Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven molecular switch that regulates gap junction size

Abstract

The proteins that form vertebrate gap junctions, the connexins, are highly regulated and have short (<2 hour) half-lives. Phosphorylation of connexin43 (Cx43) affects gap junction assembly, channel gating and turnover. After finding dramatic effects on gap junctions with Akt inhibitors, we created an antibody specific for Cx43 phosphorylated on S373, a potential Akt substrate. We found S373 phosphorylation in cells and skin or heart almost exclusively in larger gap-junctional structures that increased dramatically after wounding or hypoxia. We were able to mechanistically show that Akt-dependent phosphorylation of S373 increases gap junction size and communication by completely eliminating the interaction between Cx43 and ZO-1. Thus, phosphorylation on S373 acts as a molecular 'switch' to rapidly increase gap-junctional communication, potentially leading to initiation of activation and migration of keratinocytes or ischemic injury response in the skin and the heart, respectively.

Keywords: Akt; Connexin43; Gap junctions; Hypoxia; Wounding.

Fig. 1.

Cx43 is phosphorylated on Serine 373 in gap junctions. (A) Immunoblot detection of Cx43 in MDCK43 cell lysates using antibodies against the N-terminus (Cx43), Cx43 phosphorylated at S373 (pS373) and phosphorylated Akt ‘consensus’ sites (Akt sub). Arrows indicate the S373-phosphorylated isoform of Cx43. The fastest migrating Cx43 isoform is marked P0 and the multiple phosphoisoforms are bracketed with a P. (B–D) Cx43 immunofluorescence in MDCK43 cells using the pS373 antibody (B) and total Cx43 (C). The overlay (D) shows that the pS373 antibody signal is almost exclusively gap junctional; DAPI in blue. Scale bar: 25 µm. (E) High magnification immunofluorescence of a series of gap junctions of different size with total Cx43 (green), pS373 (red) and overlay. (F) Histogram showing size distribution of pS373 (red) and total Cx43 (green) signals. Scale bar: 2 µm.

Fig. 2.

Cx43 is phosphorylated at S373 in large gap-junctional structures and mutation of S373 to A or D can inhibit or promote gap junction formation. MDCK43, MDCK43 S373A and MDCK43 S373D cells were untreated or treated with MG132 or MK2206. (A) Immunoblot analysis of total Cx43 and pS373. Cx43 isoforms are marked with P0 and P (phosphorylated). (B) Immunofluorescence analysis with staining for total Cx43 (green), pS373 (red) and DAPI (blue); overlay images are shown. Scale bar: 25 µm. (C) Distribution of fluorescent object size after MG132 or MK2206 treatment or mutation of S373 to A or D. (D) Gap-junctional communication is increased in MDCK43 S373D and decreased in MDCK43 S373A cells compared with control MDCK43 cells expressing wild-type Cx43. Dotted lines indicate percentage recovery at 150 seconds post bleach. Data are means ± s.e.m.

Fig. 3.

Interaction of ZO-1 and Cx43 is regulated by S373 phosphorylation. (A) Coimmunoprecipitation of ZO-1 with Cx43 antibody from MDCK43 (WT), MDCK43 cells with Cx43 mutant at S365/369/373 (S3A) and MDCK43-S373D (S373D) cells treated (+) or not untreated (−) with MG132. Cx43 isoforms are marked with P0 and P (phosphorylated). (B) Tilted edge-on volume-rendered immunofluorescence colocalization of Cx43 and ZO-1 in MDCK43 (B), MDCK43 treated with MG132 (C) and MDCK43 S373D (D) cells. Cx43 is shown in green and ZO-1 in red (DAPI in blue). Grid lines are 4.06 µm apart.

Fig. 4.

TPA treatment leads to rapid phosphorylation of Cx43 at S373 and slower phosphorylation at S279/282. (A) Immunoblot analysis of a TPA treatment time course for total Cx43, Cx43 phosphorylated on S373 (pS373) and S279/282 (pS279/282). Cx43 isoforms are marked with P0 and P (phosphorylated). Data in graph on right are means ± s.e.m. of five blots. (B) Immunofluorescence analysis with staining for total Cx43 (green), pS373 (red) and DAPI (blue). Overlay images are shown. Scale bar: 25 µm.

Fig. 5.

Scratch wounding or incubation of MDCK43 cells at 1% O₂ cells increases phosphorylation of Cx43 on S373. (A) Immunoblot analysis of wounded cells over time post scratch showing increases in pS373 on Cx43 and pS308 and pS473 on Akt. The change in ratio of Akt signal from pS308 and pS473 over total Akt shows a maximum at 5 minutes. (B) Immunofluorescence of Cx43 phosphorylation at S373 (red) and total Cx43 (green) at 0 and 5 minutes post scratch with DAPI (blue). Scratched area is indicated by arrow. (C) Immunoblot analysis of cells incubated over time show increases in pS373, pS308 and pS473. Cx43 isoforms are marked with P0 and P (phosphorylated). The change in ratio of Akt signal from pS308 and pS473 over total Akt shows a maximum at 5 minutes. Data are means ± s.e.m. (D) Immunofluorescence of Cx43 phosphorylation at S373 (red) and total Cx43 (green) at 0 and 5 minutes after reduction to 1% O₂. DAPI is blue. Scale bar: 25 µm.

Fig. 6.

Cx43 is phosphorylated on S373 in murine cardiac tissue particularly after 30 minutes of hypoxia. (A) Immunoblot analysis probing for total Cx43 or pS373 in three pairs of heart tissues that had been either untreated or rendered hypoxic for 30 minutes. (B) Immunofluorescence analysis with staining of control or 30 minute hypoxic heart tissue for total Cx43 (green), pS373 (red) and DAPI (blue). Overlay images are shown. Scale bar: 25 µm.

Fig. 7.

Phosphorylation of Cx43 on S373 is increased in human skin 30 minutes post wounding. Unwounded, 30 minutes and 24 hours post-wound are shown. Total Cx43 (green), pS373 (red) and their overlay with DAPI (blue) are shown. The site of the wound is marked with an arrowhead. Scale bar: 25 µm.