Regulation of Connexin43-Protein Binding in Astrocytes in Response to Chemical Ischemia/Hypoxia
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 signal...
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| Veröffentlicht in: | The Journal of biological chemistry 2005-03, Vol.280 (9), p.7941-7948 |
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| creator | Li, Wei Hertzberg, Elliot L Spray, David C |
| description | 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 β-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 âdephosphorylatedâ
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. |
| doi_str_mv | 10.1074/jbc.M410548200 |
| format | Article |
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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 β-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 âdephosphorylatedâ
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.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M410548200</identifier><identifier>PMID: 15618229</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Animals ; Astrocytes - metabolism ; Astrocytes - pathology ; Blotting, Western ; Cell Cycle Proteins - metabolism ; Cells, Cultured ; Coloring Agents - pharmacology ; Connexin 43 - genetics ; Connexin 43 - physiology ; Cytoskeleton - metabolism ; Detergents - pharmacology ; Dual Specificity Phosphatase 1 ; Gap Junctions ; Hydrogen-Ion Concentration ; Hypoxia ; Immediate-Early Proteins - metabolism ; Immunoprecipitation ; Ischemia ; Isoquinolines - pharmacology ; Mice ; Microscopy, Fluorescence ; Mitogen-Activated Protein Kinase 1 - metabolism ; Mitogen-Activated Protein Kinase 3 - metabolism ; Octoxynol - pharmacology ; Phosphoprotein Phosphatases - metabolism ; Phosphorylation ; Phosphotyrosine - chemistry ; Protein Binding ; Protein Isoforms ; Protein Phosphatase 1 ; Protein Transport ; Protein Tyrosine Phosphatases - metabolism ; Rats ; Signal Transduction ; src-Family Kinases - metabolism</subject><ispartof>The Journal of biological chemistry, 2005-03, Vol.280 (9), p.7941-7948</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c391t-d5666c7d55926ad4c9364d46813ef21c78103e0155cf2c3571ddbd192c985e5e3</citedby><cites>FETCH-LOGICAL-c391t-d5666c7d55926ad4c9364d46813ef21c78103e0155cf2c3571ddbd192c985e5e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15618229$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Wei</creatorcontrib><creatorcontrib>Hertzberg, Elliot L</creatorcontrib><creatorcontrib>Spray, David C</creatorcontrib><title>Regulation of Connexin43-Protein Binding in Astrocytes in Response to Chemical Ischemia/Hypoxia</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>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 β-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 âdephosphorylatedâ
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.</description><subject>Animals</subject><subject>Astrocytes - metabolism</subject><subject>Astrocytes - pathology</subject><subject>Blotting, Western</subject><subject>Cell Cycle Proteins - metabolism</subject><subject>Cells, Cultured</subject><subject>Coloring Agents - pharmacology</subject><subject>Connexin 43 - genetics</subject><subject>Connexin 43 - physiology</subject><subject>Cytoskeleton - metabolism</subject><subject>Detergents - pharmacology</subject><subject>Dual Specificity Phosphatase 1</subject><subject>Gap Junctions</subject><subject>Hydrogen-Ion Concentration</subject><subject>Hypoxia</subject><subject>Immediate-Early Proteins - metabolism</subject><subject>Immunoprecipitation</subject><subject>Ischemia</subject><subject>Isoquinolines - pharmacology</subject><subject>Mice</subject><subject>Microscopy, Fluorescence</subject><subject>Mitogen-Activated Protein Kinase 1 - metabolism</subject><subject>Mitogen-Activated Protein Kinase 3 - metabolism</subject><subject>Octoxynol - pharmacology</subject><subject>Phosphoprotein Phosphatases - metabolism</subject><subject>Phosphorylation</subject><subject>Phosphotyrosine - chemistry</subject><subject>Protein Binding</subject><subject>Protein Isoforms</subject><subject>Protein Phosphatase 1</subject><subject>Protein Transport</subject><subject>Protein Tyrosine Phosphatases - metabolism</subject><subject>Rats</subject><subject>Signal Transduction</subject><subject>src-Family Kinases - metabolism</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkMtLAzEQh4MoWh9Xj7J48LY1k8ductTio1BRRMFb2CazbWSb1M0W2__eLS04l_kNfDMwHyGXQIdAS3H7PbXDFwFUCsUoPSADoIrnXMLXIRlQyiDXTKoTcprSN-1LaDgmJyALUIzpATHvOFs1VedjyGKdjWIIuPZB8PytjR36kN374HyYZX28S10b7abDtJ3eMS1jSJh1MRvNceFt1WTjZLexun3eLOPaV-fkqK6ahBf7fkY-Hx8-Rs_55PVpPLqb5JZr6HIni6KwpZNSs6JywmpeCCcKBRxrBrZUQDlSkNLWzHJZgnNTB5pZrSRK5GfkZnd32cafFabOLHyy2DRVwLhKBkrFi_7pHhzuQNvGlFqszbL1i6rdGKBmq9T0Ss2_0n7han95NV2g-8f3DnvgegfM_Wz-61s0Ux-3FgxT1GhTagH8Dxt3fRE</recordid><startdate>20050304</startdate><enddate>20050304</enddate><creator>Li, Wei</creator><creator>Hertzberg, Elliot L</creator><creator>Spray, David C</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TK</scope></search><sort><creationdate>20050304</creationdate><title>Regulation of Connexin43-Protein Binding in Astrocytes in Response to Chemical Ischemia/Hypoxia</title><author>Li, Wei ; Hertzberg, Elliot L ; Spray, David C</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c391t-d5666c7d55926ad4c9364d46813ef21c78103e0155cf2c3571ddbd192c985e5e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Animals</topic><topic>Astrocytes - metabolism</topic><topic>Astrocytes - pathology</topic><topic>Blotting, Western</topic><topic>Cell Cycle Proteins - metabolism</topic><topic>Cells, Cultured</topic><topic>Coloring Agents - pharmacology</topic><topic>Connexin 43 - genetics</topic><topic>Connexin 43 - physiology</topic><topic>Cytoskeleton - metabolism</topic><topic>Detergents - pharmacology</topic><topic>Dual Specificity Phosphatase 1</topic><topic>Gap Junctions</topic><topic>Hydrogen-Ion Concentration</topic><topic>Hypoxia</topic><topic>Immediate-Early Proteins - metabolism</topic><topic>Immunoprecipitation</topic><topic>Ischemia</topic><topic>Isoquinolines - pharmacology</topic><topic>Mice</topic><topic>Microscopy, Fluorescence</topic><topic>Mitogen-Activated Protein Kinase 1 - metabolism</topic><topic>Mitogen-Activated Protein Kinase 3 - metabolism</topic><topic>Octoxynol - pharmacology</topic><topic>Phosphoprotein Phosphatases - metabolism</topic><topic>Phosphorylation</topic><topic>Phosphotyrosine - chemistry</topic><topic>Protein Binding</topic><topic>Protein Isoforms</topic><topic>Protein Phosphatase 1</topic><topic>Protein Transport</topic><topic>Protein Tyrosine Phosphatases - metabolism</topic><topic>Rats</topic><topic>Signal Transduction</topic><topic>src-Family Kinases - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Li, Wei</creatorcontrib><creatorcontrib>Hertzberg, Elliot L</creatorcontrib><creatorcontrib>Spray, David C</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Neurosciences Abstracts</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Li, Wei</au><au>Hertzberg, Elliot L</au><au>Spray, David C</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Regulation of Connexin43-Protein Binding in Astrocytes in Response to Chemical Ischemia/Hypoxia</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2005-03-04</date><risdate>2005</risdate><volume>280</volume><issue>9</issue><spage>7941</spage><epage>7948</epage><pages>7941-7948</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><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 β-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 âdephosphorylatedâ
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.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>15618229</pmid><doi>10.1074/jbc.M410548200</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| language | eng |
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| source | PubMed Central (Open Access); MEDLINE; Alma/SFX Local Collection; EZB Electronic Journals Library |
| subjects | Animals Astrocytes - metabolism Astrocytes - pathology Blotting, Western Cell Cycle Proteins - metabolism Cells, Cultured Coloring Agents - pharmacology Connexin 43 - genetics Connexin 43 - physiology Cytoskeleton - metabolism Detergents - pharmacology Dual Specificity Phosphatase 1 Gap Junctions Hydrogen-Ion Concentration Hypoxia Immediate-Early Proteins - metabolism Immunoprecipitation Ischemia Isoquinolines - pharmacology Mice Microscopy, Fluorescence Mitogen-Activated Protein Kinase 1 - metabolism Mitogen-Activated Protein Kinase 3 - metabolism Octoxynol - pharmacology Phosphoprotein Phosphatases - metabolism Phosphorylation Phosphotyrosine - chemistry Protein Binding Protein Isoforms Protein Phosphatase 1 Protein Transport Protein Tyrosine Phosphatases - metabolism Rats Signal Transduction src-Family Kinases - metabolism |
| title | Regulation of Connexin43-Protein Binding in Astrocytes in Response to Chemical Ischemia/Hypoxia |
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