JBC Oz Biosciences

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M002410200 on April 25, 2000

J. Biol. Chem., Vol. 275, Issue 28, 21730-21736, July 14, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/28/21730    most recent
M002410200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fukuhara, S.
Right arrow Articles by Gutkind, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fukuhara, S.
Right arrow Articles by Gutkind, J. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Signaling from G Protein-coupled Receptors to ERK5/Big MAPK 1 Involves Galpha q and Galpha 12/13 Families of Heterotrimeric G Proteins

EVIDENCE FOR THE EXISTENCE OF A NOVEL Ras AND Rho-INDEPENDENT PATHWAY*

Shigetomo Fukuhara, Maria Julia Marinissen, Mario Chiariello, and J. Silvio GutkindDagger

From the Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4330

Received for publication, March 17, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The regulation of gene expression by cell surface receptors often involves the stimulation of signaling pathways including one or more members of the MAPK superfamily of serine-threonine kinases. Upon their activation in the cytosol, MAPKs can translocate to the nucleus and affect the activity of a variety of transcription factors. Recently, it has been observed that a novel member of the MAPK superfamily, ERK5, can be potently activated by transforming G protein-coupled receptors (GPCRs) and that ERK5 participates in the regulation of c-jun expression through the activation of MEF2 transcription factors. How cell surface receptors, including GPCRs, stimulate ERK5 is still poorly understood. In this study, we have used transiently transfected COS-7 cells to begin delineating the biochemical route linking GPCRs to ERK5. We show that receptors that can couple to the Gq and G12/13 families of heterotrimeric G proteins, m1 and thrombin receptors, respectively, but not those coupled to Gi, such as m2 receptors, are able to regulate the activity of ERK5. To investigate which heterotrimeric G proteins signal to ERK5, we used a chimeric system by which Galpha q- and Galpha 13-mediated signaling pathways can be conditionally activated upon ligand stimulation. Using this system, as well as the expression of activated forms of G protein subunits, we show that the Galpha q and Galpha 12/13 families of heterotrimeric G proteins, but not the Galpha i, Galpha s, and beta gamma subunits, are able to regulate ERK5. Furthermore, we provide evidence that the stimulation of ERK5 by GPCRs involves a novel signaling pathway, which is distinct from those regulated by Ras and Rho GTPases.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Mitogen-activated protein kinases (MAPKs)1 are serine-threonine protein kinases that play a central role in the transduction of environmental stimuli to the nucleus, thereby regulating the expression of genes involved in a variety of cellular processes, including cell proliferation, differentiation, programmed cell death, and neoplastic transformation (1, 2). To date, MAPKs have been classified into at least six subfamilies: p44mapk and p42mapk, also called extracellular signal-regulated kinases (ERKs) 1 and 2, respectively (referred in here as MAPK); c-Jun N-terminal kinases (JNKs), also termed stress-activated protein kinases; p38 MAPKs; ERK5, also known as big MAPK 1; and the recently identified ERK7 (3), and MOK (4) (see Ref. 5 for a review). These kinases are activated by a wide variety of extracellular stimuli such as growth factors, hormones, antigens, and cytokines and can also be stimulated in response to a diverse array of cellular stresses, such as UV irradiation, oxidative stress, and heat and osmotic shock (2).

Many cell surface receptors can effectively stimulate MAPK cascades to signal to the nucleus, including the large family of receptors that transduce signals through the activation of heterotrimeric GTP-binding proteins (G proteins) (6). For example, receptors coupled to Galpha i proteins can potently stimulate MAPK (7), and that appears to be mediated primarily through the release of beta gamma subunits (8) and the activation of a complex biochemical route involving phosphatidylinositol 3-kinases (9) and several nonreceptor and receptor tyrosine kinases (see Refs. 6 and 10 for reviews). In turn, activation of these tyrosine kinases leads to the phosphorylation of an adaptor protein, Shc, and the recruitment of the Grb2·Sos complex to the plasma membrane thus stimulating the exchange of GDP for GTP on Ras GTPases. This promotes the activation of a kinase cascade including Raf and MEK, which culminates with the activation of MAPK (1, 11). Signals generated by the activation of G protein-coupled receptors (GPCRs) can also be transmitted through Galpha i and Galpha s, which can stimulate MAPK by regulating the small GTPase Rap1 (12-14), and by Galpha q, which can activate Pyk2 and Src (15, 16), and can stimulate Raf through protein kinase C (17). Similarly, JNK and p38 MAPKs have been shown to be activated by ligands acting on GPCRs, by the release of beta gamma dimers (18) and through the Galpha q and Galpha 12/13 classes of G proteins (19, 20). However, signaling pathways from GPCRs to these MAPKs are still largely unknown, although they appear to involve the activation of the small GTPases Rac1 and Cdc42 by beta gamma dimers and RhoA and Rac1 by members of the Galpha 12/13 class of G proteins (18, 21-23).

Recently, we have observed that a novel member of the MAPK superfamily, ERK5, can be potently activated by transforming GPCRs and provided evidence that ERK5 participates in the regulation of c-jun expression by GPCRs through the activation of members of the MEF2 class of transcription factors (24). ERK5 exhibits an extended C-terminal tail, which is absent in other types of MAPKs, suggesting that the regulation and function of this kinase might be different from that of other MAPKs (25, 26). How cell surface receptors, including GPCRs, stimulate ERK5 is still unknown. In this study, we have used transfected and endogenously expressed GPCRs and the co-expression of GPCRs with chimeric G protein alpha  subunits to begin delineating the biochemical route linking GPCRs to ERK5. We show that the Galpha q and Galpha 12/13 families of heterotrimeric G proteins alpha  subunits, but not the Galpha i, Galpha s, or beta gamma subunits, are able to regulate ERK5 activity. Furthermore, we obtained evidence that the stimulation of the ERK5 cascade by GPCRs involves a novel pathway, which is distinct from those regulated by Ras and Rho GTPases.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Expression Plasmids-- PAR1, kindly provided by Dr. L.F. Brass, was subcloned into the pCEFL vector as an EcoRI fragment. DNA encoding a G13/Gi chimera, in which 5 amino acids at the C terminus of Galpha 13 were replaced by the corresponding sequence of Galpha i2, was prepared by polymerase chain reaction amplification using pcDNA3 HA-Galpha 13 (27) as a template, and the resulting DNA was subcloned into the pCEFL HA vector (24) as a BglII-EcoRI fragment. Sequences of mutagenic oligonucleotides will be made available upon request. Plasmids expressing epitope-tagged ERK5, MAPK, and JNK, pCEFL HA-ERK5, pcDNA3 HA-MAPK, and pcDNA3 HA-JNK, respectively, as well as expression plasmids for constitutively activated forms of Ras, Rho, Rac1, Cdc42, Galpha q, Galpha i2, Galpha s, Galpha 12, Galpha 13, beta  and gamma  subunits of G proteins, dominant negative mutants of Ras and Rho, the CRIB (Cdc42, Rac interactive binding) domain of PAK (PAK-N), m1 and m2 muscarinic receptors, a Gal4 fusion protein containing the transactivating domain of MEF2C and dominant negative and active mutants of MEK5, MEK5AA, and MEK5DD, respectively, were described previously (21, 24, 27, 28). A DNA plasmid encoding a Gq/Gi chimeric protein, in which 5 amino acids at the C terminus of Galpha q were replaced by the corresponding sequence of Galpha i2, was a gift from Dr. B. Conklin (29). Reporter plasmids that express the chloramphenicol acetyltransferase (CAT) gene under the control of the mutant form of the serum response element (SRE) from the c-fos promoter, lacking the ternary complex factor binding site (SREmutL) as well as an expression vector for the C3 toxin were kindly provided by Dr. R. Treisman (30).

Cell Culture and Transfection-- COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Cells were transfected using LipofectAMINE PlusTM reagent (Life Technologies, Inc.) according to the manufacturer's protocol. In each experiment, the total amount of DNA was adjusted to 3-10 µg/plate with a plasmid for green fluorescent protein.

Kinase Assays-- The ERK5 kinase activity in cells transfected with an expression plasmid for HA-ERK5 was measured as described previously (24), using 3 µg of GST·MEF2C fusion protein containing the transactivating domain of MEF2C as a substrate. MAPK and JNK activities in cells transfected with an epitope-tagged MAPK (HA-ERK2, referred in here as HA-MAPK) or JNK (HA-JNK) were determined as described previously (21), using myelin basic protein (Sigma) or bacterially expressed GST·ATF2(96) fusion protein as a substrate, respectively. The expression level of HA-ERK5, HA-MAPK, and HA-JNK in lysates from transfected cells was assessed by Western blot analysis after SDS-polyacrylamide gel electrophoresis with the specific antibody against HA (HA.11; Berkeley Antibody Company).

Reporter Gene Assays-- The transactivating activity of MEF2C and the SRE activity were determined as described previously (24, 27). Briefly, for MEF2C, COS-7 cells plated in a 24-well plate were transfected with different expression plasmids together with 2 ng of pCDNAIII-Gal4-MEF2C, a plasmid expressing a Gal4 fusion protein containing the transactivation domain of MEF2C (amino acids 161-350) as well as 50 ng of pGal4-Luc and 10 ng of pRL-null (Promega). To measure the SRE activity, COS-7 cells were transfected with the indicated plasmids together with 0.1 µg of pCDNAIII-beta -galactosidase, a plasmid expressing the enzyme beta -galactosidase, and 0.1 µg of pSREmutL, the reporter plasmid expressing a CAT gene under the control of the mutant SRE lacking a ternary complex factor binding site. After transfection, cells were cultured for ~24 h in serum-free Dulbecco's modified Eagle's medium, then stimulated with the indicated ligands for an additional 6 h, and lysed using reporter lysis buffer (Promega). Luciferase activities in cell extracts were determined using a dual luciferase assay system (Promega). CAT activity was assayed in the cell extracts by incubation at 37 °C for 1 h in the presence of 0.25 µCi of [14C]chloramphenicol (100 mCi/mmol) (ICN) and 200 µg/ml butyryl-CoA (Sigma) in 0.25 M Tris-HCl, pH 7.4. Labeled butyrylated products were extracted using a mixture of xylenes and 2,6,10,14-tetramethyl-pentadecane (ratio 1:2), and radioactivity was counted. beta -Galactosidase activity present in each sample was assayed by a colorimetric method and was used to normalize for transfection efficiency.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Coupling Specificity of G Protein-linked Receptors Stimulating ERK5 Kinase Activity-- ERK5 has been recently found to participate in the regulation of the c-jun promoter by transforming GPCRs (24). To begin exploring the nature of the pathway linking these cell surface receptors to ERK5, we first investigated which classes of GPCRs are able to stimulate ERK5 kinase activity. For these experiments, COS-7 cells were transiently transfected with expression plasmids for a HA-tagged form of ERK5, and its kinase activity was measured by an in vitro kinase assay using MEF2C fused to GST as a substrate. As shown in Fig. 1A, stimulation by carbachol, a cholinergic agonist, of transfected Galpha q-coupled m1 muscarinic receptors (31) potently activated ERK5. Similarly, stimulation with a tyrosine kinase receptor agonist, epidermal growth factor, also enhanced ERK5 kinase activity, as recently reported (32, 33). However, the stimulation of m2 muscarinic receptors, which are typical Gi-coupled receptors (34), had no effect on ERK5 activity, although it potently activated MAPK, which served as an internal control (8). ERK5 activity was also stimulated by exposure to thrombin, which acts on endogenously expressed GPCRs, and this effect was slightly enhanced by overexpression of its cognate receptors, PAR1 (Fig. 1A). Kinetics of ERK5 activation mediated by m1 and thrombin receptors were very similar, and responses were evident within 5 min after agonist addition and reached a maximal level around 10 min (Fig. 1B). As m1 and m2 muscarinic receptors couple to Galpha q and Galpha i types of heterotrimeric G proteins, respectively, and thrombin receptors can stimulate both the Galpha q and Galpha i as well as the Galpha 12/13 families of G proteins (35-38), these findings suggest that receptors coupled to Galpha q and, possibly Galpha 12/13, may harbor the ability to transduce a signal to ERK5, whereas Gi-coupled receptors do not.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 1.   Differential activation of MAPK and ERK5 by G protein-coupled receptors exhibiting distinct coupling specificity. COS-7 cells were transfected with expression plasmids for HA-ERK5 or HA-MAPK, together with plasmids expressing GFP, m1, or PAR1 receptors, as indicated, and stimulated with vehicle (c), 100 µM carbachol (Cch), 5 units/ml thrombin (Thr), or 100 ng/ml epidermal growth factor (EGF) for 10 min (A) or for the indicated time (B). Kinase reactions were performed using anti-HA immunoprecipitates from the corresponding cellular lysates. Labeled substrates are indicated. Data shown are from a representative experiment for each assay, which was repeated three to five times with similar results. Western blot (WB) analysis was performed with anti-HA antibodies using total cellular lysates. Data represent the mean ± S.E. of three to five independent experiments expressed as fold increase with respect to unstimulated cells (control).

The MEK5-ERK5 Kinase Cascade Is Involved in the Activation of MEF2C Transcriptional Activity Mediated by G Protein-coupled Receptors-- MEF2C is a physiological substrate for ERK5 (39). Thus, we next asked whether the ability to enhance the in vitro phosphorylating activity of ERK5 by GPCRs resulted in enhanced transcriptional activity of MEF2 proteins in vivo. For these experiments, we fused the transactivation domain of MEF2C to the DNA binding domain of Gal4 and tested the ability to induce the expression from a pGal4-Luc reporter plasmid, as described previously (24). As shown in Fig. 2A, expression from the Gal4-driven luciferase reporter was induced by the stimulation of m1 and thrombin receptors, but not m2 receptors, which is consistent with their abilities to stimulate ERK5 kinase activity. Furthermore, transfection of a DNA plasmid for MEK5AA, which acts as a dominant negative mutant of MEK5 (24), completely blocked the increased transcriptional activity of MEF2C elicited by thrombin and partially inhibited m1 mediated-transcriptional activation (Fig. 2B). As a control, the activation of SRE mediated by m1 and thrombin receptors was unaffected by co-expression of MEK5AA (Fig. 2B). These findings suggested that the MEK5-ERK5 kinase pathway is functionally activated by GPCRs and that this kinase cascade is involved in the activation of MEF2C by m1 and thrombin receptors.


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 2.   Stimulation of G protein-coupled receptors enhances the transcriptional activity of MEF2C through the MEK5-ERK5 signaling pathways. A, COS-7 cells were cotransfected with pcDNA3 Gal4-MEF2C, pGal4-Luc, and pRL-null, together with expression vectors for GFP and m1, m2, and PAR1 receptors, as indicated. Cells were then exposed to vehicle (c), 100 µM carbachol (Cch), and 5 units/ml thrombin (Thr) for 6 h and processed as described in "Experimental Procedures." The data represent luciferase activity normalized by the luciferase activity from Renilla reniformis present in each cellular lysate, expressed as fold induction with respect to control cells, and are the mean ± S.E. of triplicate samples from a typical experiment. Similar results were obtained in three separate experiments. B, COS-7 cells were cotransfected with pSREmutL (SRE) or with pcDNA3 Gal4-MEF2C and pGal4-Luc (MEF2C), together with the expression plasmid for m1 (left panel) or PAR1 (right panel) receptors and with increasing amount of expression plasmid for MEK5AA, as indicated, and stimulated with vehicle, 100 µM carbachol, and 5 units/ml thrombin for 6 h. Cells were processed as described under "Experimental Procedures." The data represent CAT (for SRE) and luciferase activities (for MEF2C) expressed as percentage relative to those observed in cells that did not include MEK5AA and are the mean ± S.E. of triplicate samples from a typical experiment. Similar results were obtained in three independent experiments.

Activated Forms of Galpha 12 and Galpha 13 Stimulate ERK5-- To investigate which classes of G proteins mediate ERK5 activation induced by GPCRs, we examined the effects of activated forms of Galpha subunits as well as overexpression of beta gamma subunits of heterotrimeric G proteins on ERK5 kinase activity. As shown in Fig. 3A, expression of Galpha 12QL and Galpha 13QL could induce ERK5 activation, whereas ERK5 kinase activity was not altered by the expression of Galpha qQL, Galpha i2QL, Galpha sQL, and the beta gamma subunit, thus suggesting the involvement of the Galpha 12/13 family of G proteins in GPCR-mediated signaling to ERK5. Regarding Galpha i, these observations are in line with the failure of m2 muscarinic receptors, which transduce signals through Galpha i and beta gamma subunits, to activate ERK5. In addition, ERK5 activation mediated by m1 and thrombin receptors was found to be insensitive to pertussis toxin, which ADP ribosylates and inactivates Galpha i and Galpha o (data not shown), together indicating that pertussis toxin-sensitive G proteins cannot signal to ERK5 and that they do not mediate the activation of ERK5 by thrombin and m1 receptors.


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 3.   Signaling to ERK5 through Galpha q and Galpha 12/13 families of heterotrimeric G proteins. A, effects of activated mutants of G proteins alpha  subunits and beta gamma subunits on ERK5 kinase activity. COS-7 cells were transfected with an expression plasmid for HA-ERK5 together with plasmids expressing GFP, Galpha qQL, Galpha iQL, Galpha sQL, Galpha 12QL, Galpha 13QL, or beta 1gamma 2 subunits. Kinase reactions were performed using anti-HA immunoprecipitates from the corresponding cellular lysates. Data represent the mean ± S.E. of three independent experiments, expressed as fold increase with respect to control cells. B, COS-7 cells were cotransfected with pSREmutL and pCMV-beta -galactosidase plasmid DNAs as well as with the indicated expression vectors and stimulated with vehicle (-) or 100 µM carbachol (+) for 6 h. Cells were processed as described under "Experimental Procedures." The data represent CAT activity normalized by the beta -galactosidase activity present in each cellular lysate, expressed as fold induction with respect to control cells, and are the mean ± S.E. of triplicate samples from a typical experiment. Nearly identical results were obtained in three additional experiments. C, COS-7 cells were transfected with an expression plasmid for the HA-ERK5 together with the indicated expression vectors and stimulated with vehicle (-) or 100 µM carbachol (+) for 10 min. Kinase reactions were carried out using anti-HA immunoprecipitates from the corresponding cellular lysates. Data represent the mean ± S.E. of three independent experiments, expressed as fold increase with respect to control cells.

The Use of Chimeric G Protein alpha  Subunits Reveals That the Galpha q and Galpha 12/13 Families of Heterotrimeric G Proteins Can Signal to ERK5-- Stimulation of m1 receptors, that couple to Galpha q, can activate ERK5. However, expression of an activated form of Galpha q could not enhance the ERK5 kinase activity. Interestingly, this is highly reminiscent to that observed for the MAPK pathway (8, 40). Indeed, several lines of evidence suggest the ability of Galpha q to stimulate the MAPK pathway, but expression of activated Galpha q could not induce MAPK activation (8, 40), and it even prevents the further stimulation of MAPK by a variety of stimuli (41). This discrepancy may be ascribed to the desensitization of downstream signaling pathways, such as that two days after the transfection of expression plasmids the activity of MAPK and/or ERK5 may no longer be demonstrable. To solve this problem, we took advantage of the finding that a Gq/Gi chimera, where a C-terminal region of Galpha q is replaced by the corresponding region of Galpha i, can be stimulated by Gi-coupled receptors and is able to transmit Gq-mediated signaling pathways (29). Thus, upon coexpression of this Gq/Gi chimera together with a Gi-coupled receptor, such as m2 receptors, on and off Gq-mediated signaling can now be controlled by agonist addition. Moreover, the treatment with pertussis toxin can now make it possible to specifically activate Gq-mediated signals, because Galpha i is inhibited by pertussis toxin and no longer has the ability to stimulate downstream signaling pathways. The potential usefulness of this system prompted us to design also a similar G13/Gi chimera in which the C-terminal 5 amino acids of Galpha 13 were replaced by the corresponding sequences of Galpha i. Expression of this protein was confirmed by Western blot analysis (data not shown). The functional activity of these chimeras was assessed by examining their ability to stimulate the transcriptional activation of an SRE-containing reporter plasmid, as both Galpha q and Galpha 12/13 classes of G proteins are known to activate a SRE, but not the stimulation of Galpha i (42). As previously reported, the stimulation of m2 by carbachol did not activate the SRE (42), although transcription from the SRE was potently increased by m1 receptor stimulation (Fig. 3B). However, the exposure to carbachol of cells expressing both m2 receptors and either a Gq/Gi chimera or a G13/Gi chimera significantly induced SRE activation, whereas the expression of either a Gq/Gi chimera or a G13/Gi chimera alone did not affect the SRE activity (Fig. 3B). These results indicated that these Gq/Gi and G13/Gi chimeras can be stimulated by m2 receptors and are capable of transmitting Gq- and G13-mediated signaling pathways, respectively.

We then used this system to investigate whether Galpha q and Galpha 13 have the ability to stimulate ERK5. As shown in Fig. 3C, stimulation with carbachol induced a limited activation of ERK5 in cells expressing m2 receptors. However, in cells expressing either the Gq/Gi or G13/Gi chimera, m2 receptor stimulation significantly activated ERK5 (Fig. 3C). These results indicated that both the Galpha q and Galpha 12/13 classes of heterotrimeric G proteins are able to signal to ERK5 and thus might participate in ERK5 activation by GPCRs.

Signaling from G Protein-coupled Receptors to ERK5 Does Not Involve Ras and Rho GTPases-- Gi- and Gq-coupled receptors can stimulate the Ras-MAPK pathway effectively (6). However, we found that Gi-coupled m2 receptors fail to stimulate ERK5, suggesting that the pathway linking GPCRs to ERK5 is different from that which communicates these receptors to MAPK. Indeed, we observed that activated Ras causes only a very limited increase in the enzymatic activity of ERK5, although it potently stimulates MAPK (Fig. 4). On the other hand, activation of the JNK pathway is believed to be mediated by Rac and Cdc42, two members of the Rho family of GTPases (21). However, whereas expression of activated Rac and Cdc42 strongly enhanced the kinase activity of JNK, these GTPases failed to stimulate ERK5 (Fig. 4). Similarly, expression of an activated form of Rho, which stimulates the SRE-driven reporter plasmid potently (30), also failed to enhance the enzymatic activity of ERK5. Furthermore, although some minor variations in the activity of ERK5 can be observed upon expression of these GTPases, an activated form of MEK5, MEK5DD, consistently induced ERK5 activation under these experimental conditions (Fig. 4). Thus, activation of Ras, Rho, Rac, and Cdc42 may not be sufficient to stimulate the ERK5 pathway. However, it is still possible that these small GTPases are required for GPCR-mediated ERK5 activation. To address this possibility, we used the expression of dominant interfering molecules for each of these GTPases. As shown in Fig. 5A, the activation of ERK5 mediated by m1 and thrombin receptors was not affected by the expression of a dominant negative mutant of Ras, RasN17, although this inhibitory molecule effectively inhibited MAPK activation when induced by m1 stimulation (Fig. 5B) and by thrombin (data not shown), but not by phorbol esters, as previously reported (43, 44). Thus, together these data suggest that Ras is unlikely to play a prominent role in ERK5 activation by GPCRs. For Rac and Cdc42, we used the overexpression of a molecule containing the CRIB domain of PAK fused to GST, which can specifically bind the GTP-bound forms of Rac and Cdc42 thereby inhibiting these GTPases (28, 45, 46). Indeed, expression of the CRIB domain of PAK (PAK-N) significantly inhibited JNK activation evoked by the expression of activated forms of Rac and Cdc42 and by m1 stimulation but had only a limited effect on the activation of JNK by anisomycin, which served as a control for specificity (Fig. 5C). However, PAK-N did not affect the abilities of m1 and thrombin receptors to activate ERK5 (Fig. 5A). Together, these results indicated that the small GTPases Ras, Rac, and Cdc42 are not involved in the signaling from GPCRs to ERK5.


View larger version (50K):
[in this window]
[in a new window]
  		Fig. 4.   Activation of Ras- and Rho-related GTPases is not sufficient to stimulate the ERK5 pathway. COS-7 cells were transfected with expression plasmid for HA-ERK5, HA-MAPK, or HA-JNK, together with the plasmid expressing GFP or the activated mutant of H-Ras (RasV12), RhoA (RhoQL), Rac1 (RacQL), Cdc42 (Cdc42QL), or MEK5 (MEK5DD). Kinase reactions were performed using anti-HA immunoprecipitates from the corresponding cellular lysates. Labeled substrates are indicated. Data shown are from a representative experiment for each assay. Western blot (WB) analysis was performed with anti-HA antibodies using total cell lysates (HA-ERK5 and HA-MAPK) or anti-HA immunoprecipitates (HA-JNK).


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 5.   Signaling from G protein-coupled receptors to ERK5 does not require Ras- and Rho-related GTPases. A-C, COS-7 cells were cotransfected with expression plasmid for HA-ERK5 (A), HA-MAPK (B), or HA-JNK (C) as well as with expression vectors carrying cDNAs for GFP, m1, and PAR1 receptors or the activated mutant of Rac1 (RacQL) and Cdc42 (Cdc42QL), together with plasmids encoding inhibitory molecules (RasN17, RhoN19, and PAK-N). Cells were stimulated with or without 100 µM carbachol (Cch), 5 units/ml thrombin (Thr), 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or 10 µg/ml anisomycin as indicated. Kinase reactions were performed using anti-HA immunoprecipitates from the corresponding cellular lysates. Labeled substrates are indicated. Data shown are from a representative experiment for each assay. Western blot (WB) analysis was performed with anti-HA antibodies using total cell lysates (HA-ERK5 and HA-MAPK) or anti-HA immunoprecipitates (HA-JNK). D, COS-7 cells were cotransfected with pSREmutL and pCMV-beta -galactosidase plasmid DNAs as well as with expression vectors carrying cDNAs for GFP, m1 and PAR1 receptors, and the activated mutant of Cdc42 (Cdc42QL), with or without expression plasmids for C3 toxin, as indicated. The next day, the cells were stimulated with vehicle, 100 µM carbachol (Cch) or 5 units/ml thrombin (Thr) for 6 h. Cells were processed as described under "Experimental Procedures." The data represent CAT activity normalized by the beta -galactosidase activity present in each cellular lysate, expressed as fold induction with respect to control cells, and are the mean ± S.E. of triplicate samples from a typical experiment. Similar results were obtained in three separate experiments. E, COS-7 cells were transfected with expression vectors carrying DNA for HA-ERK5 and GFP, m1 or PAR1 receptors, with or without expression plasmid for C3 toxin, and stimulated with vehicle, 100 µM carbachol (Cch), 5 units/ml thrombin (Thr), or 100 ng/ml epidermal growth factor (EGF) for 10 min. Kinase reactions were performed in anti-HA immunoprecipitates from the corresponding cellular lysates. Data represent the mean ± S.E. of three independent experiments, expressed as fold increase with respect to unstimulated cells. Autoradiograms correspond to representative experiments. Western blot (WB) analysis was performed in the corresponding cellular lysates and immunodetected with the antibody to HA.

Interestingly, both Galpha q and Galpha 12/13 classes of G proteins, but not Galpha i, have been shown to activate Rho-dependent signaling pathways (42), and recent studies suggested that Rho-specific exchange factors such as p115-RhoGEF and PDZ-RhoGEF could be directly activated by the Galpha 12/13 family of G proteins (27, 47). Together, these findings suggested the possibility that Rho may participate in signaling to ERK5. However, an activated form of Rho did not enhance the kinase activity of ERK5 (see above, Fig. 4) and that of any other member of the MAPK superfamily in this cellular setting. Nonetheless, these observations cannot rule out the possibility that Rho stimulates certain MAPKs, which might not be revealed by the expression of its activated mutants for reasons such as those described for Galpha q. Thus, to explore further the possibility that Gq- and G12/13-coupled receptors uses Rho to stimulate ERK5, we used as a more definitive approach the expression of Clostridium botulinum C3 exoenzyme, which specifically ADP ribosylates Rho thus preventing its activation (48). As shown in Fig. 5D, a C3 toxin inhibited the activation of SRE, a typical Rho-dependent response (30), by m1 and thrombin receptors, although the Cdc42-mediated activation of SRE was unaffected and served as a control. However, this toxin did not change the abilities of m1 and thrombin receptors to activate ERK5 (Fig. 5E), strongly suggesting that the activation of ERK5 by GPCRs is independent of Rho.

In conclusion, the present study demonstrates that the ERK5 pathway can be functionally activated by the stimulation of GPCRs depending on their coupling specificity and that the Gq and G12/13 families of heterotrimeric G proteins can mediate this effect. As ERK5 regulates the activity of a growing number of nuclear transcription factors, these findings may help explain the distinct ability of Gq- and G12/13-coupled receptors to promote the expression of growth related genes. Furthermore, our observations raise the possibility of the existence of a novel signaling pathway whereby GPCRs enhance the activity of ERK5. Although the precise nature of this biochemical route is still unknown, we provide evidence that the pathway linking GPCRs to the MEK5-ERK5 kinase cascade is distinct from those utilized by these cell surface receptors to stimulate MAPK, JNK, and Rho GTPases.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed: Rm. 212, Bldg. 30, Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, 30 Convent Dr., Bethesda, MD 20892-4330. Tel.: 301-496-6259; Fax: 301-402-0823; E-mail: gutkind@nih.gov.

Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M002410200

    ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; G proteins, GTP-binding proteins; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; GPCRs, G protein-coupled receptors; HA, hemagglutinin; CRIB domain, Cdc42, Rac interactive binding domain; CAT, chloramphenicol acetyltransferase; SRE, serum response element; GST, glutathione S-transferase; GFP, green fluorescent protein; PAR1, protease activated receptor 1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Marshall, C. J. (1995) Cell 80, 179-185
2. Whitmarsh, A. J., and Davis, R. J. (1996) J. Mol. Med. 74, 589-607
3. Abe, M. K., Kuo, W. L., Hershenson, M. B., and Rosner, M. R. (1999) Mol. Cell. Biol. 19, 1301-1312
4. Miyata, Y., Akashi, M., and Nishida, E. (1999) Genes Cells 4, 299-309
5. Garrington, T. P., and Johnson, G. L. (1999) Curr. Opin. Cell Biol. 11, 211-218
6. Gutkind, J. S. (1998) J. Biol. Chem. 273, 1839-1842
7. Moolenaar, W. H., Kranenburg, O., Postma, F. R., and Zondag, G. C. (1997) Curr. Opin. Cell Biol. 9, 168-173
8. Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S. (1994) Nature 369, 418-420
9. Lopez-Ilasaca, M., Crespo, P., Pellici, P. G., Gutkind, J. S., and Wetzker, R. (1997) Science 275, 394-397
10. Luttrell, L. M., Daaka, Y., and Lefkowitz, R. J. (1999) Curr. Opin. Cell Biol. 11, 177-183
11. Seger, R., and Krebs, E. G. (1995) FASEB J. 9, 726-735
12. Mochizuki, N., Ohba, Y., Kiyokawa, E., Kurata, T., Murakami, T., Ozaki, T., Kitabatake, A., Nagashima, K., and Matsuda, M. (1999) Nature 400, 891-894
13. Vossler, M. R., Yao, H., York, R. D., Pan, M. G., Rim, C. S., and Stork, P. J. (1997) Cell 89, 73-82
14. de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., and Bos, J. L. (1998) Nature 396, 474-477
15. Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S. A., and Schlessinger, J. (1996) Nature 383, 547-550
16. Igishi, T., and Gutkind, J. S. (1998) Biochem. Biophys. Res. Commun. 244, 5-10
17. Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U. R. (1993) Nature 364, 249-252
18. Coso, O. A., Teramoto, H., Simonds, W. F., and Gutkind, J. S. (1996) J. Biol. Chem. 271, 3963-3966
19. Yamauchi, J., Nagao, M., Kaziro, Y., and Itoh, H. (1997) J. Biol. Chem. 272, 27771-27777
20. Prasad, M. V., Dermott, J. M., Heasley, L. E., Johnson, G. L., and Dhanasekaran, N. (1995) J. Biol. Chem. 270, 18655-18659
21. Coso, O. A., Chiariello, M., Yu, J. C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146
22. Nagao, M., Kaziro, Y., and Itoh, H. (1999) Oncogene 18, 4425-4434
23. Collins, L. R., Minden, A., Karin, M., and Brown, J. H. (1996) J. Biol. Chem. 271, 17349-17353
24. Marinissen, M. J., Chiariello, M., Pallante, M., and Gutkind, J. S. (1999) Mol. Cell. Biol. 19, 4289-4301
25. Lee, J. D., Ulevitch, R. J., and Han, J. (1995) Biochem. Biophys. Res. Commun. 213, 715-724
26. Zhou, G., Bao, Z. Q., and Dixon, J. E. (1995) J. Biol. Chem. 270, 12665-12669
27. Fukuhara, S., Murga, C., Zohar, M., Igishi, T., and Gutkind, J. S. (1999) J. Biol. Chem. 274, 5868-5879
28. Teramoto, H., Coso, O. A., Miyata, H., Igishi, T., Miki, T., and Gutkind, J. S. (1996) J. Biol. Chem. 271, 27225-27228
29. Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., and Bourne, H. R. (1993) Nature 363, 274-276
30. Hill, C. S., Wynne, J., and Treisman, R. (1995) Cell 81, 1159-1170
31. Berstein, G., Blank, J. L., Smrcka, A. V., Higashijima, T., Sternweis, P. C., Exton, J. H., and Ross, E. M. (1992) J. Biol. Chem. 267, 8081-8088
32. Kato, Y., Tapping, R. I., Huang, S., Watson, M. H., Ulevitch, R. J., and Lee, J. D. (1998) Nature 395, 713-716
33. Kamakura, S., Moriguchi, T., and Nishida, E. (1999) J. Biol. Chem. 274, 26563-26571
34. Peralta, E. G., Ashkenazi, A., Winslow, J. W., Smith, D. H., Ramachandran, J., and Capon, D. J. (1987) EMBO J. 6, 3923-3929
35. Grand, R. J., Turnell, A. S., and Grabham, P. W. (1996) Biochem. J. 313, 353-368
36. Aragay, A. M., Collins, L. R., Post, G. R., Watson, A. J., Feramisco, J. R., Brown, J. H., and Simon, M. I. (1995) J. Biol. Chem. 270, 20073-20077
37. Majumdar, M., Seasholtz, T. M., Buckmaster, C., Toksoz, D., and Brown, J. H. (1999) J. Biol. Chem. 274, 26815-26821
38. Gohla, A., Offermanns, S., Wilkie, T. M., and Schultz, G. (1999) J. Biol. Chem. 274, 17901-17907
39. Kato, Y., Kravchenko, V. V., Tapping, R. I., Han, J., Ulevitch, R. J., and Lee, J. D. (1997) EMBO J. 16, 7054-7066
40. Faure, M., Voyno-Yasenetskaya, T. A., and Bourne, H. R. (1994) J. Biol. Chem. 269, 7851-7854
41. Qian, N. X., Winitz, S., and Johnson, G. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4077-4081
42. Mao, J., Yuan, H., Xie, W., Simon, M. I., and Wu, D. (1998) J. Biol. Chem. 273, 27118-27123
43. Seo, B., Choy, E. W., Maudsley, S., Miller, W. E., Wilson, B. A., and Luttrell, L. M. (2000) J. Biol. Chem. 275, 2239-2245
44. Ueda, Y., Hirai, S., Osada, S., Suzuki, A., Mizuno, K., and Ohno, S. (1996) J. Biol. Chem. 271, 23512-23519
45. Minden, A., Lin, A., Claret, F. X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157
46. Osada, S., Izawa, M., Koyama, T., Hirai, S., and Ohno, S. (1997) FEBS Lett. 404, 227-233
47. Hart, M. J., Jiang, X., Kozasa, T., Roscoe, W., Singer, W. D., Gilman, A. G., Sternweis, P. C., and Bollag, G. (1998) Science 280, 2112-2114
48. Aktories, K., and Hall, A. (1989) Trends Pharmacol. Sci. 10, 415-418

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
H. Morimoto, K. Kondoh, S. Nishimoto, K. Terasawa, and E. Nishida
Activation of a C-terminal Transcriptional Activation Domain of ERK5 by Autophosphorylation
J. Biol. Chem., December 7, 2007; 282(49): 35449 - 35456.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
B. C. W. Groenendijk, K. Van der Heiden, B. P. Hierck, and R. E. Poelmann
The Role of Shear Stress on ET-1, KLF2, and NOS-3 Expression in the Developing Cardiovascular System of Chicken Embryos in a Venous Ligation Model
Physiology, December 1, 2007; 22(6): 380 - 389.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. J. Martin, T. Tanos, A. B. Garcia, D. Martin, J. S. Gutkind, O. A. Coso, and M. J. Marinissen
The G{alpha}12/13 Family of Heterotrimeric G Proteins and the Small GTPase RhoA Link the Kaposi Sarcoma-associated Herpes Virus G Protein-coupled Receptor to Heme Oxygenase-1 Expression and Tumorigenesis
J. Biol. Chem., November 23, 2007; 282(47): 34510 - 34524.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
G. B. Atkins and M. K. Jain
Role of Kruppel-Like Transcription Factors in Endothelial Biology
Circ. Res., June 22, 2007; 100(12): 1686 - 1695.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
Z. Liao, C. I. Seye, G. A. Weisman, and L. Erb
The P2Y2 nucleotide receptor requires interaction with {alpha}v integrins to access and activate G12
J. Cell Sci., May 1, 2007; 120(9): 1654 - 1662.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. A. Winn, M. Van Scoyk, M. Hammond, K. Rodriguez, J. T. Crossno Jr., L. E. Heasley, and R. A. Nemenoff
Antitumorigenic Effect of Wnt 7a and Fzd 9 in Non-small Cell Lung Cancer Cells Is Mediated through ERK-5-dependent Activation of Peroxisome Proliferator-activated Receptor {gamma}
J. Biol. Chem., September 15, 2006; 281(37): 26943 - 26950.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
K. Kondoh, K. Terasawa, H. Morimoto, and E. Nishida
Regulation of nuclear translocation of extracellular signal-regulated kinase 5 by active nuclear import and export mechanisms.
Mol. Cell. Biol., March 1, 2006; 26(5): 1679 - 1690.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Liu and T. A. Voyno-Yasenetskaya
Radixin Stimulates Rac1 and Ca2+/Calmodulin-dependent Kinase, CaMKII: CROSS-TALK WITH G{alpha}13 SIGNALING
J. Biol. Chem., November 25, 2005; 280(47): 39042 - 39049.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
M. Steinhoff, J. Buddenkotte, V. Shpacovitch, A. Rattenholl, C. Moormann, N. Vergnolle, T. A. Luger, and M. D. Hollenberg
Proteinase-Activated Receptors: Transducers of Proteinase-Mediated Signaling in Inflammation and Immune Response
Endocr. Rev., February 1, 2005; 26(1): 1 - 43.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Fukuhara, A. Sakurai, H. Sano, A. Yamagishi, S. Somekawa, N. Takakura, Y. Saito, K. Kangawa, and N. Mochizuki
Cyclic AMP Potentiates Vascular Endothelial Cadherin-Mediated Cell-Cell Contact To Enhance Endothelial Barrier Function through an Epac-Rap1 Signaling Pathway
Mol. Cell. Biol., January 1, 2005; 25(1): 136 - 146.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. J. Marinissen, J.-M. Servitja, S. Offermanns, M. I. Simon, and J. S. Gutkind
Thrombin Protease-activated Receptor-1 Signals through Gq- and G13-initiated MAPK Cascades Regulating c-Jun Expression to Induce Cell Transformation
J. Biol. Chem., November 21, 2003; 278(47): 46814 - 46825.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
R. P. Thomas, M. R. Hellmich, C. M. Townsend Jr., and B. M. Evers
Role of Gastrointestinal Hormones in the Proliferation of Normal and Neoplastic Tissues
Endocr. Rev., October 1, 2003; 24(5): 571 - 599.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J.-M. Servitja, M. J. Marinissen, A. Sodhi, X. R. Bustelo, and J. S. Gutkind
Rac1 Function Is Required for Src-induced Transformation: EVIDENCE OF A ROLE FOR TIAM1 AND VAV2 IN RAC ACTIVATION BY SRC
J. Biol. Chem., September 5, 2003; 278(36): 34339 - 34346.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y.-S. Guo, J.-Z. Cheng, G.-F. Jin, J. S. Gutkind, M. R. Hellmich, and C. M. Townsend Jr.
Gastrin Stimulates Cyclooxygenase-2 Expression in Intestinal Epithelial Cells through Multiple Signaling Pathways. EVIDENCE FOR INVOLVEMENT OF ERK5 KINASE AND TRANSACTIVATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR
J. Biol. Chem., December 6, 2002; 277(50): 48755 - 48763.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Chikumi, S. Fukuhara, and J. S. Gutkind
Regulation of G Protein-linked Guanine Nucleotide Exchange Factors for Rho, PDZ-RhoGEF, and LARG by Tyrosine Phosphorylation. EVIDENCE OF A ROLE FOR FOCAL ADHESION KINASE
J. Biol. Chem., March 29, 2002; 277(14): 12463 - 12473.
[Abstract] [Full Text] [PDF]