Journal of Cell Science
Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven
molecular switch that regulates gap junction size
Clarence A. Dunn and Paul D. Lampe*
The proteins that form vertebrate gap junctions, the connexins, are
highlyregulatedandhave 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
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.
KEY WORDS: Gap junctions, Connexin43, Akt, Wounding, Hypoxia
Gap-junction-mediated intercellular communication allows the
passage of ions and small metabolites between adjacent cells
(Laird, 2010; Scemes et al., 2007; So ¨hl and Willecke, 2004). Gap
junctions are composed of integral membrane proteins from the
connexin gene family and are critically important in regulating
development, coordinated contraction of excitable cells, tissue
homeostasis, controlled cell growth and differentiation (Scemes
et al., 2007; So ¨hl and Willecke, 2004). Mutations in connexin
proteins have been linked to many diseases, including hereditary
deafness (Bergoffen et al., 1993; Kelsell et al., 1997; Laird, 2006)
and oculodentodigital dysplasia, a disease caused by connexin43
(Cx43) mutations that can lead to syndactyly, atrioseptal defects
and arrhythmias (Paznekas et al., 2003).
Cx43, which is the most ubiquitous connexin, can be
phosphorylated at 12 or more sites, with some sites apparently
increasing gap junction assembly whereas others inhibit formation
of gap junctions, or decrease channel open time (Solan and Lampe,
2009). Given the unusually short half-life (,2 hours) of Cx43 in
cell culture and tissue, Cx43 trafficking and turnover also appear to
influence gap-junctional communication. There are some specific
events that can regulate gap junction assembly and turnover.
Phosphorylation at S365 and S325, S328 or S330 positively
regulates gap junction assembly (Cooper and Lampe, 2002; Lampe
et al., 2006; Solan et al., 2007). Gap junction size is negatively
regulated by interaction with ZO-1 because a peptide mimetic of
the Cx43 ZO-1 binding domain(termeda-CT) cancompete outthe
endogenous ZO-1–Cx43 interaction (Hunter et al., 2005). a-CT
can also reduce remodeling of gap junctions and induced
arrhythmia following ventricular injury (O’Quinn et al., 2011).
ZO-1 small interfering RNA experiments also verified that loss of
ZO-1 function increases the rate of aggregation of connexons into
gap junctions (Rhett et al., 2011).
We have previously shown that ubiquitin-mediated activation
of Akt is responsible for increased gap-junctional communication
and stability following proteasomal inhibition (Dunn et al., 2012).
Here, we show that Akt activity is necessary for phosphorylation
of Cx43 at S373 and this phosphorylation event causes an
increase in gap junction size and communication levels by
limiting the interaction of ZO-1 with Cx43. Furthermore, we
show that Akt rapidly becomes activated in response to scratch-
wound injury or hypoxia, and leads to Cx43 phosphorylation at
S373. This phosphorylation event increases in cardiac tissue in
response to hypoxia and in skin in response to wounding, leading
to larger junctions. Thus, gap junction size and activity is
dynamically regulated by phosphorylation at S373 and is highly
responsive to injury, both in vitro and in vivo.
Cx43 phosphorylation on S373
Previously, we used phosphotryptic peptide/mass spectrometry
analysis to show that the C-terminal region of Cx43 (A371–I382),
containing S372 and S373, was phosphorylated in cells (Cooper
and Lampe, 2002). More recently, we showed that proteasomal
inhibition increased gap junction stability through activation of
Akt (Dunn et al., 2012). Because S373 has been described as
a possible Akt kinase substrate (Park et al., 2007), we decided
to create a phosphospecific antibody that reacts with Cx43
when it is phosphorylated at S373 (pS373). To examine Cx43
phosphorylation in cells, we used an MDCK cell line that was
null for Cx43 in the parental line (Jordan et al., 1999), which
upon stable transfection expresses Cx43 at nominal levels
(MDCK43) and routinely forms functional gap junctions (Dunn
et al., 2012). We found that untreated MDCK43 cells showed low
levels of pS373, but inhibition of the proteasome with MG132
caused a 3.260.4-fold increase (significantly different, P50.007;
n56) in the pS373 signal, primarily in a single band with reduced
SDS-PAGE mobility (Fig. 1A). Like many phosphorylated
proteins, Cx43 normally migrates as multiple bands in SDS-
PAGE owing to phosphorylation, and the pS373 band overlaid
exactly with a major Cx43 phosphoisoform (Fig. 1A, arrow) that
was increased by addition of MG132. Cell lysates from MG132-
treated cells also showed increased overall phosphorylation of
Akt substrates, as shown in cell lysates that were blotted with the
Translational Research Program, Human Biology and Public Health Sciences
Divisions, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue
North, Seattle, WA 98109, USA.
*Author for correspondence (firstname.lastname@example.org.)
Received 9 September 2013; Accepted 23 October 2013
? 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 455–464 doi:10.1242/jcs.142497
Journal of Cell Science
phospho-(Ser/Thr) Akt substrate antibody, including a minor
band that aligned with the same Cx43 and pS373 signal noted
above (Fig. 1A). Furthermore, the pS373 antibody was quite
specific for Cx43 because the only major band was eliminated if
the peptide that was used to create the antibody was preincubated
with the antibody prior to blotting (pS373/pep). These results
show that phosphorylation of Cx43 on S373, a potential Akt site,
is increased by treatment with MG132.
When our pS373 antibody was used to examine its
immunolocalization within MDCK43 cells, the pS373 signal
overlaid with the signal from a total Cx43 antibody but only
within larger, punctate, apparent gap-junctional structures
(Fig. 1B–D). A higher-resolution image of a cell–cell interface
(Fig. 1E) indicated that the pS373 signal was localized primarily
within the central regions of the larger punctate total Cx43
staining. Quantification of the size distribution of pS373 and
total Cx43 signal at over 4000 discrete fluorescent objects
confirmed that the pS373 signal was shifted to larger objects
(Fig. 1F). Note that light microscopy does not have the
resolution to determine whether these larger objects are
actually larger junctions or more tightly packed assemblages
of junctions. In either case, the increase in fluorescence does
reflect an apparent increase in area and/or density of junctional
Cx43, and we generically denote this hereafter as an increase in
gap junction size.
Proteasomal inhibition increases S373 phosphorylation, gap junction
size and gap-junctional communication
To further examine the functions of S373 phosphorylation and
Akt activity on gap junctions, we stably transfected the parental
MDCK cells with Cx43 containing mutations at S373 to mimic
(i.e. S373D) or block (i.e. S373A) phosphorylation at this site in
order to examine its effect on gap junction size compared with the
wild type in the presence of MG132 or the specific Akt1 inhibitor
MK2206. In western immunoblots (Fig. 2A), MDCK43 cells
showed a large increase in pS373 levels in the presence of
MG132 that was completely eliminated when MK2206 was
added (Fig. 2). Neither S373A nor S373D mutants showed a
response to MK2206 treatment, but both showed a reduced
increase in the slower-migrating total Cx43 isoform compared
with the control, in response to MG132. As expected, neither
lysate from the mutant cell lines showed a positive signal for the
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 mm. (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 mm.
Journal of Cell Science (2014) 127, 455–464 doi:10.1242/jcs.142497
Journal of Cell Science
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