(Received for publication, September 18, 1995; and in revised form, November 21, 1995)
From the
We have previously demonstrated that epidermal growth factor
induced a rapid, transient decrease in gap junctional communication and
increase in serine phosphorylation on the connexin-43 gap junction
protein in T51B rat liver epithelial cells. The kinase(s) responsible
for phosphorylation and specific serine targets in connexin-43 have not
been identified. There are three consensus mitogen-activated protein
(MAP) kinase serine phosphorylation sequences in the carboxyl-terminal
tail of connexin-43 and purified MAP kinase phosphorylated connexin-43 in vitro on tryptic peptides that comigrated with a subset of
peptides from connexin-43 phosphorylated in vivo in cells
treated with epidermal growth factor. These data suggested that MAP
kinase may phosphorylate connexin-43 directly in vivo. We have
utilized a glutathione S-transferase fusion protein containing
the cytoplasmic tail of connexin-43 to characterize MAP kinase
phosphorylation. Site-directed mutagenesis, phosphotryptic peptide
analysis, and peptide sequencing have confirmed that MAP kinase can
phosphorylate connexin-43 at Ser, Ser
, and
Ser
, which correspond to the consensus sites recognized
earlier. Characterization of MAP kinase-mediated phosphorylation of
connexin-43 has defined potential targets for phosphorylation in
vivo following activation of the epidermal growth factor receptor
and has provided the basis for studies of the effects of
phosphorylation, at specific molecular sites, on the regulation of gap
junctional communication.
Gap junctions are aqueous membrane channels that permit the
exchange of small (<1000 Da) regulatory ions, molecules, and
metabolites between cells. Gap junctional communication (GJC) ()allows for synchrony in events such as contraction in the
uterus and myocardium and is believed to play an important role in
regulating growth and differentiation (reviewed in 1-3). Gap
junctions form between hexameric structures of connexin molecules
(connexons) that interact with connexons in neighboring cells to form
membrane pores(4, 5) . Connexins are a conserved
family of proteins with four membrane-spanning regions and with
cytoplasmic amino and carboxyl termini, yielding one intracellular and
two extracellular loops.
GJC is known to be regulated by
posttranslational phosphorylation on connexin-43 (Cx43). Musil et
al.(6) have demonstrated that a basal level of
posttranslational phosphorylation on serine residues in Cx43 may be
essential for functional assembly and activation of gap junctions.
Up-regulation of GJC has been associated with increased levels of
cyclic AMP and increased serine phosphorylation on
Cx43(7, 8) . GJC and serine phosphorylation on Cx43
were also up-regulated in communication-deficient S180 mouse cells
following transfection with liver cell adhesion molecule
DNA(6) . In contrast, disruption of GJC has been associated
with increased tyrosine and/or serine phosphorylation on
Cx43(9, 10, 11, 12) .
Down-regulation of GJC was associated with increased serine
phosphorylation in cells expressing the ras oncogene (10) and in cells stimulated with epidermal growth factor (EGF; (13) and (14) ) and with increased tyrosine
phosphorylation in src(15) and fps(16) transformed cells. Studies from this laboratory have
demonstrated that Cx43 can serve as a direct substrate for the
pp60 tyrosine kinase(17) , however, it
is not known whether the fps kinase directly phosphorylates
Cx43 or activates a downstream tyrosine kinase responsible for Cx43
phosphorylation(16) . The possible increased serine
phosphorylation on Cx43 in cells transformed by the src and fps oncogenes is presumably mediated by the activation of
downstream serine/threonine kinases through signal transduction events.
A rapid and transient decrease in GJC, which correlated with an
increase in serine phosphorylation on Cx43, was observed in T51B rat
liver epithelial cells following stimulation of the epidermal growth
factor receptor (EGFR; (13) and (14) ). The data
suggested that the receptor tyrosine kinase activated a downstream
serine/threonine kinase(s) that phosphorylated Cx43. The identity of
this kinase(s) and the specific Cx43 sites phosphorylated have not been
characterized. EGF stimulates pathways that lead to the activation of
protein kinase C (PKC; (18) and (19) ). However,
down-regulation of 12-O-tetradecanoylphorbol
13-acetate-sensitive forms of PKC did not prevent EGF-induced
disruption of GJC or Cx43 phosphorylation (14) . EGF also
activates mitogen-activated protein kinase (MAPK) by signal
transduction events that begin with ligand activation of the EGFR and
proceed through a sequence of protein-protein interactions coupled to a
protein kinase cascade(20, 21, 22) . MAPK was
activated in EGF-treated T51B rat liver epithelial cells with kinetics
that supported a potential role for MAPK in signal transduction events
leading to Cx43 phosphorylation or perhaps in directly phosphorylating
Cx43(14) . The cytoplasmic, carboxyl-terminal tail of Cx43
possesses three putative consensus MAPK phosphorylation sequences
PX(S/T)P(23, 24, 25) , underlined in Fig. 1. Furthermore, activated MAPK may
phosphorylate Cx43 directly in vivo, since MAPK phosphorylated
Cx43 in vitro on phosphotryptic peptides that comigrated with
a subset of EGF-responsive phosphotryptic peptides obtained from Cx43
phosphorylated in vivo in EGF-stimulated cells(14) .
Figure 1:
Amino acid sequence of
the cytoplasmic carboxyl-terminal tail of Cx43 in the GST fusion
protein. The carboxyl-terminal tail of Cx43 in the GST-Cx43-CT fusion
protein begins at Val of Cx43 on the cytoplasmic side of
the membrane. Putative consensus MAPK phosphorylation sequences in Cx43
are underlined (serine sites Ser
,
Ser
, and Ser
, indicated by *). Predicted
trypsin cleavage sites are indicated by spaces between the
resulting tryptic peptides and the amino acids that are deleted in the
GST-Cx43-CT deletion mutants are enclosed in brackets (amino
acids 253-256 and 274-284). Peptide b extends from
Tyr
to Lys
.
Although it is known that GJC can be regulated by posttranslational phosphorylation on connexin, little is known about the molecular sites in the connexin molecule that are critical for regulating intercellular communication through gap junctions. The increased phosphorylation on Cx43 at specific serine sites that follows activation of the EGFR may be the direct cause of the observed functional disruption of GJC. Thus, EGF-induced phosphorylation provides an excellent system for the identification of sites in Cx43 critical to GJC and for the characterization of the signal transduction events leading from the EGF receptor to the activation of downstream serine/threonine kinases that mediate Cx43 phosphorylation. The studies presented here were carried out to further delineate the role of MAPK in Cx43 phosphorylation by identifying specific MAPK serine phosphorylation sites on Cx43. We utilized a GST (glutathione S-transferase) fusion protein containing the cytoplasmic, carboxyl-terminal tail of Cx43 (GST-Cx43-CT; (17) ) as a MAPK substrate and have identified the sites of phosphorylation by deletion and site-directed mutagenesis, phosphotryptic peptide mapping, and peptide sequence analysis. The results of these studies support the concept that MAPK phosphorylates Cx43 in vivo in response to a signal transduced by the activated EGFR and define the phosphorylation sites in Cx43 that may be directly related to the functional disruption of GJC observed in EGF-treated cells.
GST fusion proteins were expressed in Escherichia
coli (DH5) by induction with 0.1 mM isopropyl-
-D-thiogalactopyranoside for 3 h at 37
°C. Cells were lysed by brief sonication on ice in PBS with 4
mM EDTA, 1 mM benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride and then solubilized with 1% Triton
X-100. GST fusion proteins were affinity purified from clarified cell
lysates by a 2-h incubation at 4 °C with glutathione-Sepharose
4B-agarose beads (Sigma) followed by extensive washes with
PBS(17) .
MAPK-phosphorylated
full-length Cx43 and wt GST-Cx43-CT were excised from SDS-PAGE gels
(see Fig. 2), eluted, and subjected to tryptic digestion as
described under ``Materials and Methods.'' Multiple
phosphopeptides were observed on two-dimensional tryptic analysis of
Cx43 (Fig. 3A) and wt GST-Cx43-CT (Fig. 3B). Peptides a-d are labeled according to
the corresponding EGF-responsive phosphopeptides of MAPK-phosphorylated
full-length Cx43 in a previous study(14) . Phosphotryptic
peptides of wt GST-Cx43-CT comigrated (Fig. 3D) with a
subset of the tryptic peptides of Cx43 phosphorylated in vivo when T51B rat cells were treated with EGF (Fig. 3C) and also comigrated with phosphopeptides
obtained from full-length Cx43 phosphorylated by MAPK in vitro (data not shown). Control reactions, performed in the absence of
MAPK, failed to phosphorylate full-length Cx43 or wt GST-Cx43-CT,
indicating that endogenous kinases, capable of phosphorylating these
substrates, were not present in these preparations (Fig. 2, lanes 2 and 5). The GST portion of the fusion protein
was not significantly phosphorylated by MAPK (0.13% and 0.35% of
wt GST-Cx43-CT in two experiments, see Fig. 2, lane 1)
and autophosphorylation was not detected in the kinase alone control
reaction (data not shown). These data demonstrated that the major MAPK
target sites in Cx43 are located in the cytoplasmic, carboxyl-terminal
tail (Val
-Ile
) and that GST-Cx43-CT
is a suitable substrate to characterize in vitro MAPK
phosphorylation of Cx43.
Figure 2:
Phosphorylation of full-length Cx43 and wt
GST-Cx43-CT by MAPK. Wild type GST-Cx43-CT (10 µl of beads), GST
alone (10 µl), and full-length Cx43 (0.2 µg) were incubated in vitro with [-
P]ATP with or
without MAPK and then subjected to SDS-PAGE (see ``Materials and
Methods''). Lane 1, GST alone (+ MAPK); lane
2, full-length Cx43 (no MAPK); lane 3, full-length Cx43
(+ MAPK); lane 4, wt GST-Cx43-CT (+ MAPK); lane
5, wt GST-Cx43-CT (no MAPK). The His-tagged, activated rat MAPK
preparation was used for the in vitro phosphorylations
presented in this and all other figures (see ``Materials and
Methods'').
Figure 3:
Two-dimensional phosphotryptic peptide
maps of P-labeled Cx43. Panel A, recombinant
full-length Cx43 (isolated from baculovirus infected Sf9 cells)
phosphorylated by MAPK in vitro; panel B, wt
GST-Cx43-CT phosphorylated by MAPK in vitro; panel C,
Cx43 isolated from
P-labeled EGF-treated T51B rat liver
epithelial cells; panel D, mix of in vivo labeled
Cx43 from EGF-treated cells with in vitro MAPK-phosphorylated
wt GST-Cx43-CT. The origins of sample application are marked with arrowheads, and the directions of migration in both dimensions
are indicated by the arrows in the lower left corner of the figure. Phosphopeptides a-e are
indicated.
We focused on the
identification of peptide b because it is a major phosphopeptide on
two-dimensional tryptic maps of MAPK-phosphorylated full-length Cx43 (Fig. 3A) and wt GST-Cx43-CT (Fig. 3B)
and is also phosphorylated in Cx43 labeled in vivo in
EGF-treated cells (Fig. 3C). Peptide b was isolated by
HPLC from tryptic digests of MAPK-phosphorylated wt GST-Cx43-CT and
submitted for amino acid sequence analysis. Clear sequence data that
corresponded to the predicted tryptic peptide beginning at Tyr (see Fig. 1) was obtained through at least the first 15
amino acids of this peptide. The identity of peptide b is consistent
with the absence of this phosphopeptide in tryptic maps of the Cx43
274-284 deletion mutant and with MAPK phosphorylation on the
synthetic peptide that corresponded to
Cys
-Lys
. Peptide b contains two
tandem consensus MAPK phosphorylation sequences (underlined in Fig. 1; Ser
and Ser
).
Figure 4: Two-dimensional phosphotryptic peptide maps of MAPK-phosphorylated serine site mutants of GST-Cx43-CT. Panel A, wt GST-Cx43-CT; panel B, GST-Cx43-CT S255A mutant; panel C, GST-Cx43-CT S279A,S282A double mutant; panel D, GST-Cx43-CT S255A,S279A,S282A triple mutant. The sample origins are marked with arrowheads, and the directions of migration in both dimensions are indicated by the arrows in the lower left corner of the figure.
To clarify this issue, two-dimensional phosphotryptic maps of
GST-Cx43-CT double or triple mutants with consensus serine sites
altered to alanine were prepared. Eliminating both consensus MAPK sites
in peptide b (mutant S279A,S282A, Fig. 4C) left the
Ser site intact and peptides d and e were phosphorylated.
However, surprisingly, phosphopeptides b and c were both present in
this tryptic map, suggesting the presence of two additional
phosphorylation sites in the Tyr
-Lys
peptide that are phosphorylated by MAPK in the absence of the
MAPK consensus sites. Results from the phosphorylation of the Cx43
triple mutant S255A,S279A,S282A (all three consensus MAPK serine sites
altered to alanine, Fig. 4D) are consistent with these
data. Peptides d and e were not phosphorylated; however, peptide b was
phosphorylated, indicating the presence of two phosphorylation sites on
peptide b, in addition to the two consensus MAPK sites. Eliminating
both the Ser
and Ser
sites (mutant
S255A,S279A) produced a peptide map consistent with the data presented
previously; peptides d and e were absent (elimination of
Ser
) and peptides b and c were present (consistent with
phosphorylation of peptide b at Ser
and at an additional
site; data not shown).
Figure 5:
Identification of the MAPK phosphorylation
sites in peptide b isolated from wt GST-Cx43-CT. A tryptic digest of
preparative amounts of MAPK-phosphorylated wt GST-Cx43-CT (panel
A) was fractionated by HPLC and the fraction containing peptide b (panel B) was subjected to Edman degradation. The P released at each cycle was determined by Cerenkov
counting and is shown in panel C. The partial amino acid
sequence of the Tyr
-Lys
peptide b is
shown at the top of panel C. The overlapping
consensus MAPK phosphorylation sequences are underlined.
The peak at cycle 5, Asn in the
Tyr
-Lys
peptide, presumably is due to
a contaminating phosphopeptide. Although no evidence of a contaminating
peptide (other than small amounts of peptide c) was obtained on
two-dimensional peptide maps of the sample submitted to Edman
degradation (Fig. 5B), it was possible that a
contaminating peptide was undetected because it comigrated with peptide
b. It is unlikely that the contaminating peptide originated from the
carboxyl terminus of Cx43 because none of its predicted tryptic
peptides contain a potential serine or threonine phosphorylation site
at position 5 (see Fig. 1). GST was not significantly
phosphorylated by MAPK (0.13% and 0.35% of wt GST-Cx43-CT in the
absence of other MAPK substrates); thus, its level of phosphorylation
is too low to account for the radioactivity in cycle 5. Furthermore,
predicted tryptic peptides of GST do not contain phosphorylatable
residues at position 5 or recognized MAPK phosphorylation consensus
sequences. A more plausible explanation for the
P-radioactivity obtained in cycle 5 is partial hydrolysis
of the Tyr
-Lys
peptide (peptide b)
that occurred during the Edman degradation procedure. Hydrolysis of the
Thr
-Pro
bond would produce a shortened
peptide and yield phosphorylation peaks at cycles 5 and 8. This
possibility is consistent with the data (Fig. 5C) and
with the lack of evidence for other phosphopeptide contaminants in the
sample analyzed (Fig. 5B). A small increase in
radioactivity in cycle 8 (reflecting the same difference in
radioactivity compared with cycle 5 as seen between cycles 18 and 15)
is consistent with this hypothesis. Although the radioactivity at
cycles 5 and 15 was determined to be equivalent, when corrected for
repetitive losses at each cycle, the radioactivity at cycle 15 was
actually 4 times that in cycle 5, suggesting that
20% of the
peptide may have been hydrolyzed.
There are three sites in the
Tyr-Lys
peptide that are potential
candidates for alternate MAPK phosphorylation sites: Ser
,
Ser
, and Thr
. Phosphoserine was the only
radiolabeled phosphoamino acid detected in MAPK-phosphorylated wt
GST-Cx43-CT and in Cx43 phosphorylated in vivo in EGF-treated
cells(14) , consistent with phosphorylation at the consensus
MAPK serine sites. Phosphoserine was also the only radiolabeled
phosphoamino acid detected in the MAPK-phosphorylated GST-Cx43-CT
mutants with altered consensus MAPK sites in the
Tyr
-Lys
peptide (double mutant
S279A,S282A and triple mutant S255A,S279A,S282A; data not shown). Since
threonine was not phosphorylated in these mutants, the alternate sites
of MAPK phosphorylation on peptide b must be Ser
and
Ser
. All of the data presented are consistent with the
migration of peptide b as the doubly phosphorylated
Tyr
-Lys
peptide (phosphorylated at
Ser
and Ser
in wt GST-Cx43-CT) and peptide
c migrating as the singly phosphorylated form of the
Tyr
-Lys
peptide.
Earlier studies from this laboratory demonstrated an increase in serine phosphorylation on Cx43 in response to signals transduced by the activated EGF receptor(13, 14) . GJC was transiently disrupted in T51B rat liver epithelial cells following EGF treatment and coincided with increased phosphorylation on Cx43. Treating stimulated cells with okadaic acid, a serine/threonine phosphatase inhibitor, prevented both the dephosphorylation of Cx43 and the restoration of GJC. These studies suggested that phosphorylation on Cx43 at specific serine sites was directly related to the disruption of GJC. The MAPK signaling cascade is activated by EGF and leads to the activation of downstream protein kinases, such as MEK and MAPK(20, 21, 22) . MAPK was activated in EGF-treated T51B cells, and putative consensus MAPK phosphorylation sequences are present in the cytoplasmic, carboxyl-terminal tail of Cx43. Furthermore, purified MAPK phosphorylated recombinant Cx43 in vitro on serine residues in tryptic peptides that comigrated with EGF-responsive peptides obtained from Cx43 phosphorylated in vivo in the EGF-stimulated cells(14) .
In this study, we
have utilized a GST fusion protein of the carboxyl-terminal tail of
Cx43 as a substrate and demonstrated that the in vitro MAPK
phosphorylation sites in Cx43 are located in the carboxyl-terminal
tail. Tryptic peptides of MAPK-phosphorylated wt GST-Cx43-CT (Fig. 3B) comigrated (Fig. 3D) with a
subset of the tryptic peptides obtained from Cx43 phosphorylated in
vivo in EGF-treated cells (Fig. 3C) and produced
the same phosphorylation pattern as that obtained for full-length Cx43
phosphorylated by MAPK in vitro (Fig. 3A).
These data provided additional support for the potential of MAPK to
mediate phosphorylation on Cx43 in EGF-treated cells. Using a
combination of tryptic peptide analysis of MAPK-phosphorylated
GST-Cx43-CT mutants, phosphoamino acid analysis, and sequence analysis
of isolated peptides, we have identified three primary MAPK
phosphorylation sites in Cx43 at Ser, Ser
,
and Ser
. Tryptic analysis of MAPK-phosphorylated deletion
mutants indicated that the major phosphopeptides contained the
consensus MAPK sequences. Phosphopeptides d and e were absent in
tryptic maps of the MAPK-phosphorylated
253-256 deletion
mutant and the S255A serine site mutant (Fig. 4B).
Thus, Ser
is the MAPK target site in the
Ser
-Lys
peptide in wt GST-Cx43-CT.
Peptides d and e are likely to represent different tryptic digestion
products containing the Ser
-Lys
peptide, due to inefficient cleavage at Lys-Asp bonds
(Lys
-Asp
). Two phosphopeptides migrated as
peptide d, and two or three phosphopeptides migrated as peptide e (see Fig. 3B or 4A). However, with more complete
digestion, using sequencing grade TPCK-treated trypsin in the absence
of the RNase A carrier protein, the peptides migrating at positions d
and e resolved to single phosphopeptides (see Fig. 5A).
Peptide e probably represents the Ser
-Lys
peptide and peptide d the larger, less hydrophobic
Ser
-Lys
peptide. It is important to
note that phosphopeptides d and e are present in tryptic maps of Cx43
obtained from EGF-treated cells (Fig. 3C) and were
shown to be EGF-responsive peptides in the earlier study(14) ,
suggesting that Ser
may be an in vivo target for
phosphorylation on Cx43 in cells stimulated with EGF.
Two other
primary MAPK phosphorylation sites were identified in the tryptic
peptide corresponding to Tyr-Lys
. The
identity of this peptide was determined by amino acid sequence analysis
of peptide b isolated from tryptic digests of MAPK-phosphorylated wt
GST-Cx43-CT. Edman degradation confirmed that the preferred MAPK
phosphorylation sites on this peptide are the consensus MAPK sites,
Ser
and Ser
(see Fig. 5C),
and that peptide b is the doubly phosphorylated form of the
Tyr
-Lys
peptide. When one of the
consensus sites in the Tyr
-Lys
peptide is altered by the conservative substitution of alanine
for serine, MAPK phosphorylates this peptide at an alternate site and
doubly phosphorylated peptide b is still present in the tryptic maps of
the single site mutants, S279A and S282A. The phosphotryptic map of the
S279A,S282A double mutant confirmed that phosphorylation can occur at
two alternate sites on the Tyr
-Lys
peptide in the absence of both consensus MAPK sites (Fig. 4C). However, the extent of phosphorylation on
peptides b and c appears to be reduced relative to phosphorylation of
these peptides in wt GST-Cx43-CT (see Fig. 4A) and the
consensus Ser
phosphorylation site appears to be favored
relative to phosphorylation at the alternate sites in the
Tyr
-Lys
peptide (compare
phosphorylation of the S279A,S282A double mutant in Fig. 4C with phosphorylation of wt GST-Cx43-CT in Fig. 4A).
Other explanations for the presence of
phosphopeptides b and c in the S279A,S282A double mutant, such as
comigration of a contaminating peptide or phosphorylation by a
contaminating kinase, are unlikely. GST was not a substrate for MAPK,
and no candidate serine MAPK sites are present in the GST-Cx43-CT
fusion protein except for the described alternate sites, Ser and Ser
, located adjacent to a COOH-terminal
proline. Peptides b and c migrated as dimers on some two-dimensional
maps in the chromatographic dimension (see Fig. 4A and Fig. 5A). This was dependent on the extent of migration
relative to the solvent front with increasing migration resulting in a
loss of the dimeric form. Such differences in migration may be due to
oxidation differences or to hydrophobicity differences due to
phosphorylation at different sites on the peptide (33) . No
evidence of a contaminating phosphopeptide was found in the amino acid
sequence analysis of peptide b isolated from wt GST-Cx43-CT. Also,
contaminating kinases were not present in wt GST-Cx43-CT (see Fig. 2, lane 5), and two very different MAPK
preparations yielded the same phosphorylation patterns for the
site-directed mutants, even with reduced amounts of enzyme
(3-10-fold), arguing against the presence of a common
contaminating serine kinase in the MAPK preparations.
The singly
phosphorylated Tyr-Lys
peptide
appears to be a better substrate for MAPK than unphosphorylated
peptide, because the doubly phosphorylated form is apparent in tryptic
maps of all of the site-directed mutants (see Fig. 4). Peptide b
was observed on tryptic maps when either preparation of MAPK was used
and with decreased amounts of enzyme, suggesting a preference for
double phosphorylation, at tandem sites, on the
Tyr
-Lys
peptide. Peptide b was also
present in tryptic maps of Cx43 phosphorylated in vivo in
EGF-treated cells (Fig. 3C), consistent with double
phosphorylation on the Tyr
-Lys
peptide in vivo.
Since phosphoserine was the only
radiolabeled phosphoamino acid detected in the MAPK-phosphorylated
S279A,S282A double mutant, the alternate phosphorylation sites in the
Tyr-Lys
peptide are Ser
and Ser
. These sites are present in the Cx43
274-284 deletion mutant, but were not phosphorylated
(peptides b and c were missing rather than shifted in position).
Presumably, MAPK did not phosphorylate these serine sites, since the
adjacent proline residue (Pro
) was deleted. A proline
residue COOH-terminal to serine/threonine was determined to be critical
for recognition by MAPK and a NH
-terminal proline,
1-2 amino acids away, constituted an optimal phosphorylation
sequence in studies with peptide substrates(23, 25) .
Although alternate MAPK phosphorylation sites are present in the
Tyr
-Lys
peptide, phosphorylation
appears to be tightly controlled with only two sites phosphorylated in
wt GST-Cx43-CT and in Cx43 phosphorylated in vivo. Peptides b
and c migrated diagonally in relation to each other on all tryptic
maps, consistent with phosphoisomers, whereas peptide a migrated
diagonally below peptide b on some tryptic maps (see Fig. 3A and Fig. 5A) but not on others (see Fig. 3B and Fig. 4D). The identity of
peptide a is not known; however, it does not appear to be a
phosphoisomer of peptide b or to be a major phosphopeptide of Cx43
phosphorylated in vivo (Fig. 3C).
The
Ser alternate phosphorylation site in Cx43 has an
adjacent COOH-terminal proline as a minimal MAPK recognition signal.
The Ser
site (COOH-terminal proline 2 amino acids away)
does not appear to conform to a recognized MAPK phosphorylation site.
MAPK phosphorylated a synthetic peptide with an alanine residue
inserted between the phosphorylation site and the COOH-terminal proline
85-fold less effectively than a peptide with the COOH-terminal proline
residue adjacent to the phosphorylation site(23) . Amino acids
flanking Ser
in Cx43 may permit a more favorable geometry
for MAPK phosphorylation at a site 1 amino acid away from the
COOH-terminal proline or the presence of a phosphate group on
Ser
may favor subsequent phosphorylation at the adjacent
Ser
site. It is important to remember that the
phosphorylation at Ser
and Ser
observed in
these studies is induced by the elimination of the primary consensus
MAPK phosphorylation sites and probably does not reflect events
occurring in vivo. However, the potential for phosphorylation
to occur at these secondary sites must be considered in DNA
transfection experiments designed to examine the effects of these
mutations on connexin phosphorylation and function.
Ser (in the Ser
-Lys
peptide) also
has an adjacent COOH-terminal proline, but is not readily
phosphorylated by MAPK in vitro. However, this site may be
minimally phosphorylated in the absence of consensus MAPK
phosphorylation sites, since the phosphopeptide marked as d in the
MAPK-phosphorylated S255A,S279A,S282A triple mutant (Fig. 4D) comigrated with peptide d of wt GST-Cx43-CT
(data not shown). Ser
is flanked by proline and glycine
residues and may not be readily accessible to MAPK in GST-Cx43-CT.
Although there are numerous reports of posttranslational
modifications on connexin molecules associated with alterations in GJC,
little is known about the specific molecular sites in the molecule
essential for the regulation of GJC. One study in Xenopus oocytes, transfected with Cx43 mRNA, linked phosphorylation at
Tyr in Cx43 with altered GJC(12) . Oocytes
coexpressing pp60
lost the ability to
communicate, whereas GJC was not disrupted in oocytes that coexpressed
pp60
with a mutant form of Cx43 that could
not be phosphorylated at position 265 (Y265F). This study concluded
that phosphorylation at the Tyr
site on Cx43 was
sufficient for directly disrupting GJC. However, these results should
be interpreted cautiously, since events demonstrated in an oocyte
system may not reflect completely the signaling events occurring in
mammalian cells, where more than one tyrosine residue in Cx43 may be
phosphorylated, directly or indirectly by pp60
(15, 17) . Moreover, pp60
associates with the Shc adaptor protein, resulting in the
activation of the Ras/Raf signal transduction pathway and leading to
the activation of MAPK(36) .
A study by Britz-Cunningham et al.(37) demonstrated that a serine to proline mutation at position 364 of Cx43 (S364P) was associated with congenital heart defects in children. Cx43 is the main connexin expressed in heart tissue(38) , and right ventricular cardiac malformation was the primary cause of neonatal death in mice lacking the Cx43 gene(39) . The cytoplasmic, carboxyl-terminal tail of Cx43 contains consensus phosphorylation sequences for several protein kinases (40) that may regulate GJC, such as PKC, MAPK, and glycogen synthase kinase 3. L929 cells transfected with the S364P mutant of Cx43 did not exhibit the enhanced GJC observed in L929 cells transfected with wt Cx43 and differed in their responses to microinjected cAMP-dependent protein kinase and PKC(37) . Additional mutations in serine or threonine residues in the carboxyl-terminal tail of Cx43 were noted in children with visceroatrial heterotaxia syndromes; however, the functional significance of these mutations has not been characterized(37) . Nevertheless, it is clear that alterations in specific phosphorylated residues in the carboxyl-terminal tail of Cx43 can affect the regulation of GJC and the normal development of the heart.
The data presented in this study provide strong support for
MAPK's role in mediating EGF-induced phosphorylation on Cx43 and
identify the specific phosphorylated serine sites in the cytoplasmic,
carboxyl-terminal tail of the protein that may be functionally related
to the disruption of GJC. Importantly, the tryptic peptides containing
the phosphorylated Ser and Ser
/Ser
sites comigrated with major phosphopeptides of Cx43
phosphorylated in vivo, suggesting that these peptides are
phosphorylated in response to activation of the EGF receptor in intact
cells. Although alternate signaling pathways, leading to activation of
other kinases such as JNK (41, 42) or involving the
Rac and/or Rho GTPases(43, 44, 45) , have not
been excluded from acting on Cx43 by these studies, we have
demonstrated that Cx43 is a substrate for MAPK. Thus, Cx43 joins a
growing number of MAPK substrates that include ribosomal S6
kinase(46) , c-Myc(24) , c-Jun (24, 47) , myelin basic protein(48) , and the
transcription factor p62
(49) . Cx43 is also a
substrate for the pp60
tyrosine kinase(17) , the
p130
tyrosine kinase (either directly or indirectly; (16) ), and the PKC serine kinase. (
)Identification
of the protein kinases responsible for phosphorylating Cx43 at specific
sites is important to a better understanding of how GJC may be
regulated in normal development and differentiation, synchronous
contraction, and cell growth.