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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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Phosphorylation of wt or mutant Cx43 in v-Src–expressing cells. (A) Immunoprecipitation of Cx43. Confluent cells were metabolically labeled with 32Pi. Cx43 was immunoprecipitated from cell lysates with Cx43CT368 antiserum under conditions of antigen excess. The positions of multiple isoforms of phosphorylated Cx43 are shown by brackets. (B) Phosphoamino acid content of Cx43 immunoprecipitated from the cell clones. Immunoprecipitated 32P- labeled Cx43 was acid hydrolyzed and separated in two dimensions as indicated. (C) pTyr content of Cx43 from the cell clones. Cells were grown to confluence and the same amount of Cx43CT368 antiserum was used to immunoprecipitate Cx43 from cell lysates under conditions of antigen excess. The pTyr content of Cx43 was detected with a pTyr antibody (top). Quantitation of pTyr in Cx43 is shown in the bottom part of the figure. The average relative amounts of pTyr of Cx43 were obtained from scans of the Cx43 images of the top and from a second experiment. The levels of pTyr of Cx43 from wtC1 and wtS2 were normalized to 0 and 100%, respectively.
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fig4: Phosphorylation of wt or mutant Cx43 in v-Src–expressing cells. (A) Immunoprecipitation of Cx43. Confluent cells were metabolically labeled with 32Pi. Cx43 was immunoprecipitated from cell lysates with Cx43CT368 antiserum under conditions of antigen excess. The positions of multiple isoforms of phosphorylated Cx43 are shown by brackets. (B) Phosphoamino acid content of Cx43 immunoprecipitated from the cell clones. Immunoprecipitated 32P- labeled Cx43 was acid hydrolyzed and separated in two dimensions as indicated. (C) pTyr content of Cx43 from the cell clones. Cells were grown to confluence and the same amount of Cx43CT368 antiserum was used to immunoprecipitate Cx43 from cell lysates under conditions of antigen excess. The pTyr content of Cx43 was detected with a pTyr antibody (top). Quantitation of pTyr in Cx43 is shown in the bottom part of the figure. The average relative amounts of pTyr of Cx43 were obtained from scans of the Cx43 images of the top and from a second experiment. The levels of pTyr of Cx43 from wtC1 and wtS2 were normalized to 0 and 100%, respectively.

Mentions: To examine the phosphorylation of Cx43 in cells expressing Cx43 and v-Src, the cells were metabolically labeled with 32Pi, and Cx43 was immunoprecipitated from the cell lysates with equal amounts of antibody (Fig. 4 A). Phosphorylated Cx43 from the different clones was present as isoforms migrating with different mobilities as observed previously (Crow et al., 1990; Filson et al., 1990; Laird et al., 1995). More importantly, the levels of phosphorylated Cx43 in the v-Src–expressing clones were markedly increased compared with the wtC1 cells that lacked v-Src. Given that the levels of Cx43 were similar among the cell clones (Fig. 2 B), these data suggested that v-Src upregulated the phosphorylation of Cx43, consistent with previous reports (Crow et al., 1990; Filson et al., 1990; Goldberg and Lau, 1993).


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)

Phosphorylation of wt or mutant Cx43 in v-Src–expressing cells. (A) Immunoprecipitation of Cx43. Confluent cells were metabolically labeled with 32Pi. Cx43 was immunoprecipitated from cell lysates with Cx43CT368 antiserum under conditions of antigen excess. The positions of multiple isoforms of phosphorylated Cx43 are shown by brackets. (B) Phosphoamino acid content of Cx43 immunoprecipitated from the cell clones. Immunoprecipitated 32P- labeled Cx43 was acid hydrolyzed and separated in two dimensions as indicated. (C) pTyr content of Cx43 from the cell clones. Cells were grown to confluence and the same amount of Cx43CT368 antiserum was used to immunoprecipitate Cx43 from cell lysates under conditions of antigen excess. The pTyr content of Cx43 was detected with a pTyr antibody (top). Quantitation of pTyr in Cx43 is shown in the bottom part of the figure. The average relative amounts of pTyr of Cx43 were obtained from scans of the Cx43 images of the top and from a second experiment. The levels of pTyr of Cx43 from wtC1 and wtS2 were normalized to 0 and 100%, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196463&req=5

fig4: Phosphorylation of wt or mutant Cx43 in v-Src–expressing cells. (A) Immunoprecipitation of Cx43. Confluent cells were metabolically labeled with 32Pi. Cx43 was immunoprecipitated from cell lysates with Cx43CT368 antiserum under conditions of antigen excess. The positions of multiple isoforms of phosphorylated Cx43 are shown by brackets. (B) Phosphoamino acid content of Cx43 immunoprecipitated from the cell clones. Immunoprecipitated 32P- labeled Cx43 was acid hydrolyzed and separated in two dimensions as indicated. (C) pTyr content of Cx43 from the cell clones. Cells were grown to confluence and the same amount of Cx43CT368 antiserum was used to immunoprecipitate Cx43 from cell lysates under conditions of antigen excess. The pTyr content of Cx43 was detected with a pTyr antibody (top). Quantitation of pTyr in Cx43 is shown in the bottom part of the figure. The average relative amounts of pTyr of Cx43 were obtained from scans of the Cx43 images of the top and from a second experiment. The levels of pTyr of Cx43 from wtC1 and wtS2 were normalized to 0 and 100%, respectively.
Mentions: To examine the phosphorylation of Cx43 in cells expressing Cx43 and v-Src, the cells were metabolically labeled with 32Pi, and Cx43 was immunoprecipitated from the cell lysates with equal amounts of antibody (Fig. 4 A). Phosphorylated Cx43 from the different clones was present as isoforms migrating with different mobilities as observed previously (Crow et al., 1990; Filson et al., 1990; Laird et al., 1995). More importantly, the levels of phosphorylated Cx43 in the v-Src–expressing clones were markedly increased compared with the wtC1 cells that lacked v-Src. Given that the levels of Cx43 were similar among the cell clones (Fig. 2 B), these data suggested that v-Src upregulated the phosphorylation of Cx43, consistent with previous reports (Crow et al., 1990; Filson et al., 1990; Goldberg and Lau, 1993).

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