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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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Two-dimensional phosphotryptic peptide analysis of wt and mutant forms of Cx43 isolated from cells metabolically labeled with 32Pi. Phosphotryptic peptides were resolved by electrophoresis (dimension 1) followed by ascending chromatography (dimension 2). Boxes show phosphopeptides tentatively ascribed to the Y247 or the Y265 containing peptides.
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fig5: Two-dimensional phosphotryptic peptide analysis of wt and mutant forms of Cx43 isolated from cells metabolically labeled with 32Pi. Phosphotryptic peptides were resolved by electrophoresis (dimension 1) followed by ascending chromatography (dimension 2). Boxes show phosphopeptides tentatively ascribed to the Y247 or the Y265 containing peptides.

Mentions: To further characterize Cx43 phosphorylation in vivo, we employed two-dimensional phosphotryptic peptide analysis. The peptide maps obtained were complex due to potential phosphorylation on multiple serine and tyrosine sites (Fig. 5) . Although the maps did not provide additional insight into the mechanism(s) of v-Src–induced Cx43 phosphorylation, some differences were apparent, which were consistent with the mutations introduced in Cx43. Three phosphopeptides migrated in the region of peptide 4 (Fig. 1 D) and were assigned tentatively to the Y247 peptide (Fig. 1D, boxed area). These peptides migrated similarly in the Y247F and Y247F/Y265F Cx43 mutants and had a different arrangement in the wt and Y265F proteins. This difference in migration is consistent with the loss of a tyrosine phosphorylation site and the substitution of the more hydrophobic Phe residue at the Y247 site in the Y247F and Y247F/Y265F mutants. Phosphopeptides ascribed to the Y265 peptide are also boxed. The migration patterns here are more similar in the Y265F and Y247F/Y265F mutants, which have a Phe substitution on the Y265 peptide. These patterns differ from those obtained with the wt and Y247F Cx43 proteins as observed previously in our in vitro studies (Fig. 1 D).


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)

Two-dimensional phosphotryptic peptide analysis of wt and mutant forms of Cx43 isolated from cells metabolically labeled with 32Pi. Phosphotryptic peptides were resolved by electrophoresis (dimension 1) followed by ascending chromatography (dimension 2). Boxes show phosphopeptides tentatively ascribed to the Y247 or the Y265 containing peptides.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Two-dimensional phosphotryptic peptide analysis of wt and mutant forms of Cx43 isolated from cells metabolically labeled with 32Pi. Phosphotryptic peptides were resolved by electrophoresis (dimension 1) followed by ascending chromatography (dimension 2). Boxes show phosphopeptides tentatively ascribed to the Y247 or the Y265 containing peptides.
Mentions: To further characterize Cx43 phosphorylation in vivo, we employed two-dimensional phosphotryptic peptide analysis. The peptide maps obtained were complex due to potential phosphorylation on multiple serine and tyrosine sites (Fig. 5) . Although the maps did not provide additional insight into the mechanism(s) of v-Src–induced Cx43 phosphorylation, some differences were apparent, which were consistent with the mutations introduced in Cx43. Three phosphopeptides migrated in the region of peptide 4 (Fig. 1 D) and were assigned tentatively to the Y247 peptide (Fig. 1D, boxed area). These peptides migrated similarly in the Y247F and Y247F/Y265F Cx43 mutants and had a different arrangement in the wt and Y265F proteins. This difference in migration is consistent with the loss of a tyrosine phosphorylation site and the substitution of the more hydrophobic Phe residue at the Y247 site in the Y247F and Y247F/Y265F mutants. Phosphopeptides ascribed to the Y265 peptide are also boxed. The migration patterns here are more similar in the Y265F and Y247F/Y265F mutants, which have a Phe substitution on the Y265 peptide. These patterns differ from those obtained with the wt and Y247F Cx43 proteins as observed previously in our in vitro studies (Fig. 1 D).

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