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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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Fluorescent images from microinjection of the v-Src– expressing cell clones. Fluorescent images were captured and recorded at 4–6 min after microinjections of single cells with Lucifer yellow dye.
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fig6: Fluorescent images from microinjection of the v-Src– expressing cell clones. Fluorescent images were captured and recorded at 4–6 min after microinjections of single cells with Lucifer yellow dye.

Mentions: To determine the functional significance of phosphorylation at the Y247 or Y265 site in Cx43, GJC was measured in the clones by the transfer of Lucifer yellow dye 1 min after injection of the dye into the parental cells. To address the possibility of clonal variability, GJC was determined for two independently isolated clones for each experimental group. In the absence of v-Src, the substitution of Phe for Tyr at residues 247 or 265 did not disrupt the ability of Cx43 to establish functional gap junction channels (Table I). However, the expression of v-Src induced a dramatic disruption of dye transfer (∼14-fold reduction in degree of coupling and ∼5-fold change in the incidence of coupling) in cell clones coexpressing wt Cx43 compared with cell clones expressing wt Cx43 without v-Src (Table I, P < 0.01). This observation was consistent with previous results that showed that GJC was disrupted in cells containing the v-Src oncoprotein (Atkinson et al., 1981; Crow et al., 1990; Filson et al., 1990; Kurata and Lau, 1994). Importantly, dye transfer was unperturbed (P > 0.05) by the expression of v-Src in cells expressing the Y247F Cx43 mutant (247FS1 and 247FS2) or the double Cx43 mutant (dbS1 and dbS2). In addition, cells expressing v-Src together with the Y265F Cx43 mutant (265FS1 and 265FS2) exhibited high levels of GJC. Thus, v-Src either failed to disrupt, or induced a low level (∼20%) of disruption of, GJC in the cells coexpressing the Y247F and/or Y265F Cx43 mutants. Furthermore, the incidence of gap junctional coupling in cell clones expressing the Cx43 mutants in the presence or absence of v-Src was 100% in nearly all cases (Table I) in contrast to the cells expressing wt Cx43 and v-Src (18–19% coupled). The dramatic reduction of GJC in the cells expressing wt Cx43 with v-Src but not in the cells expressing Cx43 tyrosine mutants and v-Src, is also illustrated by fluorescent images captured at 4–6 min after the microinjection of single cells (Fig. 6) . These dye transfer results obtained by microinjection of single cells were substantiated by the scrape-loading technique (unpublished data). The observation that the Y247F and Y265F substitutions rendered Cx43 resistant to the disruption of GJC by v-Src strongly suggested that phosphorylation of Cx43 at these specific tyrosine sites represents a key mechanism underlying v-Src's ability to disrupt GJC in these 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)

Fluorescent images from microinjection of the v-Src– expressing cell clones. Fluorescent images were captured and recorded at 4–6 min after microinjections of single cells with Lucifer yellow dye.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Fluorescent images from microinjection of the v-Src– expressing cell clones. Fluorescent images were captured and recorded at 4–6 min after microinjections of single cells with Lucifer yellow dye.
Mentions: To determine the functional significance of phosphorylation at the Y247 or Y265 site in Cx43, GJC was measured in the clones by the transfer of Lucifer yellow dye 1 min after injection of the dye into the parental cells. To address the possibility of clonal variability, GJC was determined for two independently isolated clones for each experimental group. In the absence of v-Src, the substitution of Phe for Tyr at residues 247 or 265 did not disrupt the ability of Cx43 to establish functional gap junction channels (Table I). However, the expression of v-Src induced a dramatic disruption of dye transfer (∼14-fold reduction in degree of coupling and ∼5-fold change in the incidence of coupling) in cell clones coexpressing wt Cx43 compared with cell clones expressing wt Cx43 without v-Src (Table I, P < 0.01). This observation was consistent with previous results that showed that GJC was disrupted in cells containing the v-Src oncoprotein (Atkinson et al., 1981; Crow et al., 1990; Filson et al., 1990; Kurata and Lau, 1994). Importantly, dye transfer was unperturbed (P > 0.05) by the expression of v-Src in cells expressing the Y247F Cx43 mutant (247FS1 and 247FS2) or the double Cx43 mutant (dbS1 and dbS2). In addition, cells expressing v-Src together with the Y265F Cx43 mutant (265FS1 and 265FS2) exhibited high levels of GJC. Thus, v-Src either failed to disrupt, or induced a low level (∼20%) of disruption of, GJC in the cells coexpressing the Y247F and/or Y265F Cx43 mutants. Furthermore, the incidence of gap junctional coupling in cell clones expressing the Cx43 mutants in the presence or absence of v-Src was 100% in nearly all cases (Table I) in contrast to the cells expressing wt Cx43 and v-Src (18–19% coupled). The dramatic reduction of GJC in the cells expressing wt Cx43 with v-Src but not in the cells expressing Cx43 tyrosine mutants and v-Src, is also illustrated by fluorescent images captured at 4–6 min after the microinjection of single cells (Fig. 6) . These dye transfer results obtained by microinjection of single cells were substantiated by the scrape-loading technique (unpublished data). The observation that the Y247F and Y265F substitutions rendered Cx43 resistant to the disruption of GJC by v-Src strongly suggested that phosphorylation of Cx43 at these specific tyrosine sites represents a key mechanism underlying v-Src's ability to disrupt GJC in these 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