ACCELERATED PUBLICATION
Stress-induced Inhibition of ERK1 and ERK2 by Direct Interaction with p38 MAP Kinase*

Hong ZhangDagger , Xiaoqing ShiDagger , Maggie HampongDagger , Litsa Blanis§, and Steven PelechDagger §

From the Dagger  Department of Medicine, Koerner Pavilion, University of British Columbia, Vancouver, British Columbia V6T 1Z3 and the § Kinexus Bioinformatics Corporation, Vancouver, British Columbia V6T 1Z4, Canada

Received for publication, December 26, 2000, and in revised form, January 9, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

We have identified a direct physical interaction between the stress signaling p38alpha MAP kinase and the mitogen-activated protein kinases ERK1 and ERK2 by affinity chromatography and coimmunoprecipitation studies. Phosphorylation and activation of p38alpha enhanced its interaction with ERK1/2, and this correlated with inhibition of ERK1/2 phosphotransferase activity. The loss of epidermal growth factor-induced activation and phosphorylation of ERK1/2 but not of their direct activator MEK1 in HeLa cells transfected with the p38alpha activator MKK6(E) indicated that activated p38alpha may sequester ERK1/2 and sterically block their phosphorylation by MEK1.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Mitogen-activated protein (MAP)1 kinase modules are involved in the signal transduction of a wide variety of cellular responses in all eukaryotic organisms including proliferation, differentiation, and apoptosis (1). At least four distinct and parallel MAP kinase cascades have been identified, including extracellular signal-regulated kinases 1 and 2 (ERK1/2), the p38 MAP kinases, c-jun N-terminal or stress-activated protein kinases (JNK/SAPK), and ERK5/big MAP kinase 1 (BMK1). It is well established that ERK1/2 are typically stimulated by growth-related stimuli through the Raf1/B-MEK1/2-ERK1/2 protein kinase cascade. The JNK and p38 MAP kinases are primarily activated by stress-related signals such as heat and osmotic shock, UV irradiation, and proinflammatory cytokines by means of the MAP kinase kinases, MKK3, -4, -6, and -7 (2-4). Whereas the selective activation of distinct MAP kinase pathways in response to different extracellular stimuli has been extensively documented, there is increasing evidence for cross-talk between distinct MAP kinase pathways. A p38-dependent ERK1/2 activation was observed in several mammalian cell lines including the human embryonic kidney cell line HEK293 upon arsenite treatment (5). It was also found that inactivation of p38 by SB202190 treatment resulted in a delayed and prolonged activation of ERK1/2 in the human hepatoma cell line, HepG2 (6). In both cases, MEK1 was implicated in the activation of ERK1/2. Here we report that in HeLa and HEK293 cells, stress stimuli lead to an inhibition of ERK1/2 via p38alpha . Phosphorylated p38alpha is capable of forming a complex with ERK1/2, and it prevents their phosphorylation by MEK1/2.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Construction of pGEX-p38alpha -- The pGEX-p38alpha wild-type and p38alpha (AF) dominant negative mutant were constructed by subcloning the respective cDNA sequences into pGEX-4T vector (Amersham Pharmacia Biotech). The human p38 full-length sequences were obtained by digesting pcDNA3-flagp38alpha wild-type and p38alpha (AF) plasmids, a generous gift of Dr. J. Han (the Scripps Research Institute, La Jolla, CA). pGEX-ERK1 was obtained by subcloning human ERK1 cDNA full-length sequence into pGEX-2T vector (Amersham Pharmacia Biotech). The resulting GST fusion protein constructs were verified by DNA sequencing (7).

Expression of GST Fusion Proteins in Bacteria-- GST fusion protein plasmids were transformed into DH5alpha bacteria. Expression of GST fusion proteins was induced by 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside at 37 °C for 3 h. GST fusion proteins were purified as described previously on glutathione-agarose (Sigma) and eluted from beads with reduced glutathione; GST tag was cleaved with thrombin when necessary (8).

GST Fusion Protein Pull-down-- Two mg of rat brain lysate or 1 mg of HeLa cell lysate was mixed with 20 µg of immobilized GST-ERK1, GST-p38alpha , or GST alone on glutathione-agarose beads at 4 °C with rotation. After a 2-h incubation, beads were washed three times with 50 mM Tris (pH 8.0), 150 mM NaCl, and 1% Nonidet P-40 followed by separation of bound proteins by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to nitrocellulose membrane, and immunoblotting was performed with either anti-p38alpha or anti-ERK1-CT antibody (StressGen, Victoria, British Columbia, Canada).

In Vivo Association of ERK1 and p38-- HEK293 or HeLa cells cultured in Dulbecco's minimum essential medium (DMEM, Life Technologies, Inc.) containing 10% fetal bovine serum were grown to 50-60% confluence and transfected with pcDNA3-p38alpha using SuperFect reagent (Qiagen, Mississauga, Ontario, Canada) as per the manufacturer's instructions. For each 100-mm dish, 5 µg of plasmid DNA were introduced into cells using 30 µl of SuperFect reagent in serum-free medium. After a 3-h incubation, cells were starved in fresh serum-free DMEM. 24 h after transfection, cells were stimulated with anisomycin (Sigma) or arsenite (Sigma) alone or in combination with SB203580 (Calbiochem) in serum-free DMEM as indicated in the figure legends. Cells were lysed in 500 µl of lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, 0.5% (w/v) Nonidet P-40, 1 mM dithiothreitol (DTT), 20 mM beta -glycerophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and sonicated for 30 s. Cell debris was removed by centrifugation at 13,000 rpm for 15 min at 4 °C. Protein concentration was determined by the Bradford assay (9). Cell lysates were precleared with 40 µl of protein A-Sepharose (Amersham Pharmacia Biotech) beads and then incubated with anti-ERK1-CT antibody (1 µg/ml of lysate) at 4 °C overnight. 40 µl of protein A-Sepharose beads were then added to precipitate immunocomplexes and washed with lysis buffer three times; immunoprecipitates were analyzed by immunoblotting with anti-p38alpha antibody.

MBP Kinase Assay-- Anti-ERK1 immunoprecipitates bound to protein A beads were further washed twice in assay dilution buffer (25 mM beta -glycerophosphate, 20 mM MOPS, pH 7.2, 5 mM EGTA, 2 mM EDTA, 20 mM MgCl2, 1 mM Na3VO4, 0.25 mM DTT) and then incubated with 20 µg of MBP (Sigma) in the same buffer supplemented with 50 µM ATP and 10 µCi of [gamma -32P]ATP (Amersham Pharmacia Biotech) in a volume of 20 µl at 30 °C for 15 min. Reactions were stopped by spotting onto P81 paper. After three washes in 1% H3PO4, phosphotransferase activity was quantitated by liquid scintillation counting.

Interaction of Bacterially Expressed p38alpha and ERK1 in Vitro-- Eluted GST-p38alpha wild-type or GST-p38alpha (AF) mutant fusion protein was first incubated with bacterially expressed MKK6(E), a constitutively active form of MKK6, at 30 °C for 2 h in 1× kinase buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM DTT, 150 µM phenylmethylsulfonyl fluoride, 0.4 mM ATP). GST-p38alpha proteins were then absorbed onto to glutathione-agarose beads and incubated with bacterially expressed ERK1 at 4 °C for 2 h. Following three washes in lysis buffer, the beads were boiled in 2× Laemmli sample buffer (10), and the bound proteins were separated on SDS-PAGE. Conversely, p38alpha was first incubated with GST-MKK6(E) beads for activation under the conditions described above. GST-MKK6(E) beads were pelleted and discarded, and GST-ERK1 beads were added to the supernatants. After a 2-h incubation, the bound proteins were resolved by SDS-PAGE.


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

Evidence for the Inhibition of ERK1/2 by p38-- By using antibody specific for the phosphorylated (activated) form of ERK1/2 in Western blotting studies, we observed that anisomycin treatment resulted in decreased ERK1/2 phosphorylation. By contrast, SB203580, a specific p38 MAP kinase inhibitor, induced increased ERK1/2 phosphorylation in HeLa cells similar to that observed with epidermal growth factor (EGF) exposure (Fig. 1A). These findings are consistent with the results in HepG2 cells (6) and indicate that p38 MAP kinase somehow exerts an inhibitory effect on ERK1/2 activation. Surprisingly, an increase of myelin basic protein (MBP) phosphotransferase activity associated with anti-ERK1 immunoprecipitates was detected upon anisomycin treatment, which could be inhibited by including SB203580 in kinase assay reactions (Fig. 1B). This observation indicated that the increase of MBP phosphotransferase activity precipitated by anti-ERK1 antibody upon anisomycin treatment was at least partially due to p38 MAP kinases. The apparent contradiction between the ERK1/2 phosphorylation state in Western blotting and the MBP phosphotransferase activity associated with anti-ERK1 immunoprecipitates could be potentially reconciled if p38 is coprecipitated with ERK1/2 in response to anisomycin treatment. Previously, we reported that ERK1 was found in immunoprecipitates of a p38 homologue in immature sea star oocytes (11). Based on these observations, we postulated that the direct interaction between p38 and ERK1/2 MAP kinases might play a role in coordinating the regulation of these two distinct MAP kinase pathways.



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Fig. 1.   Effects of anisomycin and SB203580 on the ERK1/2 phosphorylation and MBP kinase activity associated with anti-ERK1 immunoprecipitates. A, HeLa cells were treated with anisomycin (10 µg/ml, 30 min), SB203580 (5 µM, 20 min), and EGF (100 ng/ml, 15 min; Calbiochem), respectively, and the phosphorylation of ERK1/2 was determined by Western blot analysis with anti-active ERK1/2 antibody (Santa Cruz Biotechology). B, MBP kinase activity was determined in anti-ERK1 immunoprecipitates from anisomycin-, SB203580-, or EGF-treated HeLa cells. The MBP kinase activity associated with anti-ERK1 immunoprecipitates from anisomycin-treated cells could be inhibited by including 5 µM SB203580 in the kinase assay reaction. The data shown are the means +/- S.E. of 4 experiments.

Interaction of ERK1/2 and p38 in Vitro-- To examine whether a physical interaction occurs between p38 and ERK1, we expressed glutathione S-transferase (GST) fusion proteins of the full-length human ERK1 and p38alpha in bacteria and used them to affinity purify proteins from a rat brain lysate. Immunoblotting with anti-p38alpha antibody revealed the presence of p38alpha protein on the GST-ERK1 beads at a level well above that bound to GST alone (Fig. 2A). Under similar conditions, GST-p38alpha was able to pull down both ERK1 and ERK2 proteins (Fig. 2B). These results indicated a specific, direct interaction between ERK1/2 and p38alpha .



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Fig. 2.   Interaction of ERK1 and p38alpha in rat brain lysate revealed by GST fusion protein pull-down experiments. A, after incubation with rat brain lysate, GST-ERK1 and GST beads were washed and immunoblotted with anti-p38alpha antibody. B, ERK1/2 were found on GST-p38alpha beads after incubation with rat brain lysate.

Interaction of ERK1/2 and p38 Is p38 Activity-dependent-- We next examined the interaction between ERK1 and p38alpha in HeLa cells treated with stimuli that specifically activate either the ERK1/2 or p38 MAP kinase pathways. The treatment of anisomycin resulted in a significant increase of p38alpha that was bound by GST-ERK1 (data not shown). No apparent difference of the amount of p38alpha protein purified by GST-ERK1 fusion protein between EGF-treated and untreated control HeLa cells was observed. Treating HeLa cells with SB203580 prior to anisomycin stimulation diminished the association of ERK1 with p38alpha . The correlation of the enhancement of the binding of ERK1 and p38alpha with p38 kinase activation indicated that the interaction between these two MAP kinases was dependent upon the p38 but not the ERK1 activity status.

We further monitored the physical interaction of these two MAP kinases in mammalian cells by coimmunoprecipitation. HeLa cells were transfected with human p38alpha full-length DNA and then treated with anisomycin 24 h later. Cell lysate was prepared and precipitated with anti-ERK1-CT antibody. Anti-ERK1 immunoprecipitates were probed with anti-p38alpha antibody in Western blot analysis for coprecipitated p38alpha . As shown in Fig. 3A, a much higher level of p38alpha protein was detected in the anti-ERK1 precipitates from anisomycin-treated HeLa cells than in those from untreated control cells. This finding was consistent with the GST fusion protein pull-down results and further confirmed the requirement of p38 kinase activity for the interaction between ERK1 and p38alpha .



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Fig. 3.   Coimmunoprecipitation of ERK1 and p38alpha in human cell lines in a p38 kinase activity-dependent manner. A, after transfection with pcDNA3-p38alpha and treatment with anisomycin (10 µg/ml, 30 min), HeLa cells were lysed, and the lysate was precipitated by anti-ERK1-CT antibody. Anti-ERK1 precipitates were immunoblotted with anti-p38alpha antibody. B, HEK293 cells were transfected with pcDNA3-p38alpha 24 h prior to treatment with arsenite (0.5 mM, 4 h) or a combination of SB203580 (5 µM, 60 min prior to arsenite) and arsenite (SB+Arsenite). Cell lysates were prepared and immunoprecipitation was performed with anti-ERK1-CT antibody. Immunoprecipitates were probed with anti-p38alpha antibody. p38alpha was present in higher levels in arsenite-treated cells than found in SB203580-treated and untreated control cells.

Similarly, interaction between ERK1 and p38alpha in HEK293 cells transfected with p38alpha was observed in a p38 activity-dependent manner (Fig. 3B). Compared with untreated control cells, an increasing level of p38alpha protein precipitated by anti-ERK1-CT antibody was found in arsenite-treated HEK293 cells. Pretreating HEK293 cells with SB203580 prior to arsenite reduced the level of p38alpha precipitated by anti-ERK1-CT to that of untreated control. These results confirmed a p38 activity-dependent association of p38alpha with endogenous ERK1 in mammalian cells.

Direct Interaction Occurs between Purified ERK1 and p38 in Vitro-- We further examined whether a direct interaction occurs between p38alpha and ERK1 using recombinant forms of these MAP kinases. Bacterially expressed ERK1 protein was mixed with GST-p38alpha wild-type or dominant negative mutant p38alpha (AF) that was preincubated with MKK6(E), a p38-specific upstream activating kinase. p38alpha (AF) is a kinase-inactive mutant with the substitution of two activating phosphorylation sites by alanine and phenylalanine (5), whereas MKK6 is constitutively activated by replacement of two activating phosphorylation sites with glutamic acid (12). The ERK1 protein was affinity-purified by p38alpha wild-type protein after its activation by MKK6(E) (Fig. 4A). No MKK6 protein was detected in the p38alpha /ERK1 complexes (data not shown), indicating that p38alpha and ERK1 can interact with each other without involvement of other proteins. Consistent with the requirement of p38 kinase activity in the formation of ERK1/p38 complexes, ERK1 precipitated by p38alpha wild-type protein was present at a higher level than that obtained with the p38alpha (AF) mutant. Similar observations were made in the converse experiment, where GST-ERK1 bound active wild-type but not inactive wild-type and dominant negative forms of p38alpha (Fig. 4B). Furthermore, Western blotting analysis with anti-phospho-p38alpha antibody revealed the p38alpha protein coprecipitated with ERK1 was in its phosphorylated form (Fig. 4C).



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Fig. 4.   Direct interaction between bacterially expressed ERK1 and p38alpha in vitro. A, following incubating with 20 µg of MKK6(E), 50 µg of eluted GST-p38alpha wild-type (WT) or GST-p38alpha (AF) was precipitated with glutathione-agarose beads. 50 µg of thrombin-digested GST-ERK1 was added to GST-p38alpha beads, and ERK1 that bound to p38 was detected by Western blotting with anti-ERK1-CT antibody. B, 50 µg of thrombin-digested p38alpha wild-type or p38alpha (AF) was first incubated with MKK6(E) and then incubated with 50 µg of GST-ERK1 immobilized on glutathione-agarose beads. GST-ERK1-bound p38alpha was detected by Western blotting with anti-p38alpha antibody. C, a similar blot to that shown in B was probed with anti-active p38alpha antibody (New England Biolabs, Beverly, MA).

p38 Does Not Suppress MEK1 Phosphorylation to Inhibit ERK1/2-- To assess the roles of p38-ERK1/2 interaction in regulating the activation of ERK1/2, we activated endogenous p38 kinases by transfecting HeLa cells with MKK6(E) DNA and then treated the cells with EGF and monitored the activation of ERK1/2 and their upstream activating kinase, MEK1. In response to EGF treatment, the phosphorylated forms of ERK1/2 were present at much lower levels in MKK6(E)-transfected cells than in nontransfected HeLa cells (Fig. 5A), indicating an inhibitory effect of active p38 on ERK1/2. Moreover, no apparent difference in the phosphorylation of MEK1 at its activation sites was observed under these two circumstances (Fig. 5B). These results indicate that p38 suppresses ERK1/2 by a mechanism independent of MEK1 phosphotransferase activity.



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Fig. 5.   p38alpha suppresses ERK1 activation by EGF without affecting MEK1 phosphorylation. HeLa cells were transfected either with pcDNA3-MKK6(E) (lane MKK6) or with pcDNA3-CAT (Control). 24 h later, both MKK6- and CAT-transfected cells were treated with EGF (100 ng/ml, 15 min). A, activation of ERK1/2 was determined by Western blotting with anti-active ERK1/2 antibody. B, phosphorylation of MEK1 was monitored by Western blotting with anti-active MEK1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

In summary, our study provides the first experimental evidence for the direct interaction between two MAP kinases lying in two distinct signaling cascades, raising the possibility that the direct interaction between p38alpha and ERK1 may play a role in coordinating activation of these two distinct MAP kinase pathways. These findings differ from those revealed by previous studies in that the cross-talk between the p38 and ERK1/2 signaling pathways was believed to mediate through upstream activating kinases of the ERK1/2 cascade. Moreover, the necessity of p38, but not ERK1, phosphotransferase activity for the interaction between ERK1 and p38alpha indicates the cross-talk between these two MAP kinase pathways is a one-way process.

Combined with previous studies, our results indicate that the communication between p38 and the ERK1/2 pathways may act through two distinct modes. Active p38 may suppress ERK1/2 phosphotransferase activity either through inhibition of upstream activating kinases of ERK1/2 or through direct interaction between p38 and ERK1/2. Based on our observation of the direct association between p38alpha and ERK1 and the inhibitory effect of p38 on ERK1/2 phosphotransferase activities independent of MEK1 phosphorylation, we hypothesize that activated p38 may sequester ERK1/2 and sterically block phosphorylation of these MAP kinases by MEK1/2. The ability of activated p38 to regulate another protein kinase allosterically is not restricted to ERK1/2. We have recently reported that activated p38 can also form a complex with casein kinase CK2 in HeLa cells, but in that instance p38 activates CK2 (13).


    ACKNOWLEDGEMENTS

We thank Dr. J. Han, the Scripps Research Institute (La Jolla, CA) for pcDNA3-flagp38alpha wild-type and p38alpha (AF) dominant negative mutant. Constructs of constitutively active MKK6(E) mutants, pGEX-MKK6(E) and pcDNA3-MKK6(E), were provided by Dr. C. Glembotski, San Diego State University and Dr. R. Davis, University of Massachusetts, respectively.


    FOOTNOTES

* This work was supported by operating grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of British Columbia and Yukon, Canada (to S. P.).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: Dept. of Medicine, Koerner Pavilion, 2211 Wesbrook Mall, University of British Columbia, Vancouver, B. C. V6T 1Z3 Canada. Tel.: 604-822-9963; Fax: 604-822-8693; E-mail: spelech@home.com.

Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.C000917200


    ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP/ERK kinase; MKK, MAP kinase kinase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's minimum essential medium; DTT, dithiothreitol; MBP, myelin basic protein; MOPS, 3-(N-morpholino)propanesulfonic acid; EGF, epidermal growth factor.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.