Limits...
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.

Show MeSH

Related in: MedlinePlus

Analysis of the phosphorylation of GST–Cx43CT by activated Src in vitro. (A) Amino acid sequence of two predicted tryptic peptides of Cx43CT (S244-K258 and Y265-K287) that contain the putative pTyr sites (Y247 and Y265) in the phosphorylated GST–Cx43CT fusion protein. Cx43CT fused to GST begins at V236 in the cytoplasmic tail of Cx43. The amino acids deleted in the Cx43CT deletion mutant are bracketed (P253LSP256). (B) Two-dimensional phosphotryptic peptide analysis of GST fusion proteins with wt or the Cx43CT deletion mutation phosphorylated by activated Src. Phosphotryptic peptides were resolved by electrophoresis (dimension 1) followed by ascending chromatography (dimension 2). Major phosphotryptic peptides are numbered. (C) Phosphoamino acid content of wt or site-directed mutant GST–Cx43CT phosphorylated by activated Src. Equal amounts of the GST–Cx43CT substrates were phosphorylated by Src, acid-hydrolyzed, and separated by electrophoresis at pH 1.9 (dimension 1) followed by electrophoresis at pH 3.5 (dimension 2). The migration positions of the unlabeled pSer, pThr, and pTyr standards are indicated. (D) Phosphotryptic peptide maps of wt and mutant GST–Cx43CT phosphorylated by activated Src. Resolved peptides were from one type of GST–Cx43CT or a mixture of wt and the Y265F GST–Cx43CT mutant. Peptides are numbered as in B.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196463&req=5

fig1: Analysis of the phosphorylation of GST–Cx43CT by activated Src in vitro. (A) Amino acid sequence of two predicted tryptic peptides of Cx43CT (S244-K258 and Y265-K287) that contain the putative pTyr sites (Y247 and Y265) in the phosphorylated GST–Cx43CT fusion protein. Cx43CT fused to GST begins at V236 in the cytoplasmic tail of Cx43. The amino acids deleted in the Cx43CT deletion mutant are bracketed (P253LSP256). (B) Two-dimensional phosphotryptic peptide analysis of GST fusion proteins with wt or the Cx43CT deletion mutation phosphorylated by activated Src. Phosphotryptic peptides were resolved by electrophoresis (dimension 1) followed by ascending chromatography (dimension 2). Major phosphotryptic peptides are numbered. (C) Phosphoamino acid content of wt or site-directed mutant GST–Cx43CT phosphorylated by activated Src. Equal amounts of the GST–Cx43CT substrates were phosphorylated by Src, acid-hydrolyzed, and separated by electrophoresis at pH 1.9 (dimension 1) followed by electrophoresis at pH 3.5 (dimension 2). The migration positions of the unlabeled pSer, pThr, and pTyr standards are indicated. (D) Phosphotryptic peptide maps of wt and mutant GST–Cx43CT phosphorylated by activated Src. Resolved peptides were from one type of GST–Cx43CT or a mixture of wt and the Y265F GST–Cx43CT mutant. Peptides are numbered as in B.

Mentions: Site-directed mutagenesis indicated that Y265 of Cx43 was a primary Tyr site targeted by v-Src upon coexpression of these proteins in Xenopus oocytes (Swenson et al., 1990). This study did not attempt to identify possible additional tyrosine phosphorylation sites in Cx43. However, the observation of two distinct pTyr-containing phosphopeptides of Cx43 in Rat-1 v-Src cells suggested that v-Src may phosphorylate Cx43 at an additional Tyr site(s) (Kurata and Lau, 1994). Because Y247, like Y265, is in a tyrosine kinase phosphorylation consensus sequence, we examined whether Y247 is another site targeted by activated Src. Glutathione S-transferase (GST) fusion proteins with wt Cx43 COOH-terminal tail (CT) or a deletion mutant that lacks residues P253-P256 (Kanemitsu et al., 1997) of Cx43CT were phosphorylated by activated Src in vitro and subjected to two-dimensional phosphotryptic peptide analysis. Since the P253-P256 deletion is located in tryptic peptide S244-K258 (Fig. 1 A), it was expected to alter the migration of this peptide. Phosphopeptide 4 detected in the map of phosphorylated wt GST–Cx43CT disappeared from the map of the phosphorylated deletion mutant GST–Cx43CT with the concomitant appearance of a slower migrating phosphopeptide, labeled 4′ (Fig. 1 B). Because this was the only difference observed in the peptide maps, we concluded that peptide 4 must be peptide S244-K258, whereas peptide 4′ represents the same peptide with the P253-P256 deletion. This conclusion was supported by a calculation of the relative peptide mobilities, which indicated that the peptide from the deletion mutant would migrate more slowly than the wt peptide in the second chromatographic dimension (Boyle et al., 1991). Because the deletion localizes in a phosphopeptide that contains a single Tyr site (Y247) (Fig. 1 A) and Src phosphorylates GST–Cx43CT only on Tyr (see below), Y247 represents another Tyr site in Cx43 that is phosphorylated by Src in vitro.


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)

Analysis of the phosphorylation of GST–Cx43CT by activated Src in vitro. (A) Amino acid sequence of two predicted tryptic peptides of Cx43CT (S244-K258 and Y265-K287) that contain the putative pTyr sites (Y247 and Y265) in the phosphorylated GST–Cx43CT fusion protein. Cx43CT fused to GST begins at V236 in the cytoplasmic tail of Cx43. The amino acids deleted in the Cx43CT deletion mutant are bracketed (P253LSP256). (B) Two-dimensional phosphotryptic peptide analysis of GST fusion proteins with wt or the Cx43CT deletion mutation phosphorylated by activated Src. Phosphotryptic peptides were resolved by electrophoresis (dimension 1) followed by ascending chromatography (dimension 2). Major phosphotryptic peptides are numbered. (C) Phosphoamino acid content of wt or site-directed mutant GST–Cx43CT phosphorylated by activated Src. Equal amounts of the GST–Cx43CT substrates were phosphorylated by Src, acid-hydrolyzed, and separated by electrophoresis at pH 1.9 (dimension 1) followed by electrophoresis at pH 3.5 (dimension 2). The migration positions of the unlabeled pSer, pThr, and pTyr standards are indicated. (D) Phosphotryptic peptide maps of wt and mutant GST–Cx43CT phosphorylated by activated Src. Resolved peptides were from one type of GST–Cx43CT or a mixture of wt and the Y265F GST–Cx43CT mutant. Peptides are numbered as in B.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Analysis of the phosphorylation of GST–Cx43CT by activated Src in vitro. (A) Amino acid sequence of two predicted tryptic peptides of Cx43CT (S244-K258 and Y265-K287) that contain the putative pTyr sites (Y247 and Y265) in the phosphorylated GST–Cx43CT fusion protein. Cx43CT fused to GST begins at V236 in the cytoplasmic tail of Cx43. The amino acids deleted in the Cx43CT deletion mutant are bracketed (P253LSP256). (B) Two-dimensional phosphotryptic peptide analysis of GST fusion proteins with wt or the Cx43CT deletion mutation phosphorylated by activated Src. Phosphotryptic peptides were resolved by electrophoresis (dimension 1) followed by ascending chromatography (dimension 2). Major phosphotryptic peptides are numbered. (C) Phosphoamino acid content of wt or site-directed mutant GST–Cx43CT phosphorylated by activated Src. Equal amounts of the GST–Cx43CT substrates were phosphorylated by Src, acid-hydrolyzed, and separated by electrophoresis at pH 1.9 (dimension 1) followed by electrophoresis at pH 3.5 (dimension 2). The migration positions of the unlabeled pSer, pThr, and pTyr standards are indicated. (D) Phosphotryptic peptide maps of wt and mutant GST–Cx43CT phosphorylated by activated Src. Resolved peptides were from one type of GST–Cx43CT or a mixture of wt and the Y265F GST–Cx43CT mutant. Peptides are numbered as in B.
Mentions: Site-directed mutagenesis indicated that Y265 of Cx43 was a primary Tyr site targeted by v-Src upon coexpression of these proteins in Xenopus oocytes (Swenson et al., 1990). This study did not attempt to identify possible additional tyrosine phosphorylation sites in Cx43. However, the observation of two distinct pTyr-containing phosphopeptides of Cx43 in Rat-1 v-Src cells suggested that v-Src may phosphorylate Cx43 at an additional Tyr site(s) (Kurata and Lau, 1994). Because Y247, like Y265, is in a tyrosine kinase phosphorylation consensus sequence, we examined whether Y247 is another site targeted by activated Src. Glutathione S-transferase (GST) fusion proteins with wt Cx43 COOH-terminal tail (CT) or a deletion mutant that lacks residues P253-P256 (Kanemitsu et al., 1997) of Cx43CT were phosphorylated by activated Src in vitro and subjected to two-dimensional phosphotryptic peptide analysis. Since the P253-P256 deletion is located in tryptic peptide S244-K258 (Fig. 1 A), it was expected to alter the migration of this peptide. Phosphopeptide 4 detected in the map of phosphorylated wt GST–Cx43CT disappeared from the map of the phosphorylated deletion mutant GST–Cx43CT with the concomitant appearance of a slower migrating phosphopeptide, labeled 4′ (Fig. 1 B). Because this was the only difference observed in the peptide maps, we concluded that peptide 4 must be peptide S244-K258, whereas peptide 4′ represents the same peptide with the P253-P256 deletion. This conclusion was supported by a calculation of the relative peptide mobilities, which indicated that the peptide from the deletion mutant would migrate more slowly than the wt peptide in the second chromatographic dimension (Boyle et al., 1991). Because the deletion localizes in a phosphopeptide that contains a single Tyr site (Y247) (Fig. 1 A) and Src phosphorylates GST–Cx43CT only on Tyr (see below), Y247 represents another Tyr site in Cx43 that is phosphorylated by Src in vitro.

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