Phosphorylation of Y247 and Y265 of Cx43CT by activated Src in vitro
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
(Kanemitsu et al., 1997
) of Cx43CT were phosphorylated by activated Src in vitro and subjected to two-dimensional phosphotryptic peptide analysis. Since the P253
deletion is located in tryptic peptide S244
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′ ( B). Because this was the only difference observed in the peptide maps, we concluded that peptide 4 must be peptide S244
, whereas peptide 4′ represents the same peptide with the P253
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) ( 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.
Figure 1. 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 (more ...)
To further examine the Src-mediated phosphorylation of Cx43 at Y247 and Y265, we generated GST–Cx43CT with a Y247F, Y265F, or Y247F/Y265F double Cx43 mutation. Phosphoamino acid analysis established that all GST–Cx43CT substrates phosphorylated by Src contained only pTyr ( C). However, data obtained in one experiment with equal amounts of GST–Cx43CT fusion proteins indicated that the Y247F and Y265F Cx43 mutants appeared to have less pTyr than wt Cx43 (86 and 63%, respectively). The Y247F/Y265F double mutant had an even lower level of pTyr than the single mutants (26% of wt), suggesting that both the Y247 and Y265 sites were required for full phosphorylation of wt Cx43CT by Src in vitro. The detection of pTyr in the Y247F/Y265F mutant suggested the presence of an additional pTyr site in wt Cx43CT induced by Src in vitro.
Two-dimensional phosphotryptic peptide mapping revealed that wt Cx43CT phosphorylated by Src in vitro contained three major phosphopeptides: 1, 4, and 5 ( D). Phosphopeptide 4 disappeared in the Y247F and Y247F/Y265F Cx43CT point mutants, indicating that it contained Y247 ( D). These data confirmed the previous conclusion obtained with the GST–Cx43CT P253-P256 deletion mutant ( B) that Src phosphorylated Cx43CT at Y247 in vitro. Compared with wt Cx43CT, phosphorylation of peptide 1 was reduced in the Y265F Cx43 mutant. This peptide appeared to migrate faster in the second dimension in the Y265F and double Cx43 mutants, possibly due to the substitution of Tyr by the more hydrophobic Phe residue ( D, labeled 1′). Analysis of a mixture of wt and Y265F Cx43CT samples confirmed this difference in migration. Taken together, these data suggested that phosphopeptide 1 contained the Y265 site ( D). Interestingly, phosphopeptide 5 still appeared in the double Cx43CT mutant, supporting the suggestion of another pTyr site in Cx43CT induced by Src in vitro. However, significant phosphorylation at an additional tyrosine site was not observed in full-length Cx43 phosphorylated in vivo (see Discussion).
Expression of Cx43 and v-Src in the different cell clones
Wt and mutant Cx43 were expressed in Cx43 knockout (KO) cells and clones with similar levels of GJC were infected with the v-Src retrovirus. To characterize the expression of v-Src in the Cx43 reexpressing cells, an in vitro kinase assay was performed on the morphologically transformed cell clones infected by pLvsrcSH. Equal amounts of antibody were used to immunoprecipitate v-Src from equal amounts of cell lysate, followed by an in vitro kinase assay of immunoprecipitated v-Src. As expected, the negative control wtC1 cells showed no v-Src kinase activity (
A, first lane). All of the other clones exhibited v-Src kinase activity with some differences in the levels of activity. One clone (indicated by an asterisk) from each set of infections by wt or mutant pBABE-cx43 and pLvsrcSH retroviruses with the most similar levels of v-Src kinase activity was chosen for further biochemical analysis. The selected clones were wtS2, 247FS2, 265FS1, and dbS2. The wtC1 clone was used as a negative control.
Figure 2. Levels of v-Src kinase activity and Cx43 in the cell clones. (A) In vitro v-Src protein kinase activities. The same amounts of anti-Src TBR serum 6-1-4 were reacted with equal amounts of cell lysate from each clone. The ability of v-Src to phosphorylate (more ...)
Immunoblotting analysis was performed to measure the expression of Cx43 in the selected clones ( B). 40 μg of whole cell lysate was loaded for each sample. Approximately equal levels of Cx43 were detected in all cell clones, indicating that the antibody recognized both the wt and the mutated Cx43 proteins and that the substitution of the Phe residue for Tyr did not affect the expression of Cx43 ( B). The antibody recognized the most abundant nonphosphorylated isoform of Cx43, and in some samples a slower migrating phosphorylated isoform was also detected.
Localization of wt and mutant Cx43 in v-Src–expressing cells
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 (
, top, arrowheads). Wt and mutant Cx43 were also localized to the plasma membrane of the clones coexpressing v-Src (, 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.
Figure 3. 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. (more ...)
Phosphorylation of wt and mutant Cx43 in v-Src–expressing cells
To examine the phosphorylation of Cx43 in cells expressing Cx43 and v-Src, the cells were metabolically labeled with 32
, and Cx43 was immunoprecipitated from the cell lysates with equal amounts of antibody (
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 ( 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
Figure 4. 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 (more ...)
Phosphoamino acid analysis demonstrated that Cx43 immunoprecipitated from 32P-labeled wtC1 cells, which did not express v-Src, contained only phosphoserine (pSer) ( B). Wt Cx43 from the v-Src expressing clone wtS2 contained pSer and pTyr ( B), suggesting that v-Src targeted wt Cx43 in these cells. However, the levels of pTyr appeared to be reduced in the Y265F, and the double Cx43 mutants coexpressed with v-Src. Finally, the Y247F Cx43 mutant appeared to contain more pTyr than the Y265F Cx43 mutant.
Because it is difficult to obtain quantitative data from phosphoamino acid analysis, immunoblotting analysis using a pTyr antibody was performed to quantify the levels of pTyr in Cx43 isolated from the various clones. Cx43 was immunoprecipitated from lysates of the different clones using the same amount of antibody under conditions of antigen excess. As expected, Cx43 from wtC1 cells did not contain pTyr, whereas Cx43 from wtS2 cells expressing v-Src contained pTyr ( C). In cells coexpressing the Y247F Cx43 mutant and v-Src, a reduced but significant amount of pTyr of Cx43 was observed (~57% of wt Cx43). However, compared with wt Cx43 the levels of pTyr in the Y265F and the double Cx43, mutants coexpressed with v-Src were greatly reduced to ~10% and 2%, respectively ( C). These results indicated that v-Src phosphorylated wt Cx43 at both the Y265 and Y247 sites in vivo, confirming the identification of these sites as Src targets in our in vitro studies.
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 ()
. 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 ( D) and were assigned tentatively to the Y247 peptide (, 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 ( D).
Figure 5. 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 (more ...)
GJC in cells expressing wt and mutant Cx43 and v-Src
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 (). 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 (, 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 () 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 ()
. 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.
Measurement of GJC in cell clones containing wt or mutant cx43 genes with or without the v-src gene
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.
Role of increased serine phosphorylation of Cx43 induced by v-Src
In this study, we demonstrated that phosphorylation of Cx43 at Y247 and Y265 was primarily responsible for the disruption of GJC by v-Src. However, Zhou et al. (1999)
reported that the ability of v-Src to induce cell uncoupling was attenuated by treating cells with the MAP kinase kinase (MEK) inhibitor, PD98059, to block MAP kinase activation. This suggested that serine phosphorylation of Cx43, perhaps by activated MAP kinase, may have contributed to the disruption of GJC by v-Src. We also used the MEK inhibitor to examine the possibility that serine phosphorylation of Cx43 might be involved in the downregulation of GJC by v-Src in mammalian cells.
WtS2 cells were treated with 100 μM MEK inhibitor for 1 h. Interestingly, this treatment failed to restore GJC. Untreated wtS2 cells communicated to an average of 1.0 neighboring cell compared with an average of 0.9 adjacent cells after treatment (
, lanes 3 and 4). The wtC1 control cells, expressing wt Cx43 without v-Src, communicated to an average of 13.1 adjacent cells (, lane 1). To confirm that the MEK inhibitor treatment inhibited MAP kinase activation, the levels of active MAP kinase were determined from equivalent amounts of whole cell lysate by immunoblotting with an antibody recognizing the phosphorylated, activated form of MAP kinase. As a positive control, wtC1 cells treated with EGF showed increased levels of active MAP kinase compared with the untreated cells (, lanes 2 and 1, respectively). Activated MAP kinase was detected in the wtS2 cells treated with DMSO alone; however, treatment of these cells with the MEK inhibitor reduced activated MAP kinase to ~5% of the untreated wtS2 cells. , lane 5, represents a single plate of wtS2 cells that communicated to an average of 0.5 neighboring cells after treatment with the MEK inhibitor. Immunoblotting the cell lysate from this single plate demonstrated that MAP kinase was reduced markedly to ~2% of the untreated wtS2 cells. In addition, we determined that the wtS2 cells treated with 100 μM MEK inhibitor for 4 h communicated to an average of 1.0 ± 0.2 (n = 20) neighboring cells and also showed significantly reduced levels of activated MAP kinase (unpublished data). Taken together, these observations strongly suggested that activated MAP kinase did not play a role in the disruption of GJC induced by v-Src in our cells.
Figure 7. The role of MAP kinase in the disruption of GJC by v-Src. Cells were untreated or treated with EGF or the MEK inhibitor before being lysed. The same amount of whole cell lysate was loaded for each sample. Activated MAP kinase was detected with an antibody (more ...)