Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down-regulation by PKC - PubMed (original) (raw)

Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down-regulation by PKC

Joell L Solan et al. J Cell Biol. 2007.

Abstract

Phosphorylation at unspecified sites is known to regulate the life cycle (assembly, gating, and turnover) of the gap junction protein, Cx43. In this paper, we show that Cx43 is phosphorylated on S365 in cultured cells and heart tissue. Nuclear magnetic resonance structural studies of the C-terminal region of Cx43 with an S365D mutation indicate that it forms a different stable conformation than unphosphorylated wild-type Cx43. Immunolabeling with an antibody specific for Cx43 phosphorylated at S365 shows staining on gap junction structures in heart tissue that is lost upon hypoxia when Cx43 is no longer specifically localized to the intercalated disk. Efficient phosphorylation at S368, an important Cx43 channel regulatory event that increases during ischemia or PKC activation, depends on S365 being unphosphorylated. Thus, phosphorylation at S365 can serve a "gatekeeper" function that may represent a mechanism to protect cells from ischemia and phorbol ester-induced down-regulation of channel conductance.

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Figures

Figure 1.

LC/ESI MS/MS of a phosphorylated peptide from Cx43. A gel band enriched for phosphorylated Cx43 was digested with trypsin and analyzed with an LTQ-FT mass spectrometer. (A) Chromatographic elution of the tryptic peptide mixture into the mass spectrometer revealed a peak at 26.02 min that contained a triply charged monoisotopic ion species (m/z 742.04611) that was suspected to be the peptide V347 to R366 of Cx43 containing a single phosphate group. (B) Tandem mass spectrometry (MS2) of this precursor ion species resulted in a fragmentation spectrum dominated by ion species m/z 710.5 that indicates the neutral loss of phosphoric acid (m/z 31.5) from the precursor. It is important to note that the deviation of m/z 1.2 from the expected value of m/z 32.7 for the neutral loss of a phosphate group is a result of the error associated with subtracting a low mass accuracy, average m/z value in B from the high mass accuracy, monoisotopic value in A. The difference in the two types of measurements is inherent with the mass measurement being performed with an LTQ-FT mass spectrometer. Tandem mass spectrometry (MS2) was performed on ion species m/z 742.04611 to generate the neutral loss species m/z 710.5 followed immediately by a second round of tandem mass spectrometry (MS3) on m/z 710.5. The resulting MS3 spectrum (C: split into 200–750 and 750–1600 m/z panels for legibility) showed numerous diagnostic b- and y-type ions to identify the Cx43 peptide as tryptic peptide V347 to R366. The observed fragment ions were compared with the expected fragment ions (D) showing that the peptide contains a dehydroalanine (“A*”) at position 365 as a result of the neutral loss of phosphoric acid in the first round of tandem mass spectrometry. The site of dehydroalanine reveals that S365 as the site of phosphorylation within the peptide (note: only the expected fragment ions that were observed in the MS3 spectrum are indicated in D).

Figure 2.

pS365 is specific for Cx43 phosphorylated on S365, recognizes predominantly the P1 isoform of Cx43 and is found in apparent gap junction plaques. (A) Western blot analysis of lysates from stably transfected MDCK cells, expressing wild type (WT), S365A or S365D mutant Cx43, were probed for total Cx43 (NT1) and with a polyclonal antibody generated to recognize Cx43 phosphorylated at S365. The phospho-antibody recognizes Cx43 in WT-expressing cells, but not when S365 is converted to alanine or aspartate. The faint bands in the 50–54-kD range are visible with multiple Cx43 antibodies, but we have been unable to identify the modification that leads to this migration shift. Overlay of NT1 (red) and pS365 (green) signals show that they are coincident. Densitometry on the individual isoforms (isoform density) shows that pS365 signal is highest in the P1 and P2 isoforms. The migration positions of molecular weight standards are indicated on the left in kD. (B) Immunofluorescence using pS365 and a monoclonal antibody for total Cx43 shows that pS365 resides predominantly in the plasma membrane (WT Cx43) and that the pS365 epitope is eliminated by conversion from serine to alanine or aspartate (S365A, S365D) (bar = 25 μm).

Figure 3.

Mutation of S365 eliminates the P1 isoform of Cx43. Lysates from two separate stable cell lines expressing wild type (WT), S365A, S365D or S364A mutant Cx43 were probed for total Cx43 (NT1) and with an antibody that recognizes Cx43 containing unmodified S364 and S365 (CT1). WT and S364A make all three Cx43 isoforms, while neither S365A nor S365D make the P1 isoform. The two tick marks on the right side of the left panel indicate the position of 50- and 36-kD standards. The right panels show a comparison of WT and S365A Cx43 expressing cells with the latter being overloaded to show a lack of the P1 isoform.

Figure 4.

NMR conformational analysis of wild-type and S365D mutant Cx43CT. 15N-HSQC spectra for wild-type Cx43CT (black) and S365D mutant (red) are presented in A (pH 5.8) and B (pH 7.5). C shows a close-up view of resonance peaks from A and B to illustrate residues that had clearly shifted at both pH 5.8 and 7.5 (top), that only shifted at pH 5.8 (middle), and that only shifted at pH 7.5 (bottom). D provides a summary of the residues affected by the S365D substitution at pH 5.8 and 7.5. The model and 15N-HSQC spectra are encoded as follows: blue, common changes between pH 5.8 and 7.5, yellow, S365D point mutant; green, regions different between the two pHs; black line, helical domains (invariant between the two pHs); red line, residues affected by the ZO-1 PDZ-2 domain; and gray boxes, residues affected by the Cx43 cytoplasmic loop domain.

Figure 5.

Differential phosphorylation of Cx43 localized at intercalated disks vs. lateral edges of myocytes in the normoxic and ischemic heart. The localization of total Cx43, pS65-Cx43, and pS368 in normoxic and hypoxic heart is shown. Note the increase in total Cx43 at the lateral edges of the myocytes and loss from the intercalated disk of the ischemic heart. Cx43 was not detectably phosphorylated at S365 in the hypoxic heart, but phosphorylation at S368 was increased mainly in the intercalated disk region (bar = 20 μm).

Figure 6.

Conversion of S365 to alanine enhances, while aspartate inhibits PMA-activated phosphorylation of S368. (A) Western blot of cell lysates from wild-type Cx43 (WT), S365A, and S365D cells treated with 100 nM PMA for 30 min (PMA) or not (CON) were probed for total Cx43 (NT1) and Cx43 phosphorylated on S368 (pS368). The migration positions of molecular weight standards are indicated on the right in kD. (B) Signals were quantified and expressed as a ratio of pS368 over NT1. Shown are results of three experiments using three separate clones (nine total measurements) for each construct. Values were normalized to control for each clone and error bars represent SEM. Western blot is representative, showing a single clone for each construct.

Figure 7.

Cx43 phosphorylated on S365 is not phosphorylated on S368. (A) Western blot of Cx43 immunoprecipitated from NRK cells treated with 100 nM PMA for 30 min (+) or untreated (−). Cx43 from lysates or Cx43 immunoprecipitated from cell lysates using antibodies to total Cx43 (α-Cx43), pS365, or pS368 were probed for total Cx43 (NT1, top) and Cx43 phosphorylated on S368 (pS368, bottom). The two tick marks on the right side indicate the position of 50- and 36-kD standards. (B) Signals were quantified and expressed as a ratio of pS368 over NT1 normalized to a value of 1 for the untreated sample immunoprecipitated and immunoblotted for pS368 to allow comparison for five separate experiments (mean ± SEM).

Figure 8.

PKC can efficiently phosphorylate wild type, but not the S365D mutant Cx43. PKC was used to phosphorylate wild-type (WT) and S365D mutant GST-Cx43CT in vitro and the proteins were separated by SDS-PAGE. (A) The level of S368 phosphorylation was determined in a Western blot with pS368 antibody (WB:pS368) and consistent loading was shown with an antibody to GST (WB:GST). The migration positions of molecular weight standards are indicated on the left in kD. (B) The level of phosphorylation was determined directly by incorporation of 32P from radiolabeled ATP and autoradiography (32P panel). Consistent loading is shown in the panel showing the Coomassie blue–stained gel (CB panel).

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Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down-regulation by PKC - PubMed (original) (raw)