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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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Localization of wt or mutant Cx43 to the plasma membrane. Top, cells lacking v-Src; bottom, cells expressing v-Src. Cells were grown to subconfluence on coverslips before being fixed and permeabilized. Cx43CT368 antiserum was used to detect Cx43. Arrowheads indicate the positions of some Cx43 gap junction plaques.
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fig3: Localization of wt or mutant Cx43 to the plasma membrane. Top, cells lacking v-Src; bottom, cells expressing v-Src. Cells were grown to subconfluence on coverslips before being fixed and permeabilized. Cx43CT368 antiserum was used to detect Cx43. Arrowheads indicate the positions of some Cx43 gap junction plaques.

Mentions: Immunofluorescence microscopy of Cx43 was performed to determine whether the conservative substitution of Phe at the Y247 and/or Y265 site or the expression of v-Src in the cells disrupted the assembly or maintenance of Cx43 gap junction plaques. Cx43 gap junction plaques were detected in the plasma membranes of wtC1 cells and in the 247FC1, 265FC1, and dbC1 cells (Fig. 3 , top, arrowheads). Wt and mutant Cx43 were also localized to the plasma membrane of the clones coexpressing v-Src (Fig. 3, bottom panel). The v-Src–expressing cells were more rounded than the non-Src cells, and some gap junctions were not clearly in focus in a particular focal plane. All of the clones also exhibited Cx43-specific reactions in intracellular locations. These results indicated that the assembly of the Cx43 mutants into gap junction plaques was not largely disrupted by the introduction of phosphorylation site mutations or by the coexpression of v-Src. Thus, wt and mutant Cx43 have the potential to establish functionally active gap junction channels, and no gross loss of gap junctional plaques was apparent in our cells stably expressing v-Src.


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)

Localization of wt or mutant Cx43 to the plasma membrane. Top, cells lacking v-Src; bottom, cells expressing v-Src. Cells were grown to subconfluence on coverslips before being fixed and permeabilized. Cx43CT368 antiserum was used to detect Cx43. Arrowheads indicate the positions of some Cx43 gap junction plaques.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Localization of wt or mutant Cx43 to the plasma membrane. Top, cells lacking v-Src; bottom, cells expressing v-Src. Cells were grown to subconfluence on coverslips before being fixed and permeabilized. Cx43CT368 antiserum was used to detect Cx43. Arrowheads indicate the positions of some Cx43 gap junction plaques.
Mentions: Immunofluorescence microscopy of Cx43 was performed to determine whether the conservative substitution of Phe at the Y247 and/or Y265 site or the expression of v-Src in the cells disrupted the assembly or maintenance of Cx43 gap junction plaques. Cx43 gap junction plaques were detected in the plasma membranes of wtC1 cells and in the 247FC1, 265FC1, and dbC1 cells (Fig. 3 , top, arrowheads). Wt and mutant Cx43 were also localized to the plasma membrane of the clones coexpressing v-Src (Fig. 3, bottom panel). The v-Src–expressing cells were more rounded than the non-Src cells, and some gap junctions were not clearly in focus in a particular focal plane. All of the clones also exhibited Cx43-specific reactions in intracellular locations. These results indicated that the assembly of the Cx43 mutants into gap junction plaques was not largely disrupted by the introduction of phosphorylation site mutations or by the coexpression of v-Src. Thus, wt and mutant Cx43 have the potential to establish functionally active gap junction channels, and no gross loss of gap junctional plaques was apparent in our cells stably expressing v-Src.

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