title: Regulation of connexin43-protein binding in astrocytes in response to chemical ischemia/hypoxia.
abstract: Connexin-protein interactions are believed to be critical for the regulation of gap junctional intercellular communication and for the function of gap junctions formed by these complexes. We have primarily used immunoprecipitation strategies to investigate whether connexin43 binds to selected signaling and cytoskeletal proteins and whether connexin43-protein binding is altered in cultured astrocytes exposed to chemical ischemia/hypoxia, a treatment that resembles ischemia in vivo. Chemical ischemia/hypoxia induced marked dephosphorylation of connexin43, which was accompanied by increased association of connexin43 with c-Src, ERK1/2, and mitogen-activated protein kinase phosphatase-1 and by decreased association between connexin43 and beta-actin. Moreover, we found that endogenous c-Src in normal astrocytes exists primarily in the Triton X-100-soluble membrane fraction, distinct from the Triton-insoluble fraction, which contains gap junctions. After chemical ischemia/hypoxia, c-Src appeared in the Triton-insoluble fraction and was co-immunoprecipitated with connexin43, suggesting that chemical ischemia/hypoxia induced translocation of c-Src to the Triton-insoluble fraction and association with connexin43. Furthermore, the "dephosphorylated" form of connexin43 was immunoprecipitated by a phosphotyrosine antibody, suggesting tyrosine phosphorylation of connexin43 by c-Src. In addition, the association between connexin43 and c-Src was blocked by inhibition of connexin43 dephosphorylation, suggesting that the interaction between connexin43 and c-Src can be regulated by alterations in the phosphorylation state of connexin43. These results identify new binding partners for connexin43 and demonstrate that interactions between connexin43 and protein kinases and phosphatases are dynamically altered as a consequence of connexin43 phosphorylation.
authors.affiliation: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Elliot L
authors.initials: EL
authors.lastname: Hertzberg
authors.forename: David C
authors.initials: DC
authors.lastname: Spray
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism* / Q000473:pathology; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004396:Coloring Agents / Q000494:pharmacology; D018031:Connexin 43 / Q000235:genetics* / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D054638:Dual Specificity Phosphatase 1; D017629:Gap Junctions; D006863:Hydrogen-Ion Concentration; D000860:Hypoxia; D017874:Immediate-Early Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D007511:Ischemia; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D054645:Protein Phosphatase 1; D021381:Protein Transport; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004396:Coloring Agents; D018031:Connexin 43; D003902:Detergents; D017874:Immediate-Early Proteins; D007546:Isoquinolines; D020033:Protein Isoforms; D019000:Phosphotyrosine; D017830:Octoxynol; C017475:lucifer yellow; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; C517020:Dusp1 protein, mouse; C517021:Dusp1 protein, rat; D017027:Protein Tyrosine Phosphatases