudan University, Shanghai, China
    J Cardiovasc Dis Res 2:206-12. 2011
    ..However, as these clinical findings were not replicated in subsequent studies, the question remains about the contribution of GJA1 mutations in human congenital heart disease (CHD)...
  4. pmc Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability
    Clarence A Dunn
    Molecular Diagnostics Program, Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
    J Biol Chem 287:2600-7. 2012
    ..This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles...
  5. pmc CASK (LIN2) interacts with Cx43 in wounded skin and their coexpression affects cell migration
    Lucrecia Marquez-Rosado
    Molecular Diagnostics Program, Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA
    J Cell Sci 125:695-702. 2012
    ....
  6. pmc MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation
    Scott R Johnstone
    Robert M Berne Cardiovascular Research Center, Charlottesville, VA 22908, USA
    Circ Res 111:201-11. 2012
    ..Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis...
  7. pmc Changes in connexin43 expression and localization during pancreatic cancer progression
    Joell L Solan
    Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, M5C800, Box 19024, Seattle, WA 98109, USA
    J Membr Biol 245:255-62. 2012
    ..These data suggest a potential role for gap junctions and connexin43 in mediating interactions between and amongst the stromal and epithelial cells in pancreatic ductal adenocarcinoma...
  8. pmc Phosphorylation of serine residues in the C-terminal cytoplasmic tail of connexin43 regulates proliferation of ovarian granulosa cells
    Paul W Dyce
    Department of Physiology and Pharmacology, The University of Western Ontario and Children s Health Research Institute, London, ON N6C 2V5, Canada
    J Membr Biol 245:291-301. 2012
    ..These results indicate that Cx43 phosphorylation by MAP kinase at serines 279 and 282 occurs in granulosa cells of early follicles and that this is involved in regulating follicle development...
  9. pmc Phosphorylation of connexin43 on S279/282 may contribute to laminopathy-associated conduction defects
    Steven C Chen
    Fred Hutchinson Cancer Research Center FHCRC, Public Health Sciences Division, 1100 Fairview Ave N, Seattle, WA 98109, USA
    Exp Cell Res 319:888-96. 2013
    ..7-fold increased levels by immunoblot. We conclude that increased pS279/282 Cx43 in the Lmna(-/-) background results in decreased cell communication and may contribute to the arrhythmic pathology in vivo...
  10. pmc Connexins and steroidogenesis in mouse Leydig cells
    Dan Li
    Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON N6A 5C1, Canada
    Can J Physiol Pharmacol 91:157-64. 2013
    ..It was concluded that connexin36, connexin43, and connexin45 are coexpressed in Leydig cells with connexins 36 and 43 contributing to gap junctions. The role of connexin45 remains to be elucidated...
  11. pmc Cardiomyocyte FGF signaling is required for Cx43 phosphorylation and cardiac gap junction maintenance
    Takashi Sakurai
    Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06511, USA
    Exp Cell Res 319:2152-65. 2013
    ..We conclude that cardiomyocyte FGF signaling is essential for cardiomyocyte homeostasis through phosphorylation of Cx43 at S325/328/330 residues which are important for the maintenance of gap junction. ..
  12. pmc Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven molecular switch that regulates gap junction size
    Clarence A Dunn
    Translational Research Program, Human Biology and Public Health Sciences Divisions, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA
    J Cell Sci 127:455-64. 2014
    ....
  13. pmc Trafficking and recycling of the connexin43 gap junction protein during mitosis
    Daniela Boassa
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, CA, USA
    Traffic 11:1471-86. 2010
    ..In late telophase, older Cx43 is segregated mainly to the plasma membrane while newer Cx43 is intracellular. This older population nucleates new gap junctions permitting rapid resumption of communication upon mitotic exit...
  14. pmc Consortin, a trans-Golgi network cargo receptor for the plasma membrane targeting and recycling of connexins
    Francisco J del Castillo
    Unite de Genetique et Physiologie de l Audition, Institut Pasteur, Paris, France
    Hum Mol Genet 19:262-75. 2010
    ..Sequence accession numbers. GenBank: Human CNST cDNA, NM_152609; mouse Cnst cDNA, NM_146105...
  15. pmc Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart
    Paul D Lampe
    Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center, Department of Pathobiology, University of Washington, 1100 Fairview Avenue N, M5C800, PO Box 19024, Seattle, WA 98109, USA
    J Cell Sci 119:3435-42. 2006
    ..These data suggest that phosphorylation of Cx43 at serine residues 325, 328 and/or 330 influences channel permselectivity and regulates the efficiency of gap junction assembly...
  16. pmc Methodologies for characterizing phosphoproteins by mass spectrometry
    Philip R Gafken
    Proteomics Facility, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
    Cell Commun Adhes 13:249-62. 2006
    ....
  17. pmc Key connexin 43 phosphorylation events regulate the gap junction life cycle
    Joell L Solan
    Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
    J Membr Biol 217:35-41. 2007
    ..However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to sort out the critical signaling pathways that regulate gap junction function...
  18. pmc The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA 92093 0608, USA
    Biochem J 408:375-85. 2007
    ..These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375-Asp379 is essential in targeting and regulation of gap junctional connexin43...
  19. pmc Gap junction remodeling and cardiac arrhythmogenesis in a murine model of oculodentodigital dysplasia
    Nellie Kalcheva
    Leon H Charney Division of Cardiology, New York University School of Medicine, New York, NY 10016, USA
    Proc Natl Acad Sci U S A 104:20512-6. 2007
    ..In summary, our data demonstrate that the I130T mutation interferes with Cx43 posttranslational processing, resulting in diminished cell-cell coupling, slowing of impulse propagation, and a proarrhythmic substrate...
  20. pmc Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down-regulation by PKC
    Joell L Solan
    Molecular Diagnostics Program and 2Proteomics Shared Resource, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
    J Cell Biol 179:1301-9. 2007
    ..Thus, phosphorylation at S365 can serve a "gatekeeper" function that may represent a mechanism to protect cells from ischemia and phorbol ester-induced down-regulation of channel conductance...
  21. pmc Connexin 43 in LA-25 cells with active v-src is phosphorylated on Y247, Y265, S262, S279/282, and S368 via multiple signaling pathways
    Joell L Solan
    Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
    Cell Commun Adhes 15:75-84. 2008
    ..These results indicate all three signaling pathways could contribute to gap junction down-regulation during src transformation in LA-25 cells...
  22. pmc Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption
    Rachael P Norris
    Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06032, USA
    Development 135:3229-38. 2008
    ..Thus, both MAP kinase-dependent gap junction closure and another redundant pathway function in parallel to ensure that meiosis resumes in response to LH...
  23. pmc Gap junction remodeling and spironolactone-dependent reverse remodeling in the hypertrophied heart
    Jiaxiang Qu
    Leon H Charney Division of Cardiology, New York University School of Medicine, 522 First Ave, Smilow 801, New York, NY 10016, USA
    Circ Res 104:365-71. 2009
    ..These data suggest a potential mechanism for some of the salutary electrophysiological and clinical effects of mineralocorticoid antagonists in myopathic hearts...
  24. pmc Connexin43 phosphorylation: structural changes and biological effects
    Joell L Solan
    Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, M5C800 Box 19024, Seattle, WA 98109, USA
    Biochem J 419:261-72. 2009
    ..The present review assesses our current understanding of the effects of phosphorylation on connexin43 structure and function that in turn regulate gap junction biology, with an emphasis on events occurring in heart and skin...
  25. pmc Oxidized phospholipid species promote in vivo differential cx43 phosphorylation and vascular smooth muscle cell proliferation
    Scott R Johnstone
    University of Virginia School of Medicine, PO Box 801394 Charlottesville VA 29908, USA
    Am J Pathol 175:916-24. 2009
    ..Taken together, these results demonstrate that post-translational phosphorylation of Cx43 could be a key factor in the pathogenesis of atherosclerosis...
  26. pmc Matricellular protein CCN3 (NOV) regulates actin cytoskeleton reorganization
    Wun Chey Sin
    Department of Cellular and Physiological Sciences and The Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada
    J Biol Chem 284:29935-44. 2009
    ..Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization...
  27. pmc Specific Cx43 phosphorylation events regulate gap junction turnover in vivo
    Joell L Solan
    Translational Research Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States
    FEBS Lett 588:1423-9. 2014
    ..g., annular junction formation or gap junctions can potentially "unzip" and be internalized/endocytosed into the cell that produced each connexin. ..

Research Grants30

  1. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013
    ..Using novel and state-of-the-art techniques, floxed mice and molecular approaches to gene regulation, we are well-positioned to lead the study of RAGE in the next cycle of this Program. ..