From Molecular Medicine, Northwest Hospital, Seattle,
Washington 98125 and the ¶ Schepens Eye Research Institute,
Harvard Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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The platelet-derived growth factor (PDGF)
mediates its cellular functions via activation of its receptor tyrosine
kinase followed by the recruitment and activation of several signaling
molecules. These signaling molecules then initiate specific signaling
cascades, finally resulting in distinct physiological effects. To
delineate the PDGF signaling pathway responsible for the disruption of
gap junctional communication (GJC), wild-type PDGF receptor The platelet-derived growth factor
(PDGF)1 induces a variety of
cellular responses, including mitogenesis, Ca2+ influx,
cell migration, etc. (1). Typical of polypeptide growth factors, the
actions of PDGF are mediated via its cellular receptor, the PDGF
receptor (PDGFR). Following the binding with PDGF, the receptor
undergoes dimerization and conformational changes that trigger the
activation of the intrinsic tyrosine kinase of the receptor, resulting
in the phosphorylation of several tyrosine residues of PDGFR In addition to inducing biological events such as mitogenesis, cell
migration, etc., PDGF is also a strong inhibitor of intercellular communication via gap junctions, or gap junctional communication (GJC)
(13-16). Gap junctions are narrow, water-filled intercellular channels
composed of a highly conserved group of membrane proteins termed
connexins (17, 18). These channels serve as conduits for the
intercellular exchange of small molecules and ions, some of which are
proposed to act as regulatory signals governing cell proliferation,
neoplastic transformation, embryonic development, etc. (19-22). Since
opening and closure of these channels will regulate the intercellular
levels of these yet to be identified signals, inhibition of GJC by
mitogens (15, 16, 23), tumor promoters (24-26), and oncogenes (27, 28)
and enhancement of GJC by antineoplastic agents (29, 30) are considered
to be critical for augmenting their respective effects.
To study the mechanism of PDGF-induced disruption of GJC, we have
developed a cell system derived from T51B rat liver epithelial cells
(15), which are devoid of any endogenous PDGFR expression, but are
GJC-competent and express high levels of a gap junctional protein,
connexin-43 (Cx43). Following retroviral infection with a construct
containing wild-type PDGFR Cell Culture and Materials--
T51B is a non-neoplastic
epithelial cell line derived from rat liver, the origin of which has
been described (35), and has been widely used for various studies (23,
36, 37). The cells were grown to confluence in a complete medium
consisting of 90% Eagle's basal medium and 10% bovine calf serum
(Colorado Serum Co., Denver, CO) and were maintained in Eagle's basal
medium alone for 18 h before treatment with PDGF (5 ng/ml, unless
specified). Recombinant human PDGF-BB was a kind gift from Dr. C. Hart
(Zymogenetics, Seattle, WA), and U73122 was purchased from Alexis
Biochemicals (San Diego, CA).
Antibodies--
Monoclonal anti-PDGFR Expression Vectors and Cell Infections--
The virus-packaging
PA317 cell lines expressing wild-type, kinase-dead, and single-site
mutant human PDGFR Analysis of Gap Junctional Communication--
Gap junctional
communication was assessed by transfer of the fluorescent dye Lucifer
yellow after single-cell microinjection performed as described
previously (15, 16). Briefly, glass micropipettes were prepared using a
Flaming/Brown micropipette puller (Sutter Instrument Co., Nevato, CA)
and back-filled with 10% solution of Lucifer yellow in 0.33 M LiCl. Injection of cells in confluent cultures was
achieved with pressure delivery by an Eppendorf microinjector. The
cells were observed under a fluorescence inverted microscope, and after
5-7 min of microinjection, the number of neighboring cells exhibiting
dye labeling was recorded. For each treatment group, a minimum of
20-30 cells in different areas of the cultures were microinjected.
Each experiment was conducted at least three times. The mean number of
communicating cells ± S.D. was calculated for each mutant or
treatment group. The values of S.D. (data not shown) were typically
<10% of the mean values.
Immunoblotting--
Western blot analyses were performed as
described previously (15, 16). Briefly, culture dishes containing
confluent cells were quickly washed with cold phosphate-buffered saline
containing 1 mM sodium orthovanadate, 5 mM
EDTA, 10 mM NaF, and 1 mM phenylmethylsulfonyl fluoride and then harvested in the same solution. The cell pellets were
lysed in 1 mM NaHCO3 containing 1 mM sodium orthovanadate, 5 mM EDTA, 10 mM NaF, and 1 mM phenylmethylsulfonyl fluoride. An equal amount (5-10 µg) of lysate protein was resolved on
polyacrylamide gels and transferred to polyvinylidene difluoride
membranes (Bio-Rad). After blocking for 16 h at 4 °C in a
solution consisting of 5% nonfat dry milk in Tris-buffered saline (20 mM Tris, pH 7.6 and 137 mM NaCl) containing
0.2% Tween 20, membranes were incubated with anti-Cx43 antibody for
1-2 h at room temperature. The membranes were then washed several
times in Tris-buffered saline containing 0.2% Tween 20, incubated with
horseradish peroxidase-conjugated donkey anti-rabbit IgG, and processed
with an enhanced chemiluminescence kit (Kirkegaard & Perry
Laboratories, Inc., Gaithersburg, MD). For anti-phosphotyrosine and
anti-PMAPK antibodies, 1% bovine serum albumin was used during the
blocking and antibody incubation steps.
Immunoprecipitation--
To detect recruitment of signal
transduction proteins by activated PDGFR, confluent cells were treated
with PDGF for 10 min and lysed with solubilizing buffer (15, 39)
containing 1% Triton X-100, 0.5% deoxycholate, 10% glycerol, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 5 mM EDTA
in Hepes-buffered saline (50 mM Hepes, pH 7.5, and 150 mM NaCl) for 30 min at 4 °C. Cell lysates, obtained
after centrifugation at 14,000 × g for 10 min, were
incubated overnight at 4 °C with a monoclonal anti-PDGFR In our previous study, we demonstrated a PDGF-induced Cx43
phosphorylation and GJC blockade in T51B cells expressing wild-type PDGFR We first infected T51B cells with the F5 construct containing
tyrosine-to-phenylalanine mutations at positions 740/751, 771, 1009, and 1021, which are required for the recruitment of PI3K, GAP, SHP-2,
and PLC Generation of the mutant F5 cDNA construct and its introduction
into virus-packaging PA317 cells and infection of T51B cells were
achieved as described previously (6, 15). Following G418 selection,
expression of the F5 mutant in infected T51B cells was confirmed (data
not shown) by Western blotting performed with anti-PDGFR (PDGFR
) and a series of PDGFR
mutants were expressed in T51B rat
liver epithelial cells. In cells expressing wild-type PDGFR
, PDGF
induced disruption of GJC and phosphorylation of a gap junctional
protein, connexin-43 (Cx43), which required activation of
mitogen-activated protein kinase, although involvement of additional
factors was also evident. In the F5 mutant lacking binding sites for
phosphatidylinositol 3-kinase, GTPase-activating protein, SHP-2, and
phospholipase C
1 (PLC
1), PDGF induced mitogen-activated protein
kinase, but failed to affect GJC or Cx43, indicating involvement of
additional signals presumably initiated by one or more of the mutated
binding sites. Examination of the single-site mutants revealed that
PDGF effects were not mediated via a single signaling component. This was confirmed by the "add-back" mutants, which showed that
restoration of either SHP-2 or PLC
1 binding was sufficient to
propagate the GJC inhibitory actions of PDGF. Further analysis showed
that activation of PLC
1 is involved in Cx43 phosphorylation, which
surprisingly failed to correlate with GJC blockade. The results of our
study demonstrate that PDGF-induced disruption of GJC can be mediated by multiple signaling pathways and requires participation of multiple components.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1-4).
Together with their adjacent sequences, these phosphotyrosine residues
form binding sites for specific proteins. In the case of PDGFR
,
critical phosphotyrosine residues include Tyr-579 and Tyr-581 (for
Src), Tyr-716 (Grb2), Tyr-740 and Tyr-751 (phosphatidylinositol
3-kinase (PI3K)), Tyr-751 (Nck), Tyr-771 (GTPase-activating protein
(GAP)), Tyr-1009 (protein-tyrosine phosphatase SHP-2 and phospholipase
C
(PLC
)), and Tyr-1021 (PLC
) (1, 4, 5). Many of these signal
transduction molecules possess intrinsic catalytic properties
(e.g. PI3K, PLC
, etc.) or act as adaptors
(e.g. Grb2) to link other molecules with enzymatic properties. Following recruitment and activation, these molecules initiate specific cascades of events to propagate the signal resulting in a particular biological response. Examination of PDGFR mutants lacking single or multiple tyrosine residues provided useful
clues regarding the roles of individual signaling molecules modulating various biological end points of PDGF treatment (6-10). Several of these studies demonstrated that PDGF effects can be mediated by multiple signaling pathways (6, 9-12).
cDNA, these cells (Kin+)
showed a PDGF-induced rapid and transient interruption of GJC and a
concomitant phosphorylation of Cx43 presumably at serine/threonine residues (15). Phosphorylation of Cx43 is believed to be causally linked with the disruption of GJC (31). Our subsequent study revealed
that a sequential activation of protein kinase C (PKC) and
mitogen-activated protein kinase (MAPK) was required for mediating these PDGF effects (16). Although MAPK was shown to be the kinase mediating the epidermal growth factor-induced phosphorylation of Cx43
(32, 33), for PDGF, we demonstrated that 1) activation of MAPK was not
sufficient for the phosphorylation of Cx43 or disruption of GJC; and 2)
phosphorylation of Cx43 was not sufficient for the GJC
blockade.2 These results
indicate that inhibition of GJC requires the participation of multiple
components, including protein kinases and Cx43-associated proteins.
Since PDGFR activation leads to multiple signaling pathways (1, 4), we
wished to determine the contribution of each signaling pathway in the
disruption of GJC. To achieve this goal, a series of PDGFR mutants
lacking single or multiple Tyr residues (6) were expressed in T51B
cells. Our results indicate that PDGF-induced disruption of GJC is
mediated via multiple pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
antibody was a generous
gift from Dr. C. Hart; polyclonal anti-PDGFR
antibody was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Cx43 antibody was
raised in rabbits by injecting a peptide corresponding to the last 16 amino acids of the C-terminal region of the Cx43 sequence (15, 16).
Antibodies against SHP-2, p85PI3K, and GAP and horseradish
peroxidase-conjugated anti-phosphotyrosine antibody were obtained from
Transduction Laboratories (Lexington, KY), and anti-PLC
antibody was
obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal
antibodies against phospho-MAPK (PMAPK) were obtained from New England
Biolabs Inc. (Beverly, MA) and Promega (Madison, WI). Horseradish
peroxidase-conjugated secondary antibodies were obtained from Jackson
ImmunoResearch Laboratories, Inc. (West Grove, PA).
cDNAs subcloned into pLXSN retroviral vector
(38) were kindly provided by Dr. J. Cooper (Fred Hutchinson Cancer
Research Center, Seattle, WA). Generation of the F5 and "add-back"
mutants has been previously described (6). Culture medium from the
virus-packaging cells was used to infect T51B cells. Following
selection with G418, populations of infected cells were obtained.
Immunoprecipitation and Western blotting techniques were utilized to
confirm the expression of functional PDGFR in these cells (see
"Results").
antibody
(5 µg/ml). The immune complexes were precipitated with protein
A-Sepharose beads (Pharmacia Biotech AB, Uppsala, Sweden). After
washing three times with solubilizing buffer and once with
Hepes-buffered saline, the immunoprecipitated material was eluted by
boiling with 40 µl of SDS-PAGE loading buffer containing 2%
-mercaptoethanol. Equal volumes were loaded for SDS-PAGE, and the
proteins were transferred to polyvinylidene difluoride membranes as
described above. To verify PDGFR activation of PDGF-treated cells,
membranes were incubated with antibodies recognizing phosphotyrosine,
PLC
, p85PI3K, SHP-2, or GAP followed by appropriate
horseradish peroxidase-conjugated secondary antibodies and processed
with an enhanced chemiluminescence kit.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Kin+) (15). These effects were not observed in
Kin
cells expressing kinase-inactive PDGFR
, indicating
that phosphorylation of tyrosine residues in PDGFR
is required for
PDGF-induced GJC blockade and Cx43 phosphorylation. As illustrated in
previous studies, tyrosine phosphorylation of PDGFR is required for the activation and recruitment of various signaling molecules, which lead
to induction of various protein kinases and phosphatases (1, 4).
Requirement of such signaling cascades in the PDGF-induced GJC blockade
and Cx43 phosphorylation was recently demonstrated by showing that
these two effects were dependent on the activation of two PDGF-induced
kinases, namely PKC and MAPK (16). Interestingly, our subsequent study
showed that factors additional to MAPK are also required,2
which suggests that PDGF-induced GJC blockade possibly involves multiple components. To define these components of the PDGF signaling pathway, our experimental strategy involves expression of a series of
PDGFR
mutants in which single or multiple tyrosine residues were
altered to phenylalanines in our T51B cell system, which is devoid of
any detectable expression of PDGFR
(15) and does not show any
PDGF-induced GJC blockade (15), activation of MAPK (16), or mitogenesis
or Ca2+ entry.3
Since these tyrosine residues, following phosphorylation, form binding
sites for specific signal transduction molecules (1, 6), substitution
of these Tyr residues with Phe will prevent signaling cascades
initiated by these signaling molecules. This approach allows us to
examine the contribution of a particular tyrosine and the corresponding
signaling molecule in the regulation of GJC/Cx43 by PDGF. Similar
strategies have identified the requirement of PI3K and PLC
in
PDGF-induced mitogenesis and neoplastic cell transformation (6,
10).
1, respectively (6). Our rationale was that by first
examining the F5 mutant, we could either confirm or eliminate the
involvement of the mutated tyrosine residues in the modulation of
GJC/Cx43 by PDGF. In case of a successful PDGF signal propagation,
remaining tyrosines such as Tyr-579 and Tyr-581 (Src site) and Tyr-716
(Grb2 site) would be examined. On the other hand, if F5 failed to
modulate GJC/Cx43 after PDGF treatment, the involvement of each
tyrosine substituted in this mutant would be investigated.
antibody
(15). To verify the functionality of the F5 mutant, PDGFR
was
immunoprecipitated from control and PDGF-treated cells and resolved by
SDS-PAGE followed by Western blotting with various antibodies
recognizing PDGFR-associated proteins (15). In Fig.
1, we show that tyrosine-to-phenylalanine
substitutions in the F5 mutant did not impair its receptor tyrosine
kinase activity, being of similar magnitude between Kin+
(wild type) and F5 cells. As expected, recruitment of GAP, PLC
1, or
PI3K (Fig. 1) or SHP-2 (data not shown) by the activated receptor was
completely abolished in the F5 mutant. This finding demonstrates that
the F5 mutant is deficient in initiating pathways via these signaling
molecules.
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Fig. 1.
Activation of PDGFR
in T51B cells expressing wild-type (Kin+) or F5
mutant PDGFR
. Confluent cells treated
with buffer (
) or 5 ng/ml PDGF (+) for 10 min and lysed, and PDGFR
was immunoprecipitated using a monoclonal anti-PDGFR
antibody. The
immunoprecipitates were separated by SDS-PAGE followed by Western
blotting and separately probed with antibodies recognizing the
indicated proteins.
After the confirmation of the characteristics of the mutant receptor,
F5 and wild-type Kin+ cells were treated with PDGF (5 ng/ml) for 20 min and then analyzed for GJC by dye microinjection and
for Cx43 by Western blotting. As observed previously (15,
16),2 PDGF treatment caused a total disruption of GJC in
Kin+ cells (Fig.
2A). In contrast, GJC in F5
cells was not significantly influenced by the same treatment,
indicating that one or more of the substituted tyrosine residues are
involved in PDGF-induced disruption of GJC. In a separate experiment,
F5 cells failed to show any disruption of GJC even when treated with a
higher (10 ng/ml) PDGF concentration for a longer (40 min) length of
time (data not shown), which suggested that the absence of PDGF action in F5 cells was due to the deleted tyrosine residues rather than a
submaximal activation of the signaling pathways. Since inhibition of
GJC was linked with the phosphorylation of Cx43 (15, 16, 25, 26, 31),
we next compared phosphorylation profiles of Cx43 in Kin+
and F5 cells. Untreated Kin+ or F5 cells contain three
characteristic Cx43 protein bands representing a minor,
non-phosphorylated form (Cx43-NP) and two highly abundant, phosphorylated forms (Cx43-P1 and Cx43-P2). Whereas the former form
represents nascent, predominantly cytoplasmic Cx43, the
post-translationally modified latter forms are believed to be required
for the assembly of functional gap junctional channels (40). As
previously reported (15, 16), PDGF treatment induced
hyperphosphorylation of Cx43, as indicated by the appearance of several
higher molecular mass Cx43 bands (Cx43-P3) in Kin+ cells
(Fig. 2A). In accord with the GJC data, PDGF treatment failed to generate any distinct high molecular mass Cx43 forms in F5
cells, indicating a lack of activation of the responsible Cx43/kinase
system. Since activation of MAPK has been shown to be required for
PDGF-induced Cx43 phosphorylation and GJC blockade (16), we also
investigated MAPK activation in Kin+ and F5 cells by
Western blotting using an antibody that specifically recognizes the
catalytically active, phospho form of MAPK (PMAPK), but not the
catalytically inactive, non-phospho form of MAPK (16).2
Previous studies showed excellent correlation between MAPK activity and
levels of PMAPK identified by similar antibodies (41, 42). The
activation of MAPK by PDGF in Kin+ and F5 cells was of
similar magnitude, as indicated by the appearance of PMAPK bands (Fig.
2A), which is consistent with a previous study in PC12 cells
(9). The absence of Cx43 phosphorylation and GJC blockade in F5 cells
despite the activation of MAPK confirms our previous observation that
although required, activation of MAPK is not sufficient in PDGF-induced
Cx43 phosphorylation or interruption of GJC.2 This result
also suggests that additional signals are required that are supposedly
provided by the one or more pathways augmented by the mutated tyrosine
residues.
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To verify that the failure of PDGF to interrupt GJC and to induce Cx43
phosphorylation in F5 cells is not due to any defects in the gap
junction structure and/or Cx43 conformation, we treated both
Kin+ and F5 cells with a tumor promoter phorbol ester,
12-O-tetradecanoylphorbol-13-acetate, a known inhibitor of
GJC and an inducer of Cx43 phosphorylation (16, 25, 26). In Fig.
2B, we demonstrate that both cell types responded
identically to 12-O-tetradecanoylphorbol-13-acetate treatment by showing a complete disruption of GJC and generation of
slow-migrating Cx43 forms. This result confirms that the lack of
PDGF-induced disruption of GJC in F5 cells is due to an impairment in
the PDGF signaling pathway resulting from the substitution of tyrosine
residues in PDGFR.
Since the mutation of five tyrosine residues resulted in the silencing
of PDGF signaling to GJC/Cx43 (Fig. 2), it is evident that one or more
of these tyrosines are involved in PDGF-induced disruption of GJC and
Cx43 phosphorylation. To understand the roles of each tyrosine and its
corresponding signaling pathway, T51B cells were retrovirally infected
with a panel of mutant PDGFR constructs in which individual
tyrosines were mutated to prevent the recruitment of a specific
signaling molecule (43). These single-site mutants include
740/751
, 771
, 1009
, and
1021
, which lack the binding sites for PI3K, GAP, SHP-2,
and PLC
1, respectively. Verification of the expressed PDGFR mutants
in the G418-resistant clones was performed by immunoprecipitating the receptor followed by Western blot analyses using the antibodies described previously (Fig. 1). In Fig. 3,
we show that although the receptor tyrosine kinase activities of these
PDGFR
mutants were equally activated by PDGF treatment, the
subsequent recruitment of signaling molecules was greatly abolished
when the corresponding binding-site tyrosine residues were mutated. For
example, PDGF-induced recruitment of GAP, but not recruitment of
PLC
1, SHP-2, or PI3K, was compromised in the 771
mutant. It is interesting that some association of PLC
1 was observed
in the mutant lacking the binding site (1021
). Since
PLC
1 can also bind at Tyr-1009 (44), we believe that the residual
recruitment of PLC
1 occurs at this site.
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After the demonstration that recruitment of specific signaling
molecules can be prevented by mutating specific tyrosine residues of
PDGFR, we then examined the effect of PDGF treatment on GJC, Cx43
phosphorylation, and activation of MAPK in cells expressing mutant
PDGFR. In Fig. 4, we show that PDGF
inhibited GJC in all of the single-site mutants, indicating that
PDGF-induced disruption of GJC is not mediated by a single signaling
component. In contrast, activation of several different routes can lead
to the same biological end point, i.e. disruption of GJC.
Interestingly, examination of the Cx43 profile revealed that similar to
the PDGF-treated Kin+ cells, multiple slow-migrating
phosphorylated forms of Cx43 appeared in most of the single-site
mutants following PDGF treatment (Fig. 4). The exception was the
1021
mutant, in which prominent phosphorylated forms of
Cx43 were not observed. This finding suggests that recruitment of
PLC
1 by activated PDGFR is involved in the PDGF-induced
phosphorylation of Cx43. Furthermore, the lack of correlation between
Cx43 phosphorylation and GJC disruption also supports our earlier
observation that the appearance of multiple phosphorylated forms of
Cx43 may not be necessary for the PDGF-induced GJC
blockade.2 Since MAPK was equally activated in all the
tested mutants after PDGF treatment, the failure of the
1021
mutant to show Cx43 phosphorylation is not due to a
lack of MAPK activation. Results obtained from the single-site mutants
suggest that PDGF activates multiple and perhaps redundant signaling
pathways leading to the disruption of GJC, which is not dependent on
the generation of Cx43 phospho forms.
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To determine which of these mutated tyrosines are involved in
augmenting the disruption of GJC, we next generated another series of
stable T51B cell lines expressing various add-back PDGFR mutants (6).
These mutants were constructed from the F5 mutant, in which single
binding sites were restored, yielding the F5+740/751, F5+771, F5+1009,
and F5+1021 mutants. Verification of these mutants was accomplished by
PDGFR immunoprecipitation and Western blotting for various signaling
molecules (Fig. 5). As expected, all the add-back mutants showed PDGF-induced receptor tyrosine phosphorylation similar to that seen in Kin+ or F5 cells, and recruitment
of specific signaling molecules (e.g. PI3K) was specifically
restored in mutants carrying the corresponding binding site
(e.g. F5+740/751).
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Examination of GJC following PDGF treatment showed that restoration of
either the SHP-2 (F5+1009)- or PLC1 (F5+1021)-binding site to the F5
mutant of PDGFR
was sufficient to mediate the inhibitory effect of
PDGF on GJC (Fig. 6). Restoration of the GAP-binding site (Tyr-771) failed to show any significant effect of
PDGF on GJC, but a partial blockade of GJC was seen when the PI3K-binding site (Tyr-740 and Tyr-751) was reintroduced into the F5
mutant. These results further support the multiplicity of PDGF
signaling pathways for the disruption of GJC by PDGF. The appearance of
the hyperphosphorylated Cx43-P3 was highly prominent in the
PDGF-treated F5+1021 mutant and only minimally present in the F5+1009
and F5+740/751 mutants (Fig. 6), which further demonstrates the lack of
correlation between Cx43 phosphorylation and GJC blockade. No
slow-migrating Cx43 forms were observed in the F5+771 mutant (Fig. 6).
Activation of MAPK remained similar in the F5+1009 and F5+1021 mutants
compared with the F5 mutant (Fig. 6), indicating that the PDGF-induced
inhibition of GJC observed in these two add-back mutants was not simply
due to an elevated level of activated MAPK, but rather to the
activation of additional components, as we previously
suggested.2 When compared with the F5 mutant, PDGF
treatment failed to activate MAPK in the F5+771 mutant, indicating that
the recruitment of GAP may have a negative impact on the MAPK
activation pathway, which is in accord with a previous study showing a
negative role of GAP in PDGF-induced mitogenesis (45). Activation of
MAPK was submaximal in F5+740/751 cells, which showed a partial
disruption of GJC. Taken together, the add-back mutants identify two
important mediators of PDGF signaling, i.e. SHP-2 and
PLC
1, which provide signals additional to MAPK activation that are
required for the inhibition of GJC by PDGF.
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The results from these studies suggest that Tyr-1021 of PDGFR, which
is the recruitment site for PLC
1 by activated PDGFR
, is involved
in the PDGF-induced hyperphosphorylation of Cx43 (Fig. 6). To examine
whether the enzymatic activity of PLC
1 is required for the
hyperphosphorylation of Cx43, we treated Kin+ cells with
U73122, a synthetic inhibitor of PLC
1 (46), prior to the treatment
with PDGF. In Fig. 7, we show that in
U73122-pretreated Kin+ cells, PDGF failed to generate the
appearance of Cx43-P3 forms, suggesting a requirement of activated
PLC
1. Unfortunately, we could not examine the effect of the
suppression of Cx43 hyperphosphorylation on GJC since U73122 showed GJC
inhibitory properties (Fig. 7). This inhibition of GJC by the
lipophilic U73122 is possibly due to its direct interaction with the
membrane organelles, which is not mediated via Cx43 phosphorylation.
Similar phosphorylation-independent inhibitory actions were observed
with other lipophilic compounds such as octanol (47) and clofibrate
(48, 49).2 Induction of MAPK was only slightly affected by
U73122 treatment, further suggesting that Cx43 hyperphosphorylation
requires participation of other signaling components downstream of
PLC
1 that are interfered by this compound.
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DISCUSSION |
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Inhibition of GJC by growth factors has been suggested to be
linked with their mitogenic actions (13-16, 23). To understand the
mechanism of PDGF-induced disruption of GJC, we have generated a cell
system derived from T51B rat liver epithelial cells to show that 1)
PDGF-induced disruption of GJC was associated with Cx43 phosphorylation
(15); 2) both of these effects were dependent on the activation of PKC
and MAPK (16); and 3) neither MAPK activation nor Cx43 phosphorylation
was sufficient to mediate inhibition of GJC.2 Together,
these findings raised the possibility that PDGF-induced disruption of
GJC required multiple components that are currently unidentified. Since
activated PDGFR initiates various signaling pathways, we examined
the importance of these signaling pathways by expressing a variety of
PDGFR mutants in T51B cells. The results presented above show that
(a) PDGF-induced disruption of GJC can be mediated via
multiple signaling pathways; (b) activation of MAPK does not
always correlate with either GJC blockade or Cx43 phosphorylation;
(c) hyperphosphorylation of Cx43 is distinct from inhibition
of GJC; and (d) activation of PLC
1 is involved in
mediating PDGF-induced Cx43 hyperphosphorylation.
Similar to many other peptide growth factors, PDGF augments its effects
by activating the tyrosine kinase activity of its cellular receptor,
PDGFR. A consequence of the induced receptor tyrosine kinase activity
is the autophosphorylation of PDGFR at distinct tyrosine residues,
which then act as binding sites for specific signaling molecules,
including enzymes such as PI3K, PLC1, and c-Src, or adaptor
molecules, including Grb2 and Nck (1, 4). Each of these molecules then
initiates specific signaling cascades leading to various cellular
responses such as mitogenesis, cell migration, Ca2+ influx,
etc. To delineate the involvement of each signaling molecule, several
investigative strategies were employed, including expression of mutant
PDGFR and microinjection of antibodies against or dominant-negative forms of signaling molecules (6, 50, 51). These studies characterized
the key signaling components in PDGF-induced mitogenesis, differentiation, neoplastic transformation, Ca2+ signaling,
Na+ channel induction, and Ras and PKC
activation (6, 7,
9-11, 50, 52-54). A common conclusion drawn in many of these studies was that PDGF actions can be mediated by multiple signaling pathways, which is in accord with our present observation that Tyr-1009 (binding
site for SHP-2) and Tyr-1021 (binding site for PLC
1) are involved in
the disruption of GJC by PDGF in the experimental T51B cell system.
Although highly likely, it remains to be demonstrated that similar
signaling molecules are also involved in cells that normally express
PDGFR. The reason for the redundancy in PDGF signaling pathways is
currently unclear. However, one can postulate that multiple signaling
pathways probably evolved as a protection against any impairment of the
favored route.
The signaling molecules identified in this study, i.e.
PLC1 and SHP-2, have been previously shown to be involved in a
variety of PDGF effects, including mitogenesis and neoplastic cell
transformation (6, 10, 50, 55). It is particularly interesting to note that gap junctions are considered to serve as conduits for the intercellular transfer of regulatory signals, and the disruption of GJC
is believed to be important in these two cellular events (21). A
partial involvement of PI3K, another signaling molecule important in
PDGF-induced mitogenesis and neoplastic transformation (6, 10), is also
in accord with this hypothesis. In contrast, a negative modulator of
PDGF signaling, GAP (45), was not involved in the disruption of GJC.
Examination of PDGF-induced cell proliferation in the PDGFR mutants,
which will provide further information linking GJC and mitogenesis, is
currently ongoing. Although their involvement is reported in many
studies, the mechanisms of action of PLC
1 and SHP-2 are not clearly
understood. In addition to their catalytic abilities, both molecules
can also act as adaptors to interact with other proteins, such as
dynamin in the case of PLC
1 (56) or Grb2/Sos1 in the case of SHP-2
(57, 58). Although the modes of their participation in PDGF-induced
disruption of GJC are currently unknown, two critical downstream
modulators of PDGF signaling can be activated by these two molecules.
Whereas diacylglycerol, a product of the catalytic action of PLC
1,
is a potent activator of PKC and subsequently of MAPK (59), recruitment
of Grb2/Sos1 by SHP-2 can also lead to the activation of MAPK (57). Our
previous demonstration of the requirement of PKC and MAPK in
PDGF-induced GJC blockade (16) supports such a mode of action. A
partial response in the PI3K add-back mutant (F5+740/751) is also in
accord with this model since PDGF-induced activation of PKC can also be
mediated by PI3K (11, 60, 61). Alternatively, PI3K may function via
activating and stabilizing PLC
(62). Although activation of PKC by
these modulators can also lead to MAPK activation (16, 59), additional
mechanisms of PKC involvement are yet to be identified.
Activation of MAPK is a common downstream step in growth factor
function (63) and is required for a variety of growth factor-induced cellular events (64-69). We (16)2 and others (33) have
shown that MAPK activation is also a required event in the disruption
of GJC and hyperphosphorylation of Cx43 by the epidermal growth factor,
PDGF, and 12-O-tetradecanoylphorbol-13-acetate. Since MAPK
is capable of phosphorylating Cx43 in vitro (32, 33), it is
generally believed that activation of MAPK is sufficient for mediating
the actions of the above-stated antagonists on GJC and Cx43. However,
by utilizing a variety of MAPK activators, we recently demonstrated
that MAPK activation does not always lead either to the disruption of
GJC or to hyperphosphorylation of Cx43,2 indicating that
factors additional to MAPK are required. The results in this study
suggest the same. For example, the degree of MAPK activation by PDGF is
of similar magnitude in Kin+ and F5 mutant cells, but GJC
blockade and Cx43 phosphorylation were seen only in Kin+
cells (Fig. 2). This result also confirms that additional signals are
required that are presumably generated by the activation of SHP-2 or
PLC1 (Fig. 6). The importance of additional signaling is also
evident in the F5+740/751 mutant, in which a partial response of PDGF
was observed despite a compromised activation of MAPK. The involvement
of cellular factors additional to MAPK has been suggested in
PDGF-mediated PC12 cell differentiation and in p21ras
desensitization of growth factor-treated cells where activation of MAPK
is required (64, 70) but is not sufficient for these biological effects
(9, 71). Based on these results, we have proposed that PDGF-induced
disruption of GJC requires a coordinated "all or none" action of
multiple components, one of which is MAPK.2 Since
restoration of either SHP-2 or PLC
1 recruitment can supplement the
signaling deficiency of the F5 mutant, it is probable that the MAPK
complementary signals can be different, although they finally converge
on the same target, i.e. disruption of GJC. The involvement
of a multicomponent signaling system was recently described for
hepatocyte growth factor/scatter factor-induced adherens junction
disassembly (72), where activation of both MAPK and PI3K was required.
The close association between antagonist-induced Cx43 phosphorylation
and GJC disruption observed in numerous studies (14-16, 23-26, 32,
33, 37) prompted the hypothesis that Cx43 phosphorylation is causally
linked with the GJC blockade (31). It is believed that the cytoplasmic
carboxyl-terminal region of Cx43, which is not essential for gap
junctional assembly (73, 74), is the primary location for GJC
regulatory phosphorylation (32, 75). In a recently proposed "ball and
chain" or "particle-receptor" model (73, 74, 76), the
carboxyl-terminal region serves as the "particle," and the
pore-forming region proximal to the plasma membrane is the
"receptor" for such a particle. Following a modification, such as
phosphorylation, the carboxyl-terminal region can directly interact
with the pore-forming region of the channel, resulting in its closure.
Alternatively, the phosphorylated carboxyl-terminal domain can bind to
an intermediary molecule to form a complex that interacts with and
closes the pore. The third possibility is that the phosphorylated
carboxyl-terminal domain acts on and modifies the intermediary
molecule, enabling it to bind to the junctional pore. In our previous
study, we demonstrated the insufficiency of gross Cx43 phosphorylation
to cause disruption of GJC,2 which strongly suggests the
requirement of intermediary molecules described in the latter two
models. This view is further supported by a lack of correlation between
the extent of Cx43 phosphorylation and degree of junctional uncoupling
in this study (Figs. 4 and 6) and in others (49, 77). Direct
association of several proteins with Cx43 (78-80) and probable
involvement of protein-protein interaction in the regulation of
Cx43-gap junctions (34, 81) are in accord with our hypothesis. The
function of PDGF-induced Cx43 phosphorylation, which is presumably
mediated by PLC1 (Figs. 4, 6, and 7), thus remains undetermined.
Since PDGF-induced disruption of GJC can occur without a gross
hyperphosphorylation of Cx43 (the 1021
mutant in Fig. 4
and the F5+1009 mutant in Fig. 6), it is possible that some, if not
most, of Cx43 phosphorylations may not be directly required for GJC
blockade. This proposal is supported by a recent study in which
epidermal growth factor treatment induced Cx43 phosphorylation, but not
GJC blockade, in fibroblasts expressing a Cx43 mutant (33). The
identification and mutational analyses of Cx43 sites that are
phosphorylated following PDGF treatment will provide further
information regarding the role of Cx43 phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank J. Cooper for PDGFR-expressing PA317 cells, C. Hart for PDGF-BB and anti-PDGFR antibody, A. Golard and D. Messner for comments and suggestions, and M. Bates for photography.
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FOOTNOTES |
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* This work was supported by National Institutes of Heath Grant CA 57064.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.
§ To whom correspondence should be addressed: Molecular Medicine, Northwest Hospital, 120 Northgate Plaza, Suite 230, Seattle, WA 98125. Tel.: 206-368-3042; Fax: 206-368-3009; E-mail: mhossain{at}nwhsea.org.
2 Hossain, M. Z., Jagdale, A. B., Ao, P., and Boynton, A. L., (1999) J. Cell. Physiol., in press.
3 A. Golard, M. Z. Hossain, R. P. Huang, A. Kazlauskas, and A. L. Boynton, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
PDGF, platelet-derived growth factor;
PDGFR, platelet-derived growth factor
receptor;
PI3K, phosphatidylinositol 3-kinase;
GAP, GTPase-activating
protein;
PLC, phospholipase C
;
GJC, gap junctional communication;
Cx43, connexin-43;
PKC, protein kinase C;
MAPK, mitogen-activated
protein kinase;
PMAPK, phosphorylated mitogen-activated protein kinase;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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