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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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The role of MAP kinase in the disruption of GJC by v-Src. Cells were untreated or treated with EGF or the MEK inhibitor before being lysed. The same amount of whole cell lysate was loaded for each sample. Activated MAP kinase was detected with an antibody recognizing phosphorylated MAP kinase. Lane 1, untreated wtC1; lane 2, wtC1 treated with 100 ng/ml EGF for 2 min; lane 3, untreated wtS2; and lanes 4 and 5, duplicate plates of wtS2 treated with 100 μM MEK inhibitor for 1 h. The same membrane used to detect active MAP kinase was stripped and reprobed with an antibody recognizing all isoforms of p42 MAP kinase. The ratios of active MAP kinase to total MAP kinase were determined and normalized to the untreated wtS2 cells set as 100%. Average GJC values obtained from multiple plates are reported for lanes 1, 3, and 4, and the GJC value from a single plate that was also used to measure the level of activated MAP kinase is shown for lane 5. The number of injections contributing to the determination of GJC is shown in brackets. Active MAP kinase was reduced to 5 and 2% of the starting level by the 1 h treatment with MEK inhibitor as shown in lanes 4 and 5, respectively.
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fig7: The role of MAP kinase in the disruption of GJC by v-Src. Cells were untreated or treated with EGF or the MEK inhibitor before being lysed. The same amount of whole cell lysate was loaded for each sample. Activated MAP kinase was detected with an antibody recognizing phosphorylated MAP kinase. Lane 1, untreated wtC1; lane 2, wtC1 treated with 100 ng/ml EGF for 2 min; lane 3, untreated wtS2; and lanes 4 and 5, duplicate plates of wtS2 treated with 100 μM MEK inhibitor for 1 h. The same membrane used to detect active MAP kinase was stripped and reprobed with an antibody recognizing all isoforms of p42 MAP kinase. The ratios of active MAP kinase to total MAP kinase were determined and normalized to the untreated wtS2 cells set as 100%. Average GJC values obtained from multiple plates are reported for lanes 1, 3, and 4, and the GJC value from a single plate that was also used to measure the level of activated MAP kinase is shown for lane 5. The number of injections contributing to the determination of GJC is shown in brackets. Active MAP kinase was reduced to 5 and 2% of the starting level by the 1 h treatment with MEK inhibitor as shown in lanes 4 and 5, respectively.

Mentions: WtS2 cells were treated with 100 μM MEK inhibitor for 1 h. Interestingly, this treatment failed to restore GJC. Untreated wtS2 cells communicated to an average of 1.0 neighboring cell compared with an average of 0.9 adjacent cells after treatment (Fig. 7 , lanes 3 and 4). The wtC1 control cells, expressing wt Cx43 without v-Src, communicated to an average of 13.1 adjacent cells (Fig. 7, lane 1). To confirm that the MEK inhibitor treatment inhibited MAP kinase activation, the levels of active MAP kinase were determined from equivalent amounts of whole cell lysate by immunoblotting with an antibody recognizing the phosphorylated, activated form of MAP kinase. As a positive control, wtC1 cells treated with EGF showed increased levels of active MAP kinase compared with the untreated cells (Fig. 7, lanes 2 and 1, respectively). Activated MAP kinase was detected in the wtS2 cells treated with DMSO alone; however, treatment of these cells with the MEK inhibitor reduced activated MAP kinase to ∼5% of the untreated wtS2 cells. Fig. 7, lane 5, represents a single plate of wtS2 cells that communicated to an average of 0.5 neighboring cells after treatment with the MEK inhibitor. Immunoblotting the cell lysate from this single plate demonstrated that MAP kinase was reduced markedly to ∼2% of the untreated wtS2 cells. In addition, we determined that the wtS2 cells treated with 100 μM MEK inhibitor for 4 h communicated to an average of 1.0 ± 0.2 (n = 20) neighboring cells and also showed significantly reduced levels of activated MAP kinase (unpublished data). Taken together, these observations strongly suggested that activated MAP kinase did not play a role in the disruption of GJC induced by v-Src in our cells.


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)

The role of MAP kinase in the disruption of GJC by v-Src. Cells were untreated or treated with EGF or the MEK inhibitor before being lysed. The same amount of whole cell lysate was loaded for each sample. Activated MAP kinase was detected with an antibody recognizing phosphorylated MAP kinase. Lane 1, untreated wtC1; lane 2, wtC1 treated with 100 ng/ml EGF for 2 min; lane 3, untreated wtS2; and lanes 4 and 5, duplicate plates of wtS2 treated with 100 μM MEK inhibitor for 1 h. The same membrane used to detect active MAP kinase was stripped and reprobed with an antibody recognizing all isoforms of p42 MAP kinase. The ratios of active MAP kinase to total MAP kinase were determined and normalized to the untreated wtS2 cells set as 100%. Average GJC values obtained from multiple plates are reported for lanes 1, 3, and 4, and the GJC value from a single plate that was also used to measure the level of activated MAP kinase is shown for lane 5. The number of injections contributing to the determination of GJC is shown in brackets. Active MAP kinase was reduced to 5 and 2% of the starting level by the 1 h treatment with MEK inhibitor as shown in lanes 4 and 5, respectively.
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Related In: Results  -  Collection

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

fig7: The role of MAP kinase in the disruption of GJC by v-Src. Cells were untreated or treated with EGF or the MEK inhibitor before being lysed. The same amount of whole cell lysate was loaded for each sample. Activated MAP kinase was detected with an antibody recognizing phosphorylated MAP kinase. Lane 1, untreated wtC1; lane 2, wtC1 treated with 100 ng/ml EGF for 2 min; lane 3, untreated wtS2; and lanes 4 and 5, duplicate plates of wtS2 treated with 100 μM MEK inhibitor for 1 h. The same membrane used to detect active MAP kinase was stripped and reprobed with an antibody recognizing all isoforms of p42 MAP kinase. The ratios of active MAP kinase to total MAP kinase were determined and normalized to the untreated wtS2 cells set as 100%. Average GJC values obtained from multiple plates are reported for lanes 1, 3, and 4, and the GJC value from a single plate that was also used to measure the level of activated MAP kinase is shown for lane 5. The number of injections contributing to the determination of GJC is shown in brackets. Active MAP kinase was reduced to 5 and 2% of the starting level by the 1 h treatment with MEK inhibitor as shown in lanes 4 and 5, respectively.
Mentions: WtS2 cells were treated with 100 μM MEK inhibitor for 1 h. Interestingly, this treatment failed to restore GJC. Untreated wtS2 cells communicated to an average of 1.0 neighboring cell compared with an average of 0.9 adjacent cells after treatment (Fig. 7 , lanes 3 and 4). The wtC1 control cells, expressing wt Cx43 without v-Src, communicated to an average of 13.1 adjacent cells (Fig. 7, lane 1). To confirm that the MEK inhibitor treatment inhibited MAP kinase activation, the levels of active MAP kinase were determined from equivalent amounts of whole cell lysate by immunoblotting with an antibody recognizing the phosphorylated, activated form of MAP kinase. As a positive control, wtC1 cells treated with EGF showed increased levels of active MAP kinase compared with the untreated cells (Fig. 7, lanes 2 and 1, respectively). Activated MAP kinase was detected in the wtS2 cells treated with DMSO alone; however, treatment of these cells with the MEK inhibitor reduced activated MAP kinase to ∼5% of the untreated wtS2 cells. Fig. 7, lane 5, represents a single plate of wtS2 cells that communicated to an average of 0.5 neighboring cells after treatment with the MEK inhibitor. Immunoblotting the cell lysate from this single plate demonstrated that MAP kinase was reduced markedly to ∼2% of the untreated wtS2 cells. In addition, we determined that the wtS2 cells treated with 100 μM MEK inhibitor for 4 h communicated to an average of 1.0 ± 0.2 (n = 20) neighboring cells and also showed significantly reduced levels of activated MAP kinase (unpublished data). Taken together, these observations strongly suggested that activated MAP kinase did not play a role in the disruption of GJC induced by v-Src in our cells.

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