Connexin 43 Interacts with Zona Occludens-1 and -2 Proteins in a Cell Cycle Stage-specific Manner*

Gap junction channels play an important role in cell growth control, secretion and embryonic development. Gap junctional communication and channel assembly can be regulated by protein-protein interaction with kinases and phosphatases. We have utilized tandem mass spectrometry (MS/MS) sequence analysis as a screen to identify proteins from cell lysates that interact with the C-terminal cytoplasmic region of connexin 43 (Cx43). MS/MS analysis of tryptic fragments yielded several proteins including zona occludens-1 (ZO-1), a structural protein previously identified to interact with Cx43, and ZO-2, a potential novel interacting partner. We confirmed the interaction of ZO-2 with Cx43 by using a combination of fusion protein “pull down,” co-immunoprecipitation, and co-localization experiments. We show that the C-terminal region of Cx43 is necessary for interaction with the PDZ2 domain of ZO-2. Far Western analysis revealed that ZO-2 can directly bind to Cx43 independent of other interacting partners. Immunofluorescence studies indicate that both ZO-1 and ZO-2 can co-localize with Cx43 within the plasma membrane at apparent gap junctional structures. We examined Cx43 interaction with ZO-1 and ZO-2 at different stages of the cell cycle and found that Cx43 had a strong preference for interaction with ZO-1 during G0, whereas ZO-2 interaction occurred approximately equally during G0 and S phases. Since essentially all of the Cx43 in G0 cells is assembled into Triton X-100-resistant junctions, Cx43-ZO-1 interaction may contribute to their stability.

• Publication year

  2005

• Venue

  Journal of Biological Chemistry

• Publication date

  2005-08-26

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1074/jbc.M506799200PMID 15980428PMCID PMC3501655
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• Cx43 shows stronger association with ZO-1 in G0, whereas ZO-2 association is similar in G0 and S phase.

  Confidence 0.96

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• ZO-2 can bind Cx43 directly without other interacting partners.

  Confidence 0.93

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• The C-terminal cytoplasmic region of Cx43 is required for binding to the PDZ2 domain of ZO-2.

  Confidence 0.95

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• MS/MS screening of Cx43-associated proteins identified ZO-1 and ZO-2 as interactors of the Cx43 C-terminal cytoplasmic region.

  Confidence 0.97

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review

• c-terminal cytoplasmic region of cx43

  protein region

  The intracellular tail segment of Cx43 used in pull-down and binding experiments.

  Aliases: Cx43 C-terminal region, Cx43 C-terminus

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• cx43

  protein

  A gap junction protein whose C-terminal cytoplasmic region was used as the bait for interaction mapping.

  Aliases: connexin 43

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• g0 phase

  cell cycle phase

  A quiescent cell-cycle stage used to compare Cx43 interactions.

  Aliases: G0

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• pdz2 domain

  protein domain

  The second PDZ module of ZO-2 that mediates binding to the Cx43 tail.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• s phase

  cell cycle phase

  The DNA-synthesis stage of the cell cycle used for comparison with G0.

  Aliases: S

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• zo-1

  protein

  A scaffolding protein examined for association with Cx43 at gap junctional sites.

  Aliases: zona occludens-1

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• zo-2

  protein

  A scaffolding protein examined as a Cx43-interacting partner in binding assays.

  Aliases: zona occludens-2

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review

• Connexin phosphorylation as a regulatory event linked to gap junction channel assembly.

  2005cited by this paper
• Connexins and cell signaling in development and disease.

  2004cited by this paper
• Modifications in the Biophysical Properties of Connexin43 Channels by a Peptide of the Cytoplasmic Loop Region

  2004cited by this paper
• Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between Binding Domains for c-Src and Zonula Occludens-1*

  2004cited by this paper
• Drebrin is a novel connexin-43 binding partner that links gap junctions to the submembrane cytoskeleton.

  2004cited by this paper
• CCN3 (NOV) Interacts with Connexin43 in C6 Glioma Cells

  2004cited by this paper
• Establishment and Characterization of Cultured Epithelial Cells Lacking Expression of ZO-1*

  2004cited by this paper
• A proposed role for ZO-1 in targeting connexin 43 gap junctions to the endocytic pathway.

  2004cited by this paper
• Gap junctions and the connexin protein family.

  2004cited by this paper
• Connexin43 Interacts with NOV

  2004cited by this paper
• Gap junctions and connexin-interacting proteins.

  2004cited by this paper
• Identification of Connexin-43 Interacting Proteins

  2003cited by this paper
• Connexin43 phosphorylation at S368 is acute during S and G2/M and in response to protein kinase C activation

  2003cited by this paper
• Role of catenins in the development of gap junctions in rat cardiomyocytes

  2003cited by this paper
• Casein Kinase 1 Regulates Connexin-43 Gap Junction Assembly*

  2002cited by this paper
• Molecular Cloning, Functional Expression, and Tissue Distribution of a Novel Human Gap Junction-forming Protein, Connexin-31.9

  2002cited by this paper
• Increased Association of ZO-1 With Connexin43 During Remodeling of Cardiac Gap Junctions

  2002cited by this paper
• Gap Junctions between Cells Expressing Connexin 43 or 32 Show Inverse Permselectivity to Adenosine and ATP*

  2002cited by this paper
• Interaction of c-Src with Gap Junction Protein Connexin-43

  2001cited by this paper
• Connexin45 directly binds to ZO‐1 and localizes to the tight junction region in epithelial MDCK cells

  2001cited by this paper
• c-Src Regulates the Interaction between Connexin-43 and ZO-1 in Cardiac Myocytes*

  2001cited by this paper
• Connexin-43 Interactions with ZO-1 and α- and β-tubulin

  2001cited by this paper
• Link of the unique oncogenic properties of adenovirus type 9 E4‐ORF1 to a select interaction with the candidate tumor suppressor protein ZO‐2

  2001cited by this paper
• Connexin45 Interacts with Zonula Occludens-1 and Connexin43 in Osteoblastic Cells*

  2001cited by this paper
• Protein kinase C-alpha and -epsilon modulate connexin-43 phosphorylation in human heart.

  2001cited by this paper
• Protein Kinase C- α and - ϵ Modulate Connexin-43 Phosphorylation in Human Heart

  2001cited by this paper
• Molecular Cloning and Characterization of Pals, Proteins Associated with mLin-7*

  2000cited by this paper
• Genetic diseases and gene knockouts reveal diverse connexin functions.

  1999cited by this paper
• PDZ domains: fundamental building blocks in the organization of protein complexes at the plasma membrane.

  1999cited by this paper
• Protein Interactions at the Tight Junction

  1999cited by this paper
• Direct analysis of protein complexes using mass spectrometry

  1999cited by this paper
• Formation of a distinct connexin43 phosphoisoform in mitotic cells is dependent upon p34cdc2 kinase.

  1998cited by this paper
• The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein.

  1998cited by this paper
• ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

  1998cited by this paper
• Direct Association of the Gap Junction Protein Connexin-43 with ZO-1 in Cardiac Myocytes*

  1998cited by this paper
• Recognition of Unique Carboxyl-Terminal Motifs by Distinct PDZ Domains

  1997cited by this paper
• Reconstitution of Mitogen-activated Protein Kinase Phosphorylation Cascades in Bacteria

  1997cited by this paper
• Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database.

  1995cited by this paper
• Tight junctions and the molecular basis for regulation of paracellular permeability.

  1995cited by this paper
• Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein

  1994cited by this paper
• Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques

  1991cited by this paper
• Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1.

  1991cited by this paper
• Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines

  1990cited by this paper
• Pdz Proteins Organize Minireview Synaptic Signaling Pathways

  year unknowncited by this paper

• Formation of tight junction-like structures of zonula occludens 2 in platelet–platelet interaction

  2025cites this paper
• Hepatic cell junctions: Pulling a double‐duty

  2024cites this paper
• Connexins in Cancer, the Possible Role of Connexin46 as a Cancer Stem Cell-Determining Protein

  2023cites this paper
• KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells

  2023cites this paper
• Hydrodynamic forces influence the gene transcription of mechanosensitive intercellular junction associated genes in corneal endothelial cells.

  2021cites this paper
• Expression patterns of ZO-1/2 and their effects on porcine oocyte in vitro maturation and early embryonic development.

  2020cites this paper
• Endothelial Glycocalyx-Mediated Intercellular Interactions: Mechanisms and Implications for Atherosclerosis and Cancer Metastasis

  2020cites this paper
• Gap junction-mediated cell-to-cell communication in oral development and oral diseases: a concise review of research progress

  2020cites this paper
• Scaffolding proteins of vertebrate apical junctions: Structure, functions and biophysics.

  2020cites this paper
• Non-canonical roles of connexins.

  2020cites this paper
• Understanding the Molecular and Cell Biological Mechanisms of Electrical Synapse Formation

  2020cites this paper
• Adenovirus targets transcriptional and posttranslational mechanisms to limit gap junction function

  2020cites this paper
• The Role of Connexin 43 in Lung Disease

  2020cites this paper
• Protective Effects of Magnesium Glycyrrhizinate on Methotrexate-Induced Hepatotoxicity and Intestinal Toxicity May Be by Reducing COX-2

  2019cites this paper
• Control of integrin-mediated mechanoresponse by binding partners and force loading rates

  2019cites this paper
• Cx43 phosphorylation–mediated effects on ERK and Akt protect against ischemia reperfusion injury and alter the stability of the stress-inducible protein NDRG1

  2019cites this paper
• Evidence of decreased gap junction coupling between astrocytes and oligodendrocytes in the anterior cingulate cortex of depressed suicides

  2019cites this paper
• Elucidating the role of sorting nexins in skeletal homeostasis

  2018cites this paper
• A Cell Junctional Protein Network Associated with Connexin-26

  2018cites this paper
• Protein–Protein Interactions with Connexin 43: Regulation and Function

  2018cites this paper
• The connexin 43 C-terminus: A tail of many tales.

  2018influential citation
• Adipokines Deregulate Cellular Communication via Epigenetic Repression of Gap Junction Loci in Obese Endometrial Cancer.

  2018cites this paper
• Lysosomal Degradation of Junctional Proteins

  2017cites this paper
• Connexin channel and its role in diabetic retinopathy

  2017cites this paper
• ZO‐2, a tight junction protein involved in gene expression, proliferation, apoptosis, and cell size regulation

  2017cites this paper
• Protective effects of connexins in atheromatous plaques in patients of carotid artery stenosis

  2017cites this paper
• Gja 1 acts downstream of Acvr 1 to regulate uterine decidualization via Hand 2 in mice

  2017cites this paper
• Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes.

  2017cites this paper
• Connexins in Cardiovascular and Neurovascular Health and Disease: Pharmacological Implications

  2017cites this paper
• Gja1 acts downstream of Acvr1 to regulate uterine decidualization via Hand2 in mice.

  2017cites this paper
• The role of IgG hypersensitivity in the pathogenesis and therapy of depressive disorders

  2016cites this paper
• Tight Junctions of the Blood-Brain Barrier - A Molecular Gatekeeper.

  2016cites this paper
• The role of connexin43-Src interaction in astrocytomas: A molecular puzzle.

  2016cites this paper
• The Role of Connexins in Wound Healing and Repair: Novel Therapeutic Approaches

  2016cites this paper
• ZO-1 expression shows prognostic value in chronic B cell leukemia.

  2016cites this paper
• Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1

  2016cites this paper
• The role of the tyrosines in the carboxyl tail of connexin43 in regulating cytoskeletal rearrangements in B-Lymphocytes

  2015cites this paper
• The connexin 43/ZO-1 complex regulates cerebral endothelial F-actin architecture and migration.

  2015cites this paper
• Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis*

  2015cites this paper
• Mutations in TJP2, encoding zona occludens 2, and liver disease

  2015cites this paper
• Influence of the scaffolding protein Zonula Occludens (ZOs) on membrane channels.

  2014cites this paper
• Biology and Regulation of Blood-Tissue Barriers

  2014cites this paper
• Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

  2014cites this paper
• Role of Zonula Occludens (ZO) proteins in regulating kidney collecting duct principal cell proliferation and adhesion

  2014cites this paper
• RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement.

  2014cites this paper
• Gap junction modulation and its implications for heart function

  2014cites this paper
• Specific Cx43 phosphorylation events regulate gap junction turnover in vivo

  2014cites this paper
• The Liver Connexin32 Interactome Is a Novel Plasma Membrane-Mitochondrial Signaling Nexus

  2013cites this paper
• Beyond cell-cell adhesion

  2013cites this paper
• The Cytoskeleton

  2013cites this paper
• Proteins and mechanisms regulating gap-junction assembly, internalization, and degradation.

  2013cites this paper
• Connexin43 mediates NF-κB signalling activation induced by high glucose in GMCs: involvement of c-Src

  2013cites this paper
• Assembly, Internalization, and Degradation Proteins and Mechanisms Regulating Gap-junction

  2013cites this paper
• Regulation of connexin‐ and pannexin‐based channels by post‐translational modifications

  2013cites this paper
• Zona occludens-2 protects against podocyte dysfunction induced by ADR in mice.

  2013cites this paper
• Translating Basic Research on Cx43 Gap Junctions into Therapies for Reducing Scarring and Cardiac Arrhythmia

  2013cites this paper
• Proteomic Approaches for the Study of Electrical Synapses and Associated Protein-Interaction Complexes

  2013cites this paper
• Emerging roles of the tight junction scaffold ZO-2

  2013cites this paper
• Gap junctions and blood-tissue barriers.

  2012cites this paper
• Molecular connexin partner remodeling orchestrates connexin traffic: From physiology to pathophysiology

  2012cites this paper
• ZO‐2, a tight junction scaffold protein involved in the regulation of cell proliferation and apoptosis

  2012cites this paper
• The connexin43 carboxyl terminus and cardiac gap junction organization.

  2012cites this paper
• Non-channel functions of connexins in cell growth and cell death.

  2012cites this paper
• Molecular characterization of the Tick-borne encephalitis virus : Environments and replication

  2012cites this paper
• Increased interaction of connexin43 with zonula occludens-1 during inhibition of gap junctions by G protein-coupled receptor agonists.

  2012cites this paper
• Gap junctional channels are parts of multiprotein complexes.

  2012cites this paper
• CASK (LIN2) interacts with Cx43 in wounded skin and their coexpression affects cell migration

  2012cites this paper
• The Molecular Role of Connexin 43 in Human Trophoblast Cell Fusion1

  2012cites this paper
• Molecular Genetics of Dupuytren´s Disease

  2012cites this paper
• Posttranslational Modifications in Connexins and Pannexins

  2012cites this paper
• Connexins and pannexins

  2011cites this paper
• Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1

  2011cites this paper
• ZO Proteins and Redox-Dependent Processes

  2011cites this paper
• Connexins and the gap in context.

  2011cites this paper
• Connexins: sensors and regulators of cell cycling.

  2011cites this paper
• The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells.

  2011cites this paper
• Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels

  2011cites this paper
• The gap junction proteome and its relationship to disease.

  2010cites this paper
• Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method.

  2010cites this paper
• Trafficking and Recycling of the Connexin43 Gap Junction Protein during Mitosis

  2010cites this paper
• New aspects of the molecular constituents of tissue barriers

  2010cites this paper
• The Dual Role of Zonula Occludens (ZO) Proteins

  2010cites this paper
• PKC inhibition increases gap junction intercellular communication and cell adhesion in human neuroblastoma

  2010cites this paper
• ZO-1 is involved in trophoblastic cell differentiation in human placenta.

  2010cites this paper
• Dexamethasone disrupts intercellular junction formation and cytoskeleton organization in human trabecular meshwork cells

  2010cites this paper
• MOLECULAR MECHANISMS OF BREAST CANCER METASTASIS: GAP JUNCTION INTERCELLULAR COMMUNICATION AND THE BONE MICROENVIRONMENT

  2010cites this paper
• Localization and trafficking of Kv channels in cardiac myocytes through the use of novel transfection methods

  2010cites this paper
• Enhanced connexin 43 expression delays intra‐mitoitc duration and cell cycle traverse independently of gap junction channel function

  2010cites this paper
• Reciprocal influence of connexins and apical junction proteins on their expressions and functions.

  2009cites this paper
• Structure of the second PDZ domain from human zonula occludens 2.

  2009influential citation
• A Historical View -- Discovering the Molecular Components of Intercellular Junctions

  2009cites this paper
• Connexin43 phosphorylation: structural changes and biological effects.

  2009cites this paper
• Connexins and pannexins: Two gap junction families mediating glioma growth control

  2009cites this paper
• Connexins: a myriad of functions extending beyond assembly of gap junction channels

  2009cites this paper
• Imaging the translocations of CLIC4 and Epac1

  2009cites this paper
• Biochemistry of Connexins

  2009cites this paper
• TLR2 Mediates Gap Junctional Intercellular Communication through Connexin-43 in Intestinal Epithelial Barrier Injury*

  2009cites this paper
• The role of gap junctions in cellular migration

  2009influential citation
• Direct association of connexin36 with zonula occludens-2 and zonula occludens-3.

  2009influential citation
• Zona occludens-2 is critical for blood-testis barrier integrity and male fertility.

  2009influential citation