Disruption of Gap Junctional Communication by the Platelet-derived Growth Factor Is Mediated via Multiple Signaling Pathways*

Mohammad Z. HossainDagger §, Ajit B. JagdaleDagger , Peng AoDagger , Andrius Kazlauskas, and Alton L. BoyntonDagger

From Dagger  Molecular Medicine, Northwest Hospital, Seattle, Washington 98125 and the  Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta  (PDGFRbeta ) and a series of PDGFRbeta mutants were expressed in T51B rat liver epithelial cells. In cells expressing wild-type PDGFRbeta , 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 Cgamma 1 (PLCgamma 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 PLCgamma 1 binding was sufficient to propagate the GJC inhibitory actions of PDGF. Further analysis showed that activation of PLCgamma 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

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 PDGFRbeta (1-4). Together with their adjacent sequences, these phosphotyrosine residues form binding sites for specific proteins. In the case of PDGFRbeta , 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 Cgamma (PLCgamma )), and Tyr-1021 (PLCgamma ) (1, 4, 5). Many of these signal transduction molecules possess intrinsic catalytic properties (e.g. PI3K, PLCgamma , 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).

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 PDGFRbeta 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

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-PDGFRbeta antibody was a generous gift from Dr. C. Hart; polyclonal anti-PDGFRbeta 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-PLCgamma 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).

Expression Vectors and Cell Infections-- The virus-packaging PA317 cell lines expressing wild-type, kinase-dead, and single-site mutant human PDGFRbeta 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").

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-PDGFRbeta 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% beta -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, PLCgamma , 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

In our previous study, we demonstrated a PDGF-induced Cx43 phosphorylation and GJC blockade in T51B cells expressing wild-type PDGFRbeta (Kin+) (15). These effects were not observed in Kin- cells expressing kinase-inactive PDGFRbeta , indicating that phosphorylation of tyrosine residues in PDGFRbeta 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 PDGFRbeta 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 PDGFRbeta (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 PLCgamma in PDGF-induced mitogenesis and neoplastic cell transformation (6, 10).

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 PLCgamma 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.

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-PDGFRbeta antibody (15). To verify the functionality of the F5 mutant, PDGFRbeta 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, PLCgamma 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.


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 1.   Activation of PDGFRbeta in T51B cells expressing wild-type (Kin+) or F5 mutant PDGFRbeta . Confluent cells treated with buffer (-) or 5 ng/ml PDGF (+) for 10 min and lysed, and PDGFRbeta was immunoprecipitated using a monoclonal anti-PDGFRbeta 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.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of PDGF treatment on GJC, Cx43, and PMAPK in Kin+ and F5 cells. Confluent cells were treated with PDGF (5 ng/ml) or 12-O-tetradecanoylphorbol-13-acetate (TPA; 25 ng/ml) for 15-20 min and then assayed for GJC by dye microinjection and examined for Cx43 and PMAPK by Western blotting. The numbers in the GJC boxes represent the mean number of communicating cells in the indicated treatment group. Immunoreactive bands representing different forms of Cx43 (40) and molecular masses (in kilodaltons) of immunoreactive PMAPK are indicated.

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 PDGFRbeta .

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 PDGFRbeta 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 PLCgamma 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 PDGFRbeta 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 PLCgamma 1, SHP-2, or PI3K, was compromised in the 771- mutant. It is interesting that some association of PLCgamma 1 was observed in the mutant lacking the binding site (1021-). Since PLCgamma 1 can also bind at Tyr-1009 (44), we believe that the residual recruitment of PLCgamma 1 occurs at this site.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 3.   Activation of PDGFRbeta in T51B cells expressing single-site PDGFRbeta mutants. Confluent cells treated with buffer (-) or PDGF (+) for 10 min were lysed, and PDGFRbeta was immunoprecipitated using a monoclonal anti-PDGFRbeta antibody. The immunoprecipitates were separated by SDS-PAGE followed by Western blotting and separately probed with antibodies against the indicated proteins.

After the demonstration that recruitment of specific signaling molecules can be prevented by mutating specific tyrosine residues of PDGFRbeta , 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 PLCgamma 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.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of PDGF treatment on GJC, Cx43, and PMAPK in Kin+ cells and single-site PDGFRbeta mutants. Confluent cells were treated with buffer (-) or PDGF (+) for 15-20 min and then analyzed for GJC, Cx43, and PMAPK as described in the legend to Fig. 2. The numbers in the GJC box represent the mean number of communicating cells in the indicated treatment group.

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).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 5.   Activation of PDGFRbeta in T51B cells expressing add-back PDGFRbeta mutants. Confluent cells treated with buffer (-) or PDGF (+) for 10 min were lysed, and PDGFRbeta was immunoprecipitated using a monoclonal anti-PDGFRbeta antibody. The immunoprecipitates were separated by SDS-PAGE followed by Western blotting and separately probed with antibodies against the indicated proteins.

Examination of GJC following PDGF treatment showed that restoration of either the SHP-2 (F5+1009)- or PLCgamma 1 (F5+1021)-binding site to the F5 mutant of PDGFRbeta 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 PLCgamma 1, which provide signals additional to MAPK activation that are required for the inhibition of GJC by PDGF.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of PDGF treatment on GJC, Cx43, and PMAPK in Kin+ cells and F5 and add-back PDGFRbeta mutants. Confluent cells were treated with buffer (-) or PDGF (+) for 15-20 min and then analyzed for GJC, Cx43, and PMAPK as described in the legend to Fig. 2. The numbers in the GJC box represent the mean number of communicating cells in the indicated treatment group.

The results from these studies suggest that Tyr-1021 of PDGFRbeta , which is the recruitment site for PLCgamma 1 by activated PDGFRbeta , is involved in the PDGF-induced hyperphosphorylation of Cx43 (Fig. 6). To examine whether the enzymatic activity of PLCgamma 1 is required for the hyperphosphorylation of Cx43, we treated Kin+ cells with U73122, a synthetic inhibitor of PLCgamma 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 PLCgamma 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 PLCgamma 1 that are interfered by this compound.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of a PLCgamma 1 inhibitor (U73122) on PDGF-induced disruption of GJC and Cx43 phosphorylation in Kin+ cells. Confluent Kin+ cells were treated with U73122 (5 µM) for 15 min before PDGF treatment. Cells were then analyzed for GJC, Cx43, and PMAPK as described in the legend to Fig. 2. The numbers in the GJC box represents the mean number of communicating cells in the indicated treatment group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PDGFRbeta 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 PLCgamma 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, PLCgamma 1, 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 PKCepsilon 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 PLCgamma 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. PLCgamma 1 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 PLCgamma 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 PLCgamma 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 PLCgamma 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 PLCgamma (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 PLCgamma 1 (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 PLCgamma 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 PLCgamma 1 (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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol 3-kinase; GAP, GTPase-activating protein; PLCgamma , phospholipase Cgamma ; 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Claesson-Welsh, L. (1994) J. Biol. Chem. 269, 32023-32026[Free Full Text]
  2. Kazlauskas, A. (1994) Curr. Opin. Genet. Dev. 4, 5-14[Medline] [Order article via Infotrieve]
  3. Heldin, C. H. (1995) Cell 80, 213-223[Medline] [Order article via Infotrieve]
  4. van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. Rev. Cell Biol. 10, 251-337[CrossRef]
  5. Kazlauskas, A., Durden, D. L., and Cooper, J. A. (1991) Cell Regul. 2, 413-425[Medline] [Order article via Infotrieve]
  6. Valius, M., and Kazlauskas, A. (1993) Cell 73, 321-334[Medline] [Order article via Infotrieve]
  7. Satoh, T., Fantl, W. J., Escobedo, J. A., Williams, L. T., and Kaziro, Y. (1993) Mol. Cell. Biol. 13, 3706-3713[Abstract]
  8. Burgering, B. M. T., Freed, E., van der Voorn, L., McCormick, F., and Bos, J. L. (1994) Cell Growth Differ. 5, 341-347[Abstract]
  9. Vaillancourt, R. V., Heasley, L. E., Zamarripa, J., Storey, B., Valius, M., Kazlauskas, A., and Johnson, G. L. (1995) Mol. Cell. Biol. 15, 3644-3653[Abstract]
  10. DeMali, K. A., Whiteford, C. C., Ulug, E. T., and Kazlauskas, A. (1997) J. Biol. Chem. 272, 9011-9018[Abstract/Free Full Text]
  11. Moriya, S., Kazlauskas, A., Akimoto, K., Hirai, S., Mizuno, K., Takenawa, T., Fukui, Y., Watanabe, Y., Ozaki, S., and Ohno, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 151-155[Abstract/Free Full Text]
  12. Montamayeur, J. P., Valius, M., Vandenheede, J., and Kazlauskas, A. (1997) J. Biol. Chem. 272, 32670-32678[Abstract/Free Full Text]
  13. Maldonado, P. E., Rose, B., and Loewenstein, W. R. (1988) J. Membr. Biol. 106, 203-210[Medline] [Order article via Infotrieve]
  14. Pelletier, D. B., and Boynton, A. L. (1994) J. Cell. Physiol. 158, 427-434[Medline] [Order article via Infotrieve]
  15. Hossain, M. Z., Ao, P., and Boynton, A. L. (1998) J. Cell. Physiol. 174, 66-77[CrossRef][Medline] [Order article via Infotrieve]
  16. Hossain, M. Z., Ao, P., and Boynton, A. L. (1998) J. Cell. Physiol. 176, 332-341[CrossRef][Medline] [Order article via Infotrieve]
  17. Kumar, N. M., and Gilula, N. B. (1996) Cell 84, 381-388[Medline] [Order article via Infotrieve]
  18. Bruzzone, R., White, T. W., and Paul, D. L. (1996) Eur. J. Biochem. 238, 1-27[Abstract]
  19. Loewenstein, W. R., and Rose, B. (1992) Semin. Cell Biol. 3, 59-79[Medline] [Order article via Infotrieve]
  20. Warner, A. E. (1992) Semin. Cell Biol. 3, 81-91[Medline] [Order article via Infotrieve]
  21. Yamasaki, H., and Naus, C. C. G. (1996) Carcinogenesis (Lond.) 17, 1199-1213[Medline] [Order article via Infotrieve]
  22. Munari-Silem, Y., and Rousset, B. (1996) Eur. J. Endocrinol. 135, 251-264[Medline] [Order article via Infotrieve]
  23. Lau, A. F., Kanemitsu, M. Y., Kurata, W. E., Danesh, S., and Boynton, A. L. (1992) Mol. Biol. Cell 3, 865-874[Abstract]
  24. Matesic, D. F., Rupp, H. L., Bonney, W. J., Ruch, R. J., and Trosko, J. E. (1994) Mol. Carcinog. 10, 226-236[Medline] [Order article via Infotrieve]
  25. Berthoud, V. M., Rook, M. B., Traub, O., Hertzberg, E. L., and Saez, J. C. (1993) Eur. J. Cell Biol. 61, 384-396
  26. Oh, S. Y., Grupen, G. S., and Murray, A. W. (1991) Biochim. Biophys. Acta 1094, 243-248[Medline] [Order article via Infotrieve]
  27. Crow, D. S., Beyer, E. C., Paul, D. L., Kobe, S. S., and Lau, A. F. (1990) Mol. Cell. Biol. 10, 1754-1763[Medline] [Order article via Infotrieve]
  28. Kurata, W. E., and Lau, A. F. (1994) Oncogene 9, 329-335[Medline] [Order article via Infotrieve]
  29. Hossain, M. Z., Wilkens, L. R., Mehta, P. P., Loewenstein, W. R., and Bertram, J. S. (1989) Carcinogenesis (Lond.) 10, 1743-1748[Abstract]
  30. Zhang, L.-X., Cooney, R. V., and Bertram, J. S. (1991) Carcinogenesis (Lond.) 12, 2109-2114[Abstract]
  31. Lau, A. F., Kurata, W. E., Kanetmitsu, M. Y., Loo, L. W. M., Warn-Cramer, B. J., Eckhart, W., and Lampe, P. D. (1996) J. Bioenerg. Biomembr. 28, 359-368[Medline] [Order article via Infotrieve]
  32. Warn-Cramer, B. J., Lampe, P. D., Kurata, W. F., Kanemitsu, M. Y., Loo, L. W. M., Eckhart, W., and Lau, A. F. (1996) J. Biol. Chem. 271, 3779-3786[Abstract/Free Full Text]
  33. Warn-Cramer, B. J., Cottrell, G. T., Burt, J. M., and Lau, A. F. (1998) J. Biol. Chem. 273, 9188-9196[Abstract/Free Full Text]
  34. TenBroek, E., Lampe, P., Taffet, S., Reynhout, J., Martyn, K., Kurata, W., Lau, A., and Johnson, R. (1998) in Proceedings of the 1997 International Gap Junctional Conference (Werner, R., ed), pp. 215-219, IOS Press, Amsterdam
  35. Swierenga, S. H. H., Whitfield, J. F., Boynton, A. L., MacManus, J. P., Rixon, R. H., Sikorska, M., Tsang, B. K., and Walker, P. R. (1980) Ann. N. Y. Acad. Sci. 349, 294-311[Abstract]
  36. Boynton, A. L., Kleine, L. P., and Whitfield, J. F. (1984) Cancer Lett. 21, 293-302[CrossRef][Medline] [Order article via Infotrieve]
  37. Kanemitsu, M. Y., and Lau, A. F. (1993) Mol. Biol. Cell 4, 837-848[Abstract]
  38. Kazlauskas, A., and Cooper, J. A. (1989) Cell 58, 1121-1133[Medline] [Order article via Infotrieve]
  39. Huang, R. P., Wu, J. X., Fan, Y., and Adamson, E. D. (1996) J. Cell Biol. 133, 211-220[Abstract]
  40. Musil, L. S., Cunningham, B. A., Edelman, G. A., and Goodenough, D. A. (1990) J. Cell Biol. 111, 2077-2088[Abstract]
  41. Gotoh, N., Toyoda, M., and Shibuya, M. (1997) Mol. Cell. Biol. 17, 1824-1831[Abstract]
  42. Cook, S. J., Beltman, J., Cadwallader, K. A., McMahon, M., and McCormick, F. (1997) J. Biol. Chem. 272, 13309-13319[Abstract/Free Full Text]
  43. Kazlauskas, A., Kashishian, A, Cooper, J. A., and Valius, M. (1992) Mol. Cell. Biol. 12, 2534-2544[Abstract]
  44. Ronnstrand, L., Mori, S., Arridsson, A. K., Eriksson, A., Wernstedt, C., Hellman, U., Claesson-Welsh, L., and Heldin, C. H. (1992) EMBO J. 11, 3911-3919[Abstract]
  45. Valius, M., Secrist, J. P., and Kazlauskas, A. (1995) Mol. Cell. Biol. 15, 3058-3071[Abstract]
  46. Tatrai, A., Lee, S. K., and Stern, P. H. (1994) Biochim. Biophys. Acta 1224, 575-582[Medline] [Order article via Infotrieve]
  47. Proulx, A., Merrifield, P. A., and Naus, C. C. (1997) Dev. Genet. 20, 133-144[CrossRef][Medline] [Order article via Infotrieve]
  48. Jansen, L. A. M., and Jongen, W. M. F. (1996) Carcinogenesis (Lond.) 17, 333-339[Abstract]
  49. Cruciani, V., Mikalsen, S. O., Vasseur, P., and Sanner, T. (1997) Int. J. Cancer 73, 240-248[CrossRef][Medline] [Order article via Infotrieve]
  50. Roche, S., McGlade, J., Jones, M., Gish, G. D., Pawson, T., and Courtneidge, S. A. (1996) EMBO J. 15, 4940-4948[Abstract]
  51. Smith, M. R., Liu, Y. L., Kim, H., Rhee, S. G., and Kung, H. F. (1990) Science 247, 1074-1077[Medline] [Order article via Infotrieve]
  52. Ma, Y. H., Reusch, H. P., Wilson, E., Escobedo, J. A., Fantl, W. J., Williams, L. T., and Ives, H. E. (1994) J Biol. Chem. 269, 30734-30739[Abstract/Free Full Text]
  53. Fanger, G. R., Vaillancourt, R. R., Heasley, L. E., Montmayeur, J. P., Johnson, G. L., and Maue, R. A. (1997) Mol. Cell. Biol. 17, 89-99[Abstract]
  54. Ridefelt, P., and Siegbahn, A. (1998) Anticancer Res. 18, 1819-1826[Medline] [Order article via Infotrieve]
  55. Rivard, N., McKenzie, F. R., Brondello, J. M., and Pouyssegur, J. (1995) J. Biol. Chem. 270, 11017-11024[Abstract/Free Full Text]
  56. Seedorf, K., Kostka, G., Lammers, R., Bashkin, P., Daly, R., Brgess, W. H., van der Bliek, A. M., Schlessinger, J., and Ullrich, A. (1994) J. Biol. Chem. 269, 16009-16014[Abstract/Free Full Text]
  57. Bennett, A. M., Tang, T. L., Sugimoto, S., Walsh, C. T., and Neel, B. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7335-7339[Abstract]
  58. Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J. H., Cooper, J. A., and Schlessinger, J. (1994) Mol. Cell. Biol. 14, 509-517[Abstract]
  59. van Dijk, M. C., Hilkmann, H., and van Blitterswijk, W. J. (1997) Biochem. J. 325, 303-307[Medline] [Order article via Infotrieve]
  60. Akimoto, K., Takahashi, R., Moriya, S., Nishioka, N., Takayanagi, J., Kimura, K., Fukui, Y., Osada, S., Mizuno, K., Hirai, S., Kazlauskas, A., and Ohno, S. (1996) EMBO J. 15, 788-798[Abstract]
  61. Le Good, J. A., Ziegler, W. H., Parekh, D. B., Alessi, D. R., Cohen, P., and Parker, P. J. (1998) Science 281, 2042-2045[Abstract/Free Full Text]
  62. Falasca, M., Logan, S. K., Lehto, V. P., Baccante, G., Lemmon, M. A., and Schlessinger, J. (1998) EMBO J. 17, 414-422[Abstract/Free Full Text]
  63. Cobb, M. H., and Goldsmith, E. J. (1995) J. Biol. Chem. 270, 14843-14846[Free Full Text]
  64. Pang, L., Sawada, T., Decker, S. J., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 13585-13588[Abstract/Free Full Text]
  65. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689[Abstract]
  66. Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., Metzger, D., and Chambon, P. (1995) Science 270, 1491-1494[Abstract]
  67. Eldar-Finkelman, H., Seger, R., Vandenheede, J. R., and Krebs, E. G. (1995) J. Biol. Chem. 270, 987-990[Abstract/Free Full Text]
  68. Xing, J., Ginty, D. D., and Greenberg, M. E. (1996) Science 273, 959-963[Abstract]
  69. Robinson, C. J. M., Scott, P. H., Alla, A. B., Jess, T., Gould, G. W., and Pelvin, R. (1996) Biochem. J. 320, 123-127[Medline] [Order article via Infotrieve]
  70. Langlois, W. J., Sasaoka, T., Saltiel, A. R., and Olefsky, J. M. (1995) J. Biol. Chem. 270, 25320-25323[Abstract/Free Full Text]
  71. Klarlund, J. K., Cherniack, A. D., McMahon, M., and Czech, M. P. (1996) J. Biol. Chem. 271, 16674-16677[Abstract/Free Full Text]
  72. Potempa, S., and Ridley, A. J. (1998) Mol. Biol. Cell 9, 2185-2200[Abstract/Free Full Text]
  73. Morely, G. E., Taffet, S. M., and Delmar, M. (1996) Biophys. J. 70, 1294-1302[Abstract]
  74. Ek-Vitorin, J. F., Calero, G., Morley, G. E., Coombs, W., Taffet, S. M., and Delmar, M. (1996) Biophys. J. 71, 1273-1284[Abstract]
  75. Swenson, K. I., Piwnica-Worms, H., McNamee, H., and Paul, D. L (1990) Cell Regul. 1, 989-1002[Medline] [Order article via Infotrieve]
  76. Delmar, M., Stergiopoulos, K., Homma, N., Ek-Vitorin, J. F., and Taffet, S. M. (1998) in Proceedings of the 1997 International Gap Junctional Conference (Werner, R., ed), pp. 8-12, IOS Press, Amsterdam
  77. Shiokawa-Sawada, M., Mano, H., Hanada, K., Kakudo, S., Kameda, T., Miyazawa, K., Nakamaru, Y., Yuasa, T., Mori, Y., Kumegawa, M., and Hakeda, Y. (1997) J. Bone Miner. Res. 12, 1165-1173[Medline] [Order article via Infotrieve]
  78. Kanemitsu, M. Y., Loo, L. W. M., Simon, S., Lau, A. F., and Eckhart, W. (1997) J. Biol. Chem. 272, 22824-22831[Abstract/Free Full Text]
  79. Toyofuku, T., Yabuki, M., Otsu, K., Kuzuya, T., Hori, M., and Tada, M. (1998) J. Biol. Chem. 273, 12725-12731[Abstract/Free Full Text]
  80. Giepmans, B. N. G., and Moolenaar, W. H. (1998) Curr. Biol. 8, 931-934[Medline] [Order article via Infotrieve]
  81. Callero, G., Kanemitsu, M., Taffet, S. M., Lau, A. F., and Delmar, M. (1998) Circ. Res. 82, 929-935[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.