From the Department of
Pharmacology/Physiology, the
Center for
Cardiovascular Research, and the §§ Department
of Anesthesiology, University of Rochester Medical Center, Rochester,
New York 14642 and the ** Department of Immunology, Scripps
Research Institute, La Jolla, California 9203
Received for publication, December 30, 2002, and in revised form, February 20, 2003
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ABSTRACT |
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The gap junction protein, Cx43,
plays a pivotal role in coupling cells electrically and metabolically,
and the putative phosphorylation sites that modulate its function are
reflected as changes in gap junction communication. Growth factor
stimulation has been correlated with a decrease in gap junction
communication and a parallel activation of ERK1/2; the inhibition of
epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was
observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5,
another MAPK family member also activated by growth factors, possesses
a phosphorylation motif similar to ERK1/2, and 2) it has been reported
that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1
activation, we investigated whether BMK1 can regulate EGF-induced Cx43
gap junction uncoupling and phosphorylation, comparing this to the role
of ERK1/2 on Cx43 function and phosphorylation induced by EGF.
Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that
BMK1, selectively activated by constitutively active MEK5 The gap junction protein connexin 43 (Cx43)1 is the main conductor
of intercellular current in ventricular myocytes (1). Gap junctions
also enable the cytoplasm of individual cells to communicate directly
and allow exchange of nutrients, ions, metabolites, and small molecules
up to 1000 Da. Diverse biological processes including cardiac function,
cellular growth, propagation of calcium waves, and embryogenesis are
regulated by gap junctional communication (GJC). It is proposed that
regulatory signaling molecules for these processes are exchanged
through gap junctions, and thus, GJC directly impacts intracellular
events (2).
Gap junctional coupling can be regulated by post-translational
phosphorylation of Cx43 (3). Phosphorylation of Cx43 by various protein
kinases regulates the assembly of gap junctions to form functional
connexons in the plasma membrane (2), connexin redirection from
the plasma membrane (3), and also alters channel open probability (5).
For example, EGF stimulates the rapid and transient disruption of GJC,
and a marked increase in the phosphorylation of Cx43, not on tyrosine,
but on serine255, serine279, and serine282 (4). EGF activation of
ERK1/2, a member of the mitogen-activated protein kinase (MAPK) family,
is thought to mediate Cx43 phosphorylation. This conclusion was
supported by the observation that several copies of the ERK1/2
consensus phosphorylation motif are present in the cytoplasmic
C-terminal tail of Cx43 and also because of the sensitivity of
GJC uncoupling to the ERK1/2 inhibitor, PD98059 (4).
JNK and p38 are stress-activated MAPKs recently demonstrated to
directly inhibit GJC (5, 6), supporting the idea that MAPK family
members appear to be instrumental in controlling GJC. EGF can potently
activate BMK1, a relatively poorly characterized member of the MAPK
family. Because the BMK1 consensus phosphorylation motif is the same as
for ERK1/2, BMK1 may in fact be the kinase that exclusively controls
cellular events, including GJC uncoupling, that were previously
credited to ERK1/2. Furthermore, because PD98059, the MAPK inhibitor
previously thought to be specific for MEK1/2, effectively inhibits
MEK5, the upstream activator of BMK1 (7), sensitivity to this
antagonist is insufficient to clearly define whether ERK1/2 or BMK1
mediates EGF-induced GJC uncoupling.
In the present study, we examined the specific role of BMK1 in
EGF-induced phosphorylation of Cx43 and inhibition of GJC by using
specific molecular reagents to manipulate the ERK1/2 or the BMK1
pathways independently in Cx43 heterologously expressed in human
embryonic kidney (HEK) 293 cells. Our data demonstrate that selective
activation of BMK1 but not ERK1/2 promotes gap junction uncoupling in
the absence of EGF. Additionally, a dominant negative form of BMK1, but
not a dominant negative form of MEK1, prevents EGF-induced inhibition
of GJC. In support of this, we also found that BMK1 binds to Cx43 and
phosphorylates preferentially on Ser-255 and a dominant negative form
of BMK1 attenuates this phosphorylation in vivo. These data
demonstrate that BMK1 kinase activity alone is both a necessary and
sufficient component in the mediation of EGF-induced Cx43 Ser-255
phosphorylation and subsequent inhibition of GJC.
Plasmids--
Dominant negative MEK1 (DN-MEK1; S218A/T222A) and
constitutively active MEK1 (CA-MEK1; S218E/T222E), CA-MEK5 Cell Culture and Transfection--
HEK293 cells (ATCC, Manassas,
VA) were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin. Cells seeded at a density of 5 × 105 cells/35-mm dish were transiently transfected with the
indicated plasmids using LipofectAMINE Plus (Invitrogen)
following the manufacturer's recommended protocol. The total plasmid
amount was adjusted to 1.5 µg/dish for single-plasmid transfections
or 3.0 µg/dish (35 mm dishes) or 10 µg/dish (100-mm dishes) for
co-transfection with multiple plasmids. Blank pCI/neo vector (Promega,
Madison, WI) was used where appropriate. Experiments were carried out
24 h post-transfection. A Cx43E stable cell line was selected with Geneticin (1 mg/ml, Invitrogen), and stable expression of the transgene
was confirmed by immunoblotting for Cx43.
Immunoprecipitation, Western Blot Analysis, and Kinases
Activity--
After treatment with reagents, the cells were washed
with phosphate-buffered saline and harvested in 0.5 ml of lysis buffer as described previously (9). Immunoprecipitation was performed as
described previously with anti-BMK1 (10), anti-GFP (Roche Applied
Science), or anti-Xpress (Invitrogen) antibody. Western blot analysis
was performed as described previously (9). In brief, the blots were
incubated for 2 h at room temperature in 3% bovine serum albumin
with the monoclonal anti-pan Cx43 antibody (Transduction Laboratories),
anti-DsRed (Clontech), anti-phosphospecific ERK1/2
(Cell Signaling, Beverly, MA), anti-ERK1 and ERK2 (Santa Cruz
Biotechnology, Santa Cruz, CA), anti-BMK1 (11), anti-Xpress, or
anti-GFP antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) in 5%
nonfat dry milk. Immunoreactive bands were visualized using enhanced
chemiluminescence (ECL, Amersham Biosciences). BMK1 activity was
measured as described previously (10, 12). To determine direct
BMK1-induced Cx43 phosphorylation and the sites phosphorylated, we
performed an in vitro kinase assay with GST, GST-MEF2C
(activation domain), GST-Cx43 C-terminal tail (Cx43CT), or GST-Cx43CT
mutants (S255A, S279A/S282A, and S255A/S279A/S282A) as the substrate.
Fluorescence Recovery after Photobleaching (FRAP) Assay for Gap
Junction Coupling--
HEK293 cells were grown on glass coverslips to
70% confluence and loaded with 10 µM calcein blue-AM
(CBAM, Molecular Probes, Eugene, OR) in external solution (in
mM: 140 NaCl, 3 CaCl2, 1 MgCl2, 2.8 KCl, 10 HEPES buffer, pH 7.4) by incubating the cells with the
solution at 37 °C for 10 min. Calcein blue-AM (molecular mass, 465 daltons; excitation 360 nm and emission 440 nm) was used as the intercellular tracer to minimize interference from the GFP
and DsRed fluorescent tags. After loading, the cells were washed and
maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum at room temperature. The FRAP
assay, described elsewhere (13), was performed on cells visualized
under a Nikon Diaphot inverted epifluorescent microscope equipped with
a Noran OZ (Noran Instruments, Middleton, WI) confocal apparatus. The
recovery phase of the normalized fluorescence intensity plots, taking
into account background photobleaching, was fitted with a
mono-exponential function F(t) = F0 (1 MALDI-TOF Mass Spectrometry Analysis of Cx43 Phosphorylation by
BMK1--
Tryptic digestion of pooled gel slices containing
immunoprecipitated Cx43 after endogenous BMK1 was activated was
subjected to enzymatic cleavage for the generation of peptide
fragments. Pieces were washed with 100 mM ammonium
bicarbonate, reduced (dithiothreitol) and alkylated (iodoacetamide),
and then dehydrated via acetonitrile evaporation. The gel pieces were
re-swollen with 25 mM ammonium bicarbonate containing ~ 0.2 µg of enzyme to achieve a substrate/enzyme ratio of ~ 10:1. ZipTip tippets (Millipore, Bedford, MA), packed with C18 matrix,
were utilized to clean and concentrate peptide samples prior to
analysis. Tips were washed with acetonitrile before peptides were bound
and then eluted with either acetonitrile or matrix solution. ZipTip use
affords a recovery of 50-70% in a 1 µl volume. Digested protein was
mixed with the matrix Materials--
PD98059 (50 µM) was from Cell
Signaling Technology, epidermal growth factor (100 ng/ml, human
recombinant) from Invitrogen, and hydrogen peroxide (500 µM) from Sigma.
Statistical Analysis--
Statistical analysis was performed
using Student's t test as appropriate. A p value
of less than 0.05 was considered appropriate for accepting or rejecting
the null hypothesis.
EGF-induced Cx43 Phosphorylation Is Inhibited by PD98059--
We
examined whether extracellular EGF can activate signal transduction
pathways leading to Cx43 phosphorylation. HEK293 cells do not express
Cx43 but do express all of the necessary components for activation of
MAP kinases by EGF (15, 16) and thus are a good model system for the
study of events mediated by this pathway. First we confirmed that
HEK293 cells do not express detectable endogenous Cx43, which is
demonstrated in an overexposed Western blot described previously (15)
(Fig. 1A). Next, HEK293 cells were transiently transfected with Cx43 cDNA fused at the C terminus to EGFP cDNA; the C-terminal EGFP tag on Cx43 (Cx43E) allowed us to
detect plasma membrane targeting of the fusion protein (15). EGF (100 ng/ml) stimulation of the transfected cells resulted in an
electrophoretic mobility shift on Cx43 but not EGFP alone, demonstrating that any electrophoretic mobility shift on Cx43E is not
an artifact of phosphorylation of the EGFP tag (Fig. 1B). Indeed, if the mobility shift was because of phosphorylation of Cx43E,
it should be collapsed by treatment with alkaline phosphatase (2). We
determined that EGF-induced Cx43E mobility shift was abolished by
alkaline phosphatase treatment (Fig. 1C).
We examined EGF activation of the four best-characterized MAP kinase
family members by immunoblotting for the activated forms of ERK1/2,
JNK, and p38 and by in vitro kinase assay for BMK1. EGF
activated all MAP kinases examined (not shown). Because EGF-induced GJC
uncoupling and Cx43 phosphorylation is blocked by PD98059, we examined
the sensitivity of MAPK activation to this antagonist. PD98059
inhibited EGF-induced Cx43E mobility shift and ERK1/2 and BMK1
activation (Fig. 1D) but not p38 or JNK activation (data not
shown). These data suggest that both ERK1/2 and BMK1 are potential kinases responsible for the EGF-induced Cx43 phosphorylation and its
functional uncoupling. Because PD98059 sensitivity alone is not
sufficient to discriminate between responses evoked by ERK1/2 and BMK1,
we developed more selective molecular reagents to manipulate the two
pathways independently.
Effects of ERK1/2 and BMK1 Activation on EGF-induced
Cx43 Functional Uncoupling of Gap Junctions--
To circumvent the
limitations of drug non-specificity, we created highly specific
molecular reagents to selectively activate and inhibit the ERK1/2 and
BMK1 pathways. DN-MEK1 and CA-MEK1 were created, and their
specificity was detected. We did not detect BMK1 activation in
CA-MEK1-transfected cells, although ERK1/2 was potently activated (Fig.
2A). DN-MEK1 blocked
EGF-activated ERK1/2 but did not inhibit EGF-induced BMK1 activation,
suggesting the specificity in inhibiting the ERK1/2 pathway (Fig.
2B). CA-MEK5
We sought to determine the consequences of EGF-induced Cx43
phosphorylation through ERK1/2 or BMK1 activation in order to establish
functional significance, because Cx43 phosphorylation has been reported
to inhibit GJC (4). To further evaluate the role of ERK1/2 and BMK1 in
EGF-induced GJC uncoupling, we used CA-MEK1, DN-MEK1, CA-MEK5 BMK1 Phosphorylates and Associates with Cx43--
Having
demonstrated that BMK1 activation promoted GJC uncoupling, we
hypothesized that BMK1 could possibly directly phosphorylate Cx43. We
transfected CA-MEK5
To further determine the role of BMK1 as a possible "Cx43 kinase"
in vivo, we investigated the possible association between BMK1 and Cx43. We transfected a Cx43E-expressing stable cell line with
epitope-tagged BMK1. Immunoprecipitation of Cx43 by the epitope tag and
immunoblotting for the epitope tag on BMK1 revealed robust Cx43 and
BMK1 association (Fig. 4B) but not with GFP and BMK1 (Fig. 4C). This protein-protein association was decreased
when BMK1 was activated by CA-MEK5 The BMK1 Phosphorylation Site on the C Terminus of Cx43 Is Ser-255
but Not Ser-279 or Ser-282 in Vitro and in Vivo--
It has
been reported that Ser-255, Ser-279, and Ser-282 are the preferred
ERK1/2 phosphorylation sites on Cx43 (4). To evaluate the preferred
phosphorylation site on Cx43 by BMK1, we performed tryptic peptide
mapping using MALDI-TOF mass spectrometry after immunoprecipitated
endogenous BMK1 was activated by CA-MEK5
To support this result, we generated three GST-Cx43CT peptides
with mutations in the proline-directed serines that are potential BMK1
phosphorylation sites (Ser-255, Ser-279, and Ser-282). We transfected
CA-MEK5
To investigate the role of BMK1 on Cx43 Ser-255 phosphorylation
in vivo, we studied full-length Cx43 point mutants (S255A, S279A/S282A, or S255A/S279A/S282A). Again, the C-terminal GFP tag on
Cx43 demonstrated that all Cx43 mutants express equally and target to
the plasma membrane. We then co-transfected mutants of Cx43E with
CA-MEK1 or CA-MEK5
As shown in Fig. 6A,
transfection of both CA-MEK5 In the present study, we investigated the role of the activated
MEK5-BMK1 module in Cx43 phosphorylation and subsequent inhibition of
GJC. The major finding of this study is that BMK1 associates with Cx43
and phosphorylates Ser-255 on Cx43, and BMK1 kinase activation is
sufficient to inhibit GJC in an in vivo model. In contrast,
CA-MEK1 did not inhibit GJC, and DN-MEK1 could not relieve EGF-induced
gap junction inhibition. Our functional study suggests that activated
ERK1/2 alone is not a sufficient step for growth factor-induced gap
junction phosphorylation and uncoupling, which is in agreement with
other studies (17-20).
To our knowledge our work is the first to document Cx43 as a downstream
target of BMK1. Earlier work has suggested that ERK1/2 can
phosphorylate Cx43 at Ser-255, Ser-279, and Ser-282, all residues that
are found within the MAP kinase phosphorylation consensus motif (21),
and we confirmed the inhibition of Cx43 phosphorylation by the MEK
inhibitor, PD98059. However, recent studies indicate that PD98059 quite
effectively inhibits MEK5, a specific upstream MEK for BMK1 (12, 22,
23). As such, previously reported functional effects credited to
EGF-induced phosphorylation of Cx43 by ERK1/2 (4, 24) may be, in part
or entirely, through the MEK5/BMK1 pathway. Through the use of dominant
negative and constitutively active molecular reagents with greater
specificity than pharmacological reagents, we have demonstrated that
activated BMK1 leads directly to Cx43 phosphorylation in
vitro and in vivo and to the inhibition of GJC.
Cx43 phosphorylation by selective BMK1 activation and consequent
inhibition of GJC but insensitivity to MEK1 manipulations indicate that
activated BMK1 but not ERK1/2 mediates the EGF-induced Cx43 gap
junction uncoupling. This is supported by highly specific molecular
manipulations showing a lack of cross-talk between the ERK1/2 and BMK1
signaling pathways and thus a delineation of responses elicited by
these two pathways when activated by EGF (Fig. 2).
Our data also demonstrate that EGF-induced ERK1/2 activation and
consequent Cx43 phosphorylation, in isolation, may not be a direct
correlate for inhibition of GJC in vivo. ERK1/2 activation by CA-MEK1 co-transfection with the triple Cx43 mutant
(S255A/S279A/S282A) could still induce electrophoretic mobility shift,
and so the existence of alternative ERK1/2 phosphorylation sites on
Cx43 in addition to Ser-255, Ser-279, and Ser-282 is apparent.
The fact that we did not observe this same result with activated BMK1 may indicate a greater specificity for this protein kinase.
DN-MEK1 or DN-BMK1 can significantly inhibit phosphorylation of the
double Cx43 mutant (S279A/S282A) after EGF stimulation, and thus
Ser-255 is clearly targeted by each of these activated kinases. It
remains unresolved as to why BMK1 but not ERK1/2 can regulate
EGF-induced Cx43 gap junctional uncoupling through Ser-255 phosphorylation alone. Cx43 can be phosphorylated in vivo on
multiple residues by ERK1/2, but because only activated BMK1 inhibits
Cx43 function, ERK1/2 phosphorylation elsewhere may affect BMK1
regulation of Cx43. A coordinated effort between activated ERK2 and
activated BMK1 in signaling pathways has been reported (25). However, BMK1 but not ERK1/2 association with Cx43 was demonstrated, and so it
is quite possible that BMK1 binding coupled with BMK1-induced Cx43
phosphorylation on Ser-255 has an equally important effect in
controlling GJC. Moorby and Patel (26) reported that single mutants of
Ser-255, Ser-279, and Ser-282 independently prevented GJC blockade
following treatment of the cells with platelet-derived growth
factor, suggesting the important role for each independent phosphorylated serine of Cx43 in controlling its gating properties. It
is noteworthy that the gating of Cx43 and gap junctions can indeed be
achieved by phosphorylation-independent mechanisms, particularly via
protein-protein interactions such as the interaction of Cx43 with the
tight junction protein ZO-1 and the oncogenic product v-Src (15, 27).
Such protein-to-protein interaction with connexins, recently referred
to as the "nexus," is an emerging mechanism that could regulate
GJC independently (28). Because the C-terminal region of BMK1 interacts
with the transcription factor MEF2 to potently promote its
transcriptional activation independently from phosphorylation (29), it
is possible that the physical interaction of BMK1 with Cx43 may
influence GJC. Peracchia et al. (30) have shown that the
calcium-binding protein, calmodulin, can directly gate Cx32 in the
so-called "cork" model. Cx43 not only co-localizes with the v-Src
tyrosine kinase to plasma membrane regions of the cell, but it also
appeared to interact directly with v-Src (31, 32). This is particularly
intriguing because we demonstrate here that basally active
overexpressed BMK1 and endogenous BMK1 complex with Cx43 and that this
association is decreased substantially when BMK1 is activated. Because
we were unable to demonstrate Cx43 and ERK1/2 association, it is reasonable that the profound effect of BMK1 on GJC could be assigned in
part to Cx43 association as well as to BMK1 phosphorylation. Additional
studies will be required for the further investigation of the role of
BMK1/Cx43 protein-protein interaction in regulating GJC.
, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap
junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on
Ser-255 in vitro and in vivo, but not on
S279/S282, which are reported as the consensus phosphorylation
sites for MAPK. Furthermore, by co-immunoprecipitation, we found
that BMK1 directly associates with Cx43 in vivo. These data
indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43
gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(S311D/S315D), Cx43 mutants (S255A, S279A/S282A, S255A/S279A/S282A)
were created using the two-step PCR method for mutagenesis. ERK5b, a
splice variant of full-length BMK1, was used as the selective inhibitor of BMK1 (8) and designated DN-BMK1. Cx43-EGFP, DN-MEK1-DsRed, CA-MEK1-DsRed, and CA-MEK5
-DsRed fusion constructs were created by a
C-terminal fusion with enhanced green fluorescent reporter protein
(EGFP) or DsRed cDNA (Clontech, Palo Alto, CA).
The integrity of all engineered constructs was verified by automatic
sequencing. Cx43 (wild type) cDNA was a kind gift from Dr. Camillo
Peracchia (University of Rochester). Experiments were carried out
24 h after transient transfection.
exp
t/
rec) using Origin, version 6.0 (Origin Laboratories, Northampton, MA).
F(t) is the normalized fluorescence at time
t, F0 is the initial pre-bleach
fluorescence intensity, and
rec is the mono-exponential recovery time constant. The average
rec is given as
mean ± S.E., and statistical analysis was performed with the
StatView 4.0 package (Abacus Concepts, Berkeley, CA).
-cyano-4-hydroxycinnamic acid, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis was performed in the University of Rochester Protein/Peptide Core Facility as described previously (14). Mass fingerprinting analysis and determination of phosphorylation were performed initially by
MS-FIT.2 The data base search
was considered significant if the protein was ranked as the best hit
with a sequence coverage of more than 30%. Significance was defined as
a molecular weight search (MOWSE) score of at least 1e+003 (MS-FIT) or a difference in
probability of 10
3 from the first to the second protein
candidate (ProFound).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
BMK1 and ERK1/2 are potential mediators for
EGF-induced phosphorylation of Cx43 in vivo. A,
Cx43 expression in HEK293 cells, which have no endogenous Cx43.
IB, immunoblotting. B, EGF (100 ng/ml, 20 min)
induces Cx43 (wild type) and Cx43E (EGFP-tagged) mobility shifts but no
mobility shift of EGFP alone. C, EGF-induced Cx43E mobility
shift is fully reversed by alkaline phosphatase treatment.
D, PD98059 (50 µM, 1 h) collapses
EGF-induced Cx43E mobility shift. ERK1/2 and BMK1 activation are also
diminished. Representative blots are from at least three independent
experiments.
, which potently activates endogenous BMK1,
does not activate ERK1/2 (Fig. 2C), supporting its
specificity. Finally, ERK5b, a splice variant of full-length BMK1, was
used as the selective inhibitor of BMK1 (8) because we discovered that
a DN-MEK5 construct described previously (12) was not quite as
efficient in inhibiting BMK1 in our cell type under our particular
conditions. ERK5b is referred to as DN-BMK1. We found that unlike
wild-type BMK1 (ERK5a), ERK5b has no kinase activity (Fig. 2D,
panel 1), although it can specifically inhibit BMK1-induced MEF2C
transcriptional activity (Fig. 2D, panel 2) without
affecting EGF-induced ERK1/2 activation (Fig. 2D, panel 3).
This gives us confidence that the most downstream component of the
signaling pathway can be inhibited effectively.
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Fig. 2.
No cross-talk exists between the BMK1 and
ERK1/2 pathways. A, CA-MEK1 (S218E/T222E)
selectively activates ERK1/2 without activating BMK1 (shown as
phosphorylation of the BMK1 substrate, MEF2C, in an in vitro
kinase assay). IB, immunoblotting; IP,
immunoprecipitation. B, DN-MEK1 (S218A/T222A) selectively
inhibits EGF-activated ERK1/2 without affecting BMK1 activity.
C, CA-MEK5 (S311D/S315D) selectively activates BMK1
without altering ERK1/2 activity. D, panel 1,
in vitro kinase assay demonstrates wild-type BMK1
autophosphorylation by CA-MEK5
. DN-BMK1, conversely, is kinase-dead.
Panel 2, DN-BMK1 but not wild-type BMK1 inhibits activated
BMK1-induced MEF2C transcriptional activity. Results are represented as
the mean ± S.E., and an asterisk denotes statistical
significance. Panel 3, DN-BMK1 inhibits EGF-activated BMK1
without altering ERK1/2 activity. Representative blots are from at
least three independent experiments.
, and
DN-BMK1 to selectively activate or inhibit the two pathways. With the
exception of DN-BMK1, these constructs were C-terminal DsRed fusion
proteins, allowing clear visual confirmation of successful
co-expression with Cx43E. Typically, we obtained 80-90% transfection
efficiency, determined from co-localization of the GFP and DsRed
reporter fluorescence (data not shown). The direct functional coupling
between cells was studied using the FRAP assay. A typical cell cluster
selected for FRAP assay exhibited Cx43E in punctate arrangements at the
plasma membrane (Fig. 3A). Ultraviolet laser bleaching of the selected cell resulted in the loss
of fluorescence with a subsequent time-dependent recovery of this fluorescence as the reporter molecule diffused back through gap
junctions coupled to neighboring cells (Fig. 3B). A
representative fluorescence recovery curve for control and
EGF-stimulated Cx43E-expressing HEK293 cells is shown in Fig.
3C. Quantitation of cell-to-cell coupling revealed that EGF
inhibited GJC (Fig. 3D, lane 2), which was
apparent from the increase in the mean recovery time constant. Co-transfection of DN-BMK1 prevented this inhibition (lane
3), but in contrast, DN-MEK1 had no effect on the EGF-induced
uncoupling (lane 4). CA-MEK5
, which activated endogenous
BMK1, blocked GJC in the absence of EGF stimulation, but CA-MEK1 failed
to inhibit GJC (lanes 5 and 6, respectively). In
conjunction with the biochemical data demonstrating the specificity of
the reagents we used, these findings suggest a novel BMK1-mediated
signaling pathway that regulates GJC. BMK1 activation by EGF is
apparently a hitherto unrecognized critical component in Cx43 gap
junction uncoupling.
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Fig. 3.
EGF-induced activation of BMK1 is sufficient
for functional inhibition of GJC. A, under
fluorescent microscopy using fluorescein isothiocyanate
(FITC), HEK293 cells transfected with Cx43E demonstrated
punctuate, often linear arrangements of connexons along the plasma
membrane. B, fluorescence of the photobleached cell
(arrowheads) recovered over a 5-min time period as
the reporter molecule diffused through gap junctions from surrounding
cells (top panels). With EGF stimulation (bottom
panels), the fluorescence recovery was slow. C,
fluorescence recovery after photobleaching exhibited a mono-exponential
time course; the absolute fluorescence corrected for background
bleaching was fitted to a first order rate equation (see
"Experimental Procedures"). The symbols are
the corrected fluorescence values, with fitted mono-exponential
recovery time course. D, summary bar diagrams of recovery
time constants for EGF-stimulated cells transfected with the denoted
constructs. All results are represented as mean ± S.E.
(n = 9-24 cells). *, denotes statistical
significance.
to constitutively activate endogenous BMK1, and
this was immunoprecipitated with an anti-BMK1 antibody. An in
vitro kinase assay was performed between activated BMK1 and GST
(negative control), GST-MEF2C (activation domain, positive control),
and GST-Cx43 (C-terminal region) as substrates. Activated BMK1 can
phosphorylate GST-MEF2C and GST-Cx43 but not GST (Fig. 4A).
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Fig. 4.
BMK1 phosphorylates Cx43 on the C terminus
in vitro and associates with Cx43 in
vivo. A, in vitro kinase assay
demonstrates 32P incorporation into GST-Cx43 (amino
acids 230-382, left panel) and GST-MEF2C (activation
domain, right panel) but not into GST (negative control).
Endogenous BMK1 was activated by CA-MEK5 transfection and then
immunoprecipitated from HEK293 cells immediately before the in
vitro kinase assay was performed. Ponceau S staining of the
nitrocellulose membrane demonstrates the position of the proteins after
separation by SDS-PAGE. IP, immunoprecipitation.
B, Cx43E coimmunoprecipitates with basally active BMK1 but
less so when BMK1 is activated by CA-MEK5
transfection. A stably
expressing Cx43E HEK293 cell line was transfected with Xpress-tagged
BMK1 without (
) or with CA-MEK5
(+). Cx43E was immunoprecipitated
with anti-GFP antibody and immunoblotted with anti-Xpress antibody.
C, additional control (GFP alone co-expressed with
Xpress-tagged BMK1) shows lack of association and confirms the
Cx43-BMK1 association demonstrated. The total cell lysate
(TCL) lanes demonstrate the presence of input Cx43E proteins
for both conditions. D, a complementary experiment where
BMK1 was immunoprecipitated with anti-Xpress antibody and Cx43E was
immunoblotted with anti-GFP antibody. A loading control is shown at the
bottom of each autoradiogram. These data are representative
of four separate experiments. E, panel 1, Cx43E
was immunoprecipitated with anti-GFP antibody, and endogenous BMK1 was
immunoblotted with anti-BMK1 antibody. BMK1 is very potently activated
by H2O2 as we reported previously, and thus it
was used to activate BMK1 (10). Panel 2, Cx43E was
immunoprecipitated with anti-GFP antibody, and endogenous ERK1/2 was
immunoblotted with anti-ERK1/2 antibody. Cx43 and ERK1/2 association
was not apparent basally or when ERK1/2 or BMK1 activation status was
altered by EGF stimulation ± DN-MEK1 or DN-BMK1. The
TCL lanes demonstrate the presence of input of total
proteins for each condition.
co-transfection. This Cx43 and BMK1 association was a reciprocal phenomenon and was detected by
repeating the co-immunoprecipitation experiment using antibodies against the epitope tag on each protein in a reverse manner (Fig. 4D, IP, BMK1, and IB, Cx43). In HEK293
cells, endogenous inactive BMK1 also co-immunoprecipitated with Cx43,
and this association was absent when BMK1 was activated (Fig.
4E, panel 1), but all attempts to
co-immunoprecipitate either active or inactive ERK1/2 with Cx43 proved
unsuccessful (Fig. 4E, panel 2). These data
suggest a novel role for BMK1 in regulating Cx43 GJC via
phosphorylation and possibly also suggest association with Cx43.
transfection and incubated
with GST-Cx43CT in an in vitro kinase reaction.
Computer-assisted proteomic analysis revealed two tryptic peptide ions
with mass/charge ratios of 1877.7906 and 3155.3995, corresponding to
the C-terminal fragmented peptides spanning amino acids 244-258
and 265-293, respectively. Furthermore, we observed that only the
amino acid region 244-258 was modified by phosphorylation on a single
serine residue. Threonine and tyrosine residues within that region were
not modified, and there were no covalent modifications within the amino
acid 265-293 tryptic-digested region. Because Ser-255 is the only
potential BMK1 phosphorylation site within that region, we suspected
that it might be the preferred phosphorylation site for BMK1, even
though two other potential BMK1 phosphorylation sites were available
for phosphorylation.
to constitutively activate endogenous BMK1, which was
immunoprecipitated with an anti-BMK1 antibody. As before, an in
vitro kinase assay with mutant GST-Cx43 (C-terminal region, amino
acids 230-382) as a substrate was performed. As shown in Fig.
5, we found that the double mutant
(S279A/S282A) was potently phosphorylated by BMK1. However,
activated BMK1 failed to phosphorylate Cx43 single (S255A) or triple
mutant (S255A/S279A/S282A) peptides. Our in vitro kinase
assay strongly suggests that Ser-255 is the only residue that is
accessible for BMK1-mediated phosphorylation, which is consistent with
our MALDI-TOF data.
View larger version (51K):
[in a new window]
Fig. 5.
In vitro BMK1 phosphorylation of
Cx43 on the C terminus is on Serine 255. In vitro
kinase of single (S255A), double (S279A/S282A), and triple
(S255A/S279A/S282A) mutants of Cx43-CT demonstrates the loss of
32P incorporation when Ser-255 is mutated to alanine.
Endogenous BMK1 was activated by CA-MEK5 transfection and then was
immunoprecipitated (IP) from HEK293 cells immediately before
the in vitro kinase assay was performed.
to activate endogenous ERK1/2 or BMK1,
respectively, in HEK293 cells.
and CA-MEK1 can phosphorylate Cx43E.
When we co-transfected the Cx43 (S255A) mutant with CA-MEK1 or
CA-MEK5
, we observed that only CA-MEK1 induced a modest
electrophoretic mobility shift (Fig. 6B). When Cx43
(S279A/S282A) was co-transfected with CA-MEK1 or CA-MEK5
, we
observed that both CA-MEK1 and CA-MEK5
induced an electrophoretic shift (Fig. 6C, panel 1). Moreover,
co-transfection of Cx43 (S279A/S282A) with a null vector and
stimulation with EGF yielded a reproducibly small electrophoretic
mobility shift on Cx43. This shift was abolished when EGF stimulation
was repeated in cells co-transfected with DN-BMK1 or DN-MEK1 (Fig.
6C, panel 1), and co-transfection of wild-type
Cx43 with DN-BMK1 together with CA-MEK1 showed that Cx43E
phosphorylation was still present (Fig. 6C, panel
2). Finally, co-transfection of Cx43 (S255A/S279A/S282A) with
CA-MEK1 or CA-MEK5
demonstrated an electrophoretic mobility shift
only when CA-MEK1 was transfected (Fig. 6D). These findings
strongly suggest that activated BMK1 induces Cx43 phosphorylation
preferentially at Ser-255 but not at Ser-279 or Ser-282 in
vivo. In contrast, ERK1/2 may phosphorylate Cx43 at several sites
in addition to Ser-255, Ser-279, and Ser-282.
View larger version (42K):
[in a new window]
Fig. 6.
In vivo BMK1 phosphorylation of
Cx43 on the C terminus is on serine 255. A, wild-type Cx43;
B, single mutant (S255A); C, panel 1, double
mutant (S279A/S282A); panel 2, wild-type Cx43. D,
triple mutants (S255A/S279A/S282A) of Cx43 were co-transfected with
null vector and stimulated with EGF ± DN-BMK1 or DN-MEK1 or
co-transfected with CA-MEK1 or CA-MEK5 without EGF stimulation to
selectively activate endogenous ERK1/2 and BMK1, respectively.
Ser-255, which appears to be the only BMK1 phosphorylation site
on Cx43 in vivo, is signified by the appearance of a
phosphorylation-induced electrophoretic mobility shift. These data are
representative of three independent experiments. IB,
immunoblotting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical support of Nancy C. Ward with some of the molecular reagents and Dr. Bradford C. Berk for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by American Heart Association Grant AHA 0110123T (to S. J. C.), New York State Department of Health Grant SCRIB C016891(to J. Y.), and National Institutes of Health/NHLBI Grant RO1 HL66919-02 (to J. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ In partial fulfillment of the requirements for the Ph.D. degree.
¶ These authors contributed equally to this work.
To whom correspondence may be addressed: Center for
Cardiovascular Research, University of Rochester Medical Center,
Rochester, NY 14642. Tel.: 585-273-1686; Fax: 585-275-9895; E-mail:
Jun-ichi_Abe@urmc.rochester.edu.
¶¶ To whom correspondence may be addressed: Dept. of Anesthesiology, Columbia University Physicians and Surgeons, 630 West 168th Street, New York, NY 10032. Tel.: 212-342-0023; Fax: 212-305-0777; E-mail: Jy2029@columbia.edu.
Published, JBC Papers in Press, March 12, 2003, DOI 10.1074/jbc.M213283200
2 On the Web at Prospector.ucsf.edu.
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ABBREVIATIONS |
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The abbreviations used are: Cx43, connexin 43; Cx43E, Cx43 with a C-terminal EGFP tag; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; BMK1, big MAP kinase 1; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; DsRed, Distal Red protein; GJC, gap junctional communication; FRAP, fluorescence recovery after photobleaching; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; DN, dominant negative; CA, constitutively active; EGF, epidermal growth factor; GST, glutathione S-transferase; MEF2C, myocyte elongation factor 2C; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; HEK, human embryonic kidney; JNK, c-Jun N-terminal kinase; PD98059, MEK1 antagonist.
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