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v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication.

Lin R, Warn-Cramer BJ, Kurata WE, Lau AF - J. Cell Biol. (2001)

Bottom Line: When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo.Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src.We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.

View Article: PubMed Central - PubMed

Affiliation: Molecular Carcinogenesis Section, Cancer Research Center of Hawaii, Honolulu, HI 96813, USA.

ABSTRACT
The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.

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Levels of v-Src kinase activity and Cx43 in the cell clones. (A) In vitro v-Src protein kinase activities. The same amounts of anti-Src TBR serum 6-1-4 were reacted with equal amounts of cell lysate from each clone. The ability of v-Src to phosphorylate the heavy chain of IgG in an immune complex kinase assay was examined. Clone wtC1 infected by the pLXSH vector alone served as a negative control. Asterisks indicate the clones chosen for further biochemical analysis. (B) Expression of Cx43. 40 μg of whole cell lysates was resolved by SDS-PAGE and electrotransferred to Immobilon-P membranes.
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fig2: Levels of v-Src kinase activity and Cx43 in the cell clones. (A) In vitro v-Src protein kinase activities. The same amounts of anti-Src TBR serum 6-1-4 were reacted with equal amounts of cell lysate from each clone. The ability of v-Src to phosphorylate the heavy chain of IgG in an immune complex kinase assay was examined. Clone wtC1 infected by the pLXSH vector alone served as a negative control. Asterisks indicate the clones chosen for further biochemical analysis. (B) Expression of Cx43. 40 μg of whole cell lysates was resolved by SDS-PAGE and electrotransferred to Immobilon-P membranes.

Mentions: Wt and mutant Cx43 were expressed in Cx43 knockout (KO) cells and clones with similar levels of GJC were infected with the v-Src retrovirus. To characterize the expression of v-Src in the Cx43 reexpressing cells, an in vitro kinase assay was performed on the morphologically transformed cell clones infected by pLvsrcSH. Equal amounts of antibody were used to immunoprecipitate v-Src from equal amounts of cell lysate, followed by an in vitro kinase assay of immunoprecipitated v-Src. As expected, the negative control wtC1 cells showed no v-Src kinase activity (Fig. 2 A, first lane). All of the other clones exhibited v-Src kinase activity with some differences in the levels of activity. One clone (indicated by an asterisk) from each set of infections by wt or mutant pBABE-cx43 and pLvsrcSH retroviruses with the most similar levels of v-Src kinase activity was chosen for further biochemical analysis. The selected clones were wtS2, 247FS2, 265FS1, and dbS2. The wtC1 clone was used as a negative control.


v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication.

Lin R, Warn-Cramer BJ, Kurata WE, Lau AF - J. Cell Biol. (2001)

Levels of v-Src kinase activity and Cx43 in the cell clones. (A) In vitro v-Src protein kinase activities. The same amounts of anti-Src TBR serum 6-1-4 were reacted with equal amounts of cell lysate from each clone. The ability of v-Src to phosphorylate the heavy chain of IgG in an immune complex kinase assay was examined. Clone wtC1 infected by the pLXSH vector alone served as a negative control. Asterisks indicate the clones chosen for further biochemical analysis. (B) Expression of Cx43. 40 μg of whole cell lysates was resolved by SDS-PAGE and electrotransferred to Immobilon-P membranes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196463&req=5

fig2: Levels of v-Src kinase activity and Cx43 in the cell clones. (A) In vitro v-Src protein kinase activities. The same amounts of anti-Src TBR serum 6-1-4 were reacted with equal amounts of cell lysate from each clone. The ability of v-Src to phosphorylate the heavy chain of IgG in an immune complex kinase assay was examined. Clone wtC1 infected by the pLXSH vector alone served as a negative control. Asterisks indicate the clones chosen for further biochemical analysis. (B) Expression of Cx43. 40 μg of whole cell lysates was resolved by SDS-PAGE and electrotransferred to Immobilon-P membranes.
Mentions: Wt and mutant Cx43 were expressed in Cx43 knockout (KO) cells and clones with similar levels of GJC were infected with the v-Src retrovirus. To characterize the expression of v-Src in the Cx43 reexpressing cells, an in vitro kinase assay was performed on the morphologically transformed cell clones infected by pLvsrcSH. Equal amounts of antibody were used to immunoprecipitate v-Src from equal amounts of cell lysate, followed by an in vitro kinase assay of immunoprecipitated v-Src. As expected, the negative control wtC1 cells showed no v-Src kinase activity (Fig. 2 A, first lane). All of the other clones exhibited v-Src kinase activity with some differences in the levels of activity. One clone (indicated by an asterisk) from each set of infections by wt or mutant pBABE-cx43 and pLvsrcSH retroviruses with the most similar levels of v-Src kinase activity was chosen for further biochemical analysis. The selected clones were wtS2, 247FS2, 265FS1, and dbS2. The wtC1 clone was used as a negative control.

Bottom Line: When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo.Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src.We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.

View Article: PubMed Central - PubMed

Affiliation: Molecular Carcinogenesis Section, Cancer Research Center of Hawaii, Honolulu, HI 96813, USA.

ABSTRACT
The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.

Show MeSH
Related in: MedlinePlus