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Volume 270, Number 13, Issue of March 31, 1995 pp. 7420-7426
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pro-inflammatory Cytokines and Environmental Stress Cause p38 Mitogen-activated Protein Kinase Activation by Dual Phosphorylation on Tyrosine and Threonine (*)

(Received for publication, November 29, 1994; and in revised form, January 27, 1995)

Joël Raingeaud (1),  Shashi Gupta (1),  Jeffrey S. Rogers (1),  Martin Dickens (1),  Jiahuai Han (3),  Richard J. Ulevitch (3),  Roger J. Davis (1) (2)(§)

From the  (1)Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, the (2)Howard Hughes Medical Institute, Worcester, Massachusetts 01605, and the (3)Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.

INTRODUCTION

Several MAP (^1)kinase signal transduction pathways have been molecularly characterized(1) . At least four genetically distinct signaling pathways have been defined in the yeast Saccharomyces cerevisiae(2) . One pathway leads to activation of the FUS3 and KSS1 MAP kinases and is required for the response to mating pheromone(3) . A second MAP kinase pathway (MPK1) functions during cell wall biosynthesis(4, 5) . A third genetically defined MAP kinase pathway (HOG1) is involved in osmoregulation(6) . The fourth MAP kinase pathway (SMK1) is required for the control of sporulation(7) . Significantly, these MAP kinase pathways appear to function independently because mutations that disrupt one pathway do not alter signal transduction mediated by the other pathways(2) . This independent function may arise from the substrate specificity of the MAP kinase cascades. In addition, it has been established that there is an important role for tethering proteins (e.g. STE5) that bind multiple components of the MAP kinase cascade to create a functional signal transduction module(8, 9) .

Although detailed information is available for yeast, the organization of MAP kinase pathways in mammals is more poorly understood. The ERK group of MAP kinases is activated by growth factors via a Ras-dependent signal transduction pathway(10) . In contrast, the JNK group of MAP kinases (also designated SAPK) is activated by pro-inflammatory cytokines and environmental stress(11, 12, 13, 14, 15, 16, 17) . JNK activation is also observed during co-stimulation of T lymphocytes(18) . Importantly, the signal transduction pathways that lead to ERK and JNK activation are biochemically and functionally distinct(11) .

Recently, a novel mammalian MAP kinase (p38) was identified by Han et al.(19) . This MAP kinase isoform has been implicated in the mechanism of activation of MAPKAP kinase-2 (20, 21) and the expression of pro-inflammatory cytokines(22) . Homologs of p38 MAP kinase (CSBP1 and CSBP2) have been identified in human tissues(22) . A p38 MAP kinase homolog (MPK2) has also been identified in Xenopus laevis(20) . The purpose of this study was to examine the mechanism of p38 activation and to establish the relationship of the p38 MAP kinase pathway to the ERK and JNK signal transduction pathways.

EXPERIMENTAL PROCEDURES

Materials

Tumor necrosis factor alpha and interleukin-1alpha were from Genzyme Corp. Lipolysaccharide (LPS) was isolated from lyophilized Salmonella minesota Re595 bacteria as described (23) . Phorbol myristate acetate was from Sigma. EGF was purified from mouse salivary glands(24) . The monoclonal antibodies M2 and PY20 were obtained from IBI-Kodak and ICN, respectively. [P]ATP was prepared using a Gammaprep A kit (Promega Biotech) and [P]phosphate (DuPont NEN). Recombinant ATF2 proteins have been described(25) . GST-IkappaB was provided by Dr. D. Baltimore (Massachusetts Institute of Technology). GST-c-Myc(26) , GST-EGF-R (residues 647-688)(27) , and GST-c-Jun (11) fusion proteins have been described. GST-p38 MAP kinase was prepared using the expression vector pGEX and a polymerase chain reaction fragment containing the coding region of the p38 MAP kinase cDNA. The GST fusion proteins were purified by affinity chromatography using gluthathione-agarose(28) . Polyclonal antibodies that recognize JNK and p38 MAP kinase were raised in rabbits using GST-p38 and GST-JNK1 as antigens.

The plasmid pCMV-Flag-JNK1 (11) and the expression vectors for human MKP-1 (CL100) and PAC-1 (29) have been described. The plasmid pCMV-Flag-p38 MAP kinase was prepared using the expression vector pCMV5 (30) and the p38 cDNA. The Flag epitope (-Asp-Tyr-Lys-Asp-Asp-Asp-Aps-Lys-; Immunex Corp.) was inserted between codons 1 and 2 of p38 by insertional overlapping polymerase chain reaction(31) . A similar polymerase chain reaction procedure was employed to replace Thr and Tyr with Ala and Phe, respectively. The sequence of all plasmids was confirmed by automated sequencing using an Applied Biosystems model 373A machine.

Tissue Culture

COS-1 and HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% calf serum (Life Technologies, Inc.). Chinese hamster ovary cells expressing human CD14 (32) were maintained in Ham's F-12 medium supplemented with 5% fetal bovine serum (Life Technologies, Inc.). Transient transfection assays were performed using the lipofectamine reagent according to the manufacturer's recommendations (Life Technologies, Inc.). Phosphate labeling was performed by incubation of cells (4 h) in phosphate-free modified Eagle's medium (Flow Laboratories Inc.) supplemented with 1 mCi/ml [P]phosphate (DuPont NEN) and 1% fetal bovine serum.

Western Blot Analysis

Proteins were fractionated by SDS-PAGE, electrophoretically transferred to an Immobilon-P membrane, and probed with monoclonal antibodies to phosphotyrosine (PY20) and the Flag epitope (M2). Immunecomplexes were detected using enhanced chemiluminescence (Amersham International PLC).

Immunoprecipitation

The cells were solubilized with lysis buffer (20 mM Tris (pH 7.4), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 2 mM pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin) and centrifuged at 15,000 times g for 15 min at 4 °C. The epitope-tagged protein kinases were immunoprecipitated by incubation 1 h at 4 °C with the M2 antibody pre-bound to protein-G Sepharose (Pharmacia Biotech Inc.) for 15 min at 22 °C. Endogenous p38 and JNK was immunoprecipitated with polyclonal antibodies pre-bound to protein-A Sepharose (Pharmacia Biotech Inc.) 1 h. The immunoprecipitates were washed twice with lysis buffer.

Binding Assays

Recombinant GST-ATF2 fusion proteins (5 µg) pre-bound to gluthathione-agarose beads were incubated with cell lysates (80 µg) in 500 µl of lysis buffer. After 1 h of incubation at 4 °C, the beads were washed five times with lysis buffer. Protein kinases in the cell lysate and bound to the beads were detected by Western blot analysis.

Protein Phosphorylation

Kinase assays were performed using immunoprecipitates of p38 MAP kinase and JNK. The immunecomplexes were washed twice with kinase buffer (25 mM Hepes (pH 7.4), 25 mM beta-glycerophosphate, 25 mM MgCl(2), 2 mM dithiothreitol, 0.1 mM orthovanadate). The assays were initiated by the addition of 1 µg of substrate protein and 50 µM [-P]ATP (10 Ci/mmol) in a final volume of 25 µl. The reactions were terminated after 30 min at 30 °C by addition of Laemmli sample buffer. The phosphorylation of the substrate proteins was examined after SDS-PAGE by autoradiography and PhosphorImager (Molecular Dynamics Inc.) analysis. Phosphoamino acid analysis was performed by partial acid hydrolysis and thin layer electrophoresis(11) .

Immunocytochemistry

Coverslips (22 times 22 mm No. 1; Gold Seal Cover Glass; Becton Dickinson) were pre-treated by boiling in 0.1 N HCl for 10 min, rinsed in distilled water, autoclaved, and coated with 0.01% poly-L-lysine (Sigma). The coverslips were placed at the bottom of 35-mm multiwell tissue culture plates (Becton Dickinson). Transfected COS-1 cells were plated directly on the coverslips and allowed to adhere overnight in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum (Life Technologies, Inc.). 24 h post-transfection, the cells were rinsed once and incubated at 37 °C for 30 min in 25 mM Hepes (pH 7.4), 137 mM NaCl, 6 mM KCl, 1 mM MgCl(2), 1 mM CaCl(2), 10 mM glucose. The cells were rinsed once with phosphate-buffered saline and the coverslips removed from the tissue culture wells. Cells were fixed in fresh 4% paraformaldehyde in phosphate-buffered saline for 15 min at 22 °C. The cells were permeabilized with 0.25% Triton X-100 in phosphate-buffered saline for 5 min and washed three times in DWB solution (150 mM NaCl, 15 mM sodium citrate (pH 7.0), 2% horse serum, 1% (w/v) bovine serum albumin, 0.05% Triton X-100) for 5 min. The primary antibody (M2 anti-FLAG monoclonal antibody, Eastman-Kodak Co., New Haven, CT) was diluted 1:250 in DWB and applied to the cells in a humidified environment at 22 °C for 1 h. The cells were washed three times and fluorescein isothiocyanate-conjugated goat anti-mouse Ig secondary antibody (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) was applied at a 1:250 dilution for 1 h at 22 °C in a humidified environment. The cells were washed three times in DWB and mounted onto slides with Gel-Mount (Biomeda Corp., Foster City, CA) for immunofluorescence analysis.

Control experiments were performed to assess the specificity of the observed immunofluorescence. No fluorescence was detected when the transfected cells were stained in the absence of the primary M2 monoclonal antibody. In addition, we did not observe fluorescence in experiments using mock-transfected cells. Together, these data demonstrate that the immunofluorescence observed detects the epitope-tagged p38 MAP kinase.

Digital Imaging Microscopy and Image Restoration

Digital images of the fluorescence distribution in single cells were obtained using a Nikon 60x Planapo objective (numerical aperture = 1.4) on a Zeiss IM-35 microscope equipped for epifluorescence as described previously(33, 34) . Images of various focal planes were obtained with a computer controlled focus mechanism and a thermoelectrically cooled charged-coupled device camera (model 220; Photometrics Ltd., Tucson, AZ). The exposure of the sample to the excitation source was determined by a computer-controlled shutter and wavelength selector system (MVI, Avon, MA). The charge-coupled device camera and microscope functions were controlled by a microcomputer, and the data acquired from the camera were transferred to a Silicon Graphics model 4D/GTX workstation (Mountainview, CA) for image processing. Images were corrected for non-uniformities in sensitivity and for the dark current of the charge coupled device detector. The callibration of the microscopy blurring was determined by measuring the instrument's point spread function as a series of optical sections at 0.125-µm intervals of a 0.3-µm diameter fluorescently labeled latex bead (Molecular Probes Inc.). The image restoration algorithm used is based upon the theory of ill-posed problems and obtains quantitative dye density values within the cell that are substantially more accurate than those in an unprocessed image (33, 34) . After image processing, individual optical sections of cells were inspected and analyzed using computer graphics software on a Silicon Graphics workstation.

RESULTS

Substrate Specificity of p38 MAP Kinase

The p38 MAP kinase shares amino acid sequence similarity with the MAP kinase family of proteins including ERK, JNK, and HOG1(1) . In order to characterize the enzymatic activity of p38, we employed recombinant p38 expressed in Escherichia coli to examine the phosphorylation of several proteins that have been demonstrated to be substrates for the ERK and/or JNK groups of MAP kinases. In initial studies we examined the phosphorylation of the ERK substrates myelin basic protein (35) and the EGF-R(36) . It was observed that p38 phosphorylated both of these proteins (Fig. 1A). In contrast, phosphorylation of the JNK substrate c-Jun (11, 12, 14) was not detected. These data indicate that the substrate specificity of p38 is similar to the ERK group of MAP kinases. However, not all ERK substrates were phosphorylated by p38. For example, the ERK substrates cytoplasmic phospholipase A(2) (cPLA(2)) (37) and c-Myc (26) were not phosphorylated by p38 (Fig. 1A). Together, these data demonstrate that the substrate specificity of p38 differs from both the ERK and JNK groups of MAP kinases.

Figure 1: Substrate specificity of p38 MAP kinase. Panel A, substrate phosphorylation by p38 MAP kinase was examined by incubation of bacterially-expressed p38 MAP kinase with different proteins and [-P]ATP. The mutated ATF2 protein (mATF2) was created by substitution of the phosphorylation sites Thr-69 and Thr-71 with Ala. The phosphorylation reaction was terminated after 30 min by addition of Laemmli sample buffer. The phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. The rate phosphorylation of the substrate proteins was quantitated by PhosphorImager analysis. The relative phosphorylation of ATF2, myelin basic protein (MPB), EGF-R, and IkappaB was 1.0, 0.23, 0.04, and 0.001, respectively. Panel B, cell extracts expressing epitope-tagged JNK1 and p38 MAP kinase were incubated with a GST fusion protein containing the activation domain of ATF2 (residues 1-109) immobilized on gluthathione-agarose. The supernatant was removed and the agarose was washed extensively. Western blot analysis of the supernantant and agarose-bound fractions with the M2 monoclonal antibody was used to detect the protein kinases by enhanced chemiluminescence detection. Control experiments were performed using immobilized GST.


Although the ERK substrates myelin basic protein and the EGF-R were phosphorylated by p38, it was unclear whether these proteins represent preferred substrates for this protein kinase. We therefore tested several additional proteins as potential substrates for p38. This analysis demonstrated a low level of phosphorylation of IkappaB (Fig. 1A). However, the transcription factor ATF2 was found to be an excellent p38 substrate. Phosphorylation of ATF2 caused by p38 resulted in an electrophoretic mobility shift during polyacrylamide gel electrophoresis. The site(s) of phosphorylation were mapped to the NH(2)-terminal activation domain of ATF2 by deletion analysis (data not shown). Interestingly, JNK phosphorylates ATF2 on Thr-69 and Thr-71(25) . We therefore tested the hypothesis that p38 phosphorylates ATF2 on the same sites. It was found that the replacement of Thr-69 and Thr-71 with Ala residues blocked the phosphorylation of ATF2 caused by p38 (Fig. 1A). We conclude that p38 phosphorylates ATF2 within the NH(2)-terminal activation domain on Thr-69 and Thr-71. Significantly, the phosphorylation of ATF2 on these sites causes increased transcriptional activity(25) . Thus, the transcription factor ATF2 is a potential target of signal transduction by p38 MAP kinase and JNK.

It is known that JNK binds to the activation domain of the substrate c-Jun(11, 12, 38, 39, 40) . By analogy to JNK, it is possible that p38 MAP kinase binds to ATF2. To test this hypothesis, we incubated cell extracts with immobilized GST or GST-ATF2 (activation domain; residues 1-109). The complexes were extensively washed and the bound protein kinases were detected by Western blotting. This analysis demonstrated that both p38 MAP kinase and JNK bind to the ATF2 activation domain (^2)(Fig. 1B).

p38 MAP Kinase Is Activated by Pro-inflammatory Cytokines and Environmental Stress

Treatment of cultured cells with EGF or phorbol ester causes maximal activation of the ERK subgroup of MAP kinases(41, 42) . However, these treatments cause only a small increase in JNK protein kinase activity(11, 12, 14) . Significantly, EGF and phorbol ester caused only a modest increase in p38 protein kinase activity ( Fig. 2and Fig. 3). Together, these data indicate that the regulation of p38 may be more similar to JNK than ERK. This hypothesis was confirmed by investigation of the effect of JNK activators on p38 protein kinase activity. It was observed that environmental stress (UV radiation and osmotic shock) caused a marked increase in the activity of both p38 and JNK ( Fig. 4and Fig. 5). It was also observed that p38 and JNK were activated in cells treated with pro-inflammatory cytokines (tumor necrosis factor and interleukin-1) or endotoxic LPS (Fig. 6Fig. 7Fig. 8). Together, these data indicate that p38 MAP kinase, like JNK, is activated by a stress-induced signal transduction pathway. However, activation of p38 MAP kinase by alternative pathways is not excluded by these data.

Figure 2: Phorbol ester weakly activates p38 MAP kinase. The activity of p38 MAP kinase and JNK was measured in immunecomplex protein kinase assays using [-P]ATP and ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE by autoradiography. The figure shows the effect of treatment of HeLa cells with 10 nM phorbol myristate acetate. The rate of phosphorylation was quantitated by PhosphorImager analysis and is presented as the JNK and p38 protein kinase activity relative to control cells treated without agonist (1.0).


Figure 3: EGF weakly activates p38 MAP kinase. The activity of p38 MAP kinase and JNK was measured in immunecomplex protein kinase assays using [-P]ATP and ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE by autoradiography. The figure shows the effect of treatment of HeLa cells with 10 nM EGF. The rate of phosphorylation was quantitated by PhosphorImager analysis and is presented as the JNK and p38 protein kinase activity relative to control cells treated without agonist (1.0).


Figure 4: UV radiation activates p38 MAP kinase. The activity of p38 MAP kinase and JNK was measured in immunecomplex protein kinase assays using [-P]ATP and ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE by autoradiography. The figure shows the effect of treatment of HeLa cells with 40 J/m^2 UV-C. The rate of phosphorylation was quantitated by PhosphorImager analysis and is presented as the JNK and p38 protein kinase activity relative to control cells treated without agonist (1.0).


Figure 5: Osmotic stress activates p38 MAP kinase. The activity of p38 MAP kinase and JNK was measured in immunecomplex protein kinase assays using [-P]ATP and ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE by autoradiography. The figure shows the effect of treatment of HeLa cells with 300 mM sorbitol. The rate of phosphorylation was quantitated by PhosphorImager analysis and is presented as the JNK and p38 protein kinase activity relative to control cells treated without agonist (1.0).


Figure 6: Interleukin-1 activates p38 MAP kinase. The activity of p38 MAP kinase and JNK was measured in immunecomplex protein kinase assays using [-P]ATP and ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE by autoradiography. The figure shows the effect of treatment of HeLa cells with 10 ng/ml interleukin-1. The rate of phosphorylation was quantitated by PhosphorImager analysis and is presented as the JNK and p38 protein kinase activity relative to control cells treated without agonist (1.0).


Figure 7: Tumor necrosis factor activates p38 MAP kinase. The activity of p38 MAP kinase and JNK was measured in immunecomplex protein kinase assays using [-P]ATP and ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE by autoradiography. The figure shows the effect of treatment of HeLa cells with 10 ng/ml tumor necrosis factor alpha. The rate of phosphorylation was quantitated by PhosphorImager analysis and is presented as the JNK and p38 protein kinase activity relative to control cells treated without agonist (1.0).


Figure 8: LPS activates p38 MAP kinase. The activity of p38 MAP kinase and JNK1 was examined using Chinese hamster ovary cells that express human CD14. The effect of treatment of the cells with 10 ng/ml LPS is presented. The protein kinase activity was measured in immunecomplex protein kinase assays using [-P]ATP and ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE by autoradiography. The rate of phosphorylation was quantitated by PhosphorImager analysis and is presented as the JNK and p38 protein kinase activity relative to control cells treated without agonist (1.0).


p38 MAP Kinase Is Activated by Dual Phosphorylation on Tyr and Thr

ERKs and JNKs are activated by dual phosphorylation within the motifs Thr-Glu-Tyr and Thr-Pro-Tyr, respectively(11, 43) . In contrast, the p38 MAP kinase contains the related sequence Thr-Gly-Tyr (19, 20) . To test whether this motif is relevant to the activation of p38, we examined the effect of the replacement of Thr-Gly-Tyr with Ala-Gly-Phe. The wild-type and mutant forms of p38 were expressed at similar levels (Fig. 9A). Western blot analysis using an antiphosphotyrosine antibody demonstrated that exposure to UV radiation caused an increase in the Tyr phosphorylation of p38 (Fig. 9A). The increased Tyr phosphorylation was confirmed by phosphoamino acid analysis of p38 isolated from [P]phosphate-labeled cells (Fig. 9B). This analysis also demonstrated that UV radiation caused increased Thr phosphorylation of p38 (Fig. 9B). Significantly, the increased phosphorylation on Tyr and Thr was blocked by mutation of the dual phosphorylation motif Thr-Gly-Tyr (Fig. 4, A and B). To examine the signficance of the dual phosphorylation of p38, we measured the protein kinase activity of the wild-type and mutated enzymes. UV radiation caused a marked increase in the activity of wild-type (Thr-Gly-Tyr) p38 (Fig. 9C). In contrast, the mutated (Ala-Gly-Phe) p38 was found to be catalytically inactive (Fig. 9C). Together, these data demonstrate that p38 is activated by dual phosphorylation within the motif Thr-Gly-Tyr.

Figure 9: Dual phosphorylation on Thr and Tyr is required for p38 MAP kinase activation. Panel A, COS-1 cells expressing wild-type (Thr-Gly-Tyr) or mutated (Ala-Gly-Phe) p38 MAP kinase were treated without and with UV-C (40 J/m^2). The cells were harvested 30 min following exposure to UV-C radiation. Control experiments were performed using mock-transfected cells. The level of expression of epitope-tagged p38 MAP kinase and the state of Tyr phosphorylation of p38 MAP kinase was examined by Western blot analysis using the M2 monoclonal antibody and the phosphotyrosine monoclonal antibody PY20. Immune complexes were detected by enhanced chemiluminescence. Panel B, the p38 MAP kinase was isolated from cells metabolically-labeled with [P]phosphate by immunoprecipitation with the M2 monoclonal antibody and SDS-PAGE. The p38 MAP kinase phosphorylation was examined by phosphoamino acid analysis. Panel C, the p38 MAP kinase was isolated from the COS-1 cells by immunoprecipitation. Protein kinase activity was measured in the immune complex using [-P]ATP and GST-ATF2 as substrates. The phosphorylated GST-ATF2 was detected after SDS-PAGE by autoradiography.


p38 MAP Kinase Is Inhibited by Dual Specificity MAP Kinase Phosphatases

It has recently been demonstrated that ERK activity is regulated by the mitogen-induced dual specificity phosphatases MKP1 and PAC1(29, 44) . The activation of p38 by dual phosphorylation (Fig. 9) suggests that p38 MAP kinase may also be regulated by dual specificity phosphatases. We therefore examined the effect of MKP1 and PAC1 on p38 MAP kinase activation. It was observed that the expression of PAC1 or MKP1 inhibited p38 activity (Fig. 10). The inhibitory effect of MKP1 was greater than PAC1. In contrast, a catalytically inactive mutant phosphatase did not inhibit p38 MAP kinase (Fig. 10). Control experiments demonstrated that these phosphatases did not alter the level of expression of p38 MAP kinase (data not shown). Together, these data demonstrate that p38 MAP kinase can be regulated by the dual specificity phosphatases PAC1 and MKP1.

Figure 10: MAP kinase phosphatase inhibits p38 MAP kinase activation. The effect of expression of human MKP1 and PAC1 on p38 MAP kinase activity is presented. The cells were treated without and with 40 J/m^2 UV-C. Control experiments were performed using mock-transfected cells (control) and cells transfected with the catalytically inactive mutated phosphatase mPAC1 (Cys/Ser). The activity of p38 MAP kinase was measured with an immunecomplex protein kinase assay employing [-P]ATP and GST-ATF2 as substrates.


Subcellular Distribution of p38 MAP Kinase

The subcellular distribution of p38 MAP kinase was examined by indirect immunofluorescence microscopy. Epitope-tagged p38 MAP kinase was detected using the M2 monoclonal antibody. Control experiments demonstrated that no staining of mock-transfected cells was detected. However, specific staining of cells transfected with epitope-tagged p38 MAP kinase was observed (Fig. 11). The p38 MAP kinase was detected at the cell surface, in the cytoplasm, and in the nucleus. Marked changes in cell surface and nuclear p38 MAP kinase were not observed following UV irradiation, but an increase in the localization of cytoplasmic p38 MAP kinase to the perinuclear region was detected (Fig. 11). Together, these data demonstrate that p38 MAP kinase is present in both the nuclear and cytoplasmic compartments of cells and that activation by UV irradiation does not cause marked redistribution of p38 MAP kinase from the cytoplasm to the nucleus. (^3)The absence of nuclear redistribution of p38 MAP kinase contrasts with observations reported for the ERK group of MAP kinases(45, 46, 47, 48) . The ERKs are present in the cytoplasm of quiescent cells and translocate into the nucleus following activation(45, 46, 47, 48) .

Figure 11: Subcellular distribution of p38 MAP kinase. Epitope-tagged p38 MAP kinase was expressed in COS cells. The cells were treated without (Panel A) or with (Panel B) 40 J/m^2 UV radiation and then incubated for 60 min at 37 °C. The p38 MAP kinase was detected by indirect immunofluorescence using the M2 monoclonal antibody. The images were acquired by digital imaging microscopy and processed for image restoration.


DISCUSSION

The requirement of dual phosphorylation for activation establishes that p38 is a member of the MAP kinase group of signal transducing proteins(1) . However, the absence of detectable phosphorylation of cPLA(2), c-Myc, and c-Jun together with the strong phosphorylation of ATF2 indicates that the substrate specificity of p38 differs from both the JNK(11, 12, 13, 14, 16, 17, 25) and ERK(41, 42) subgroups of MAP kinase. It is therefore likely that the p38 MAP kinase signal transduction pathway has a distinct function in the cell. Indeed, it has recently been established that p38 may function in a signal transduction pathway that leads to phosphorylation of small heat shock proteins (20, 21) and increased expression of inflammatory cytokines(22) .

EGF and phorbol ester are potent activators of the ERK signal transduction pathway(10) . However, we found that these treatments did not cause a marked increase in p38 protein kinase activity. These data indicate that the mechanism of activation of p38 is not identical to the ERK group of MAP kinases. In contrast, the pattern of activation of p38 was found to be similar to JNK, a MAP kinase that is potently activated by pro-inflammatory cytokines and environmental stress(1) . Thus p38, like JNK, may be regulated, in part, by a stress-activated signal transduction pathway (Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8). This conclusion is consistent with the observation that both p38 and JNK1 are able to complement a defect in the expression of the HOG1 stress-activated MAP kinase in yeast(15, 19) . Although p38 and JNK both appear to be activated by a stress-induced signal transduction pathway, a significant question remains concerning the organization of these pathways.

The comparison of the regulation of p38 and JNK activation reveals a marked similarity between these protein kinases, but differences in the time course and extent of activation were also observed (Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8). These differences indicate that the p38 and JNK pathways may be distinct. Indeed, p38 and JNK could represent parallel stress-activated signal transduction pathways(49) . Alternatively, it is possible that p38 and JNK are activated by a common pathway. A rigorous test of these hypotheses requires the molecular cloning of the dual specificity kinase kinases that activate p38 and JNK. Recently, two MAP kinase kinases (MKK3 and MKK4) that activate p38 MAP kinase have been identified(49) . MKK3 is specific for p38 MAP kinase(49) . In contrast, MKK4 activates both JNK (49, 50) and p38 MAP kinase(49) . Thus, p38 and JNK are activated by related MAP kinase kinases (Fig. 12).

Figure 12: Mammalian MAP kinases form an integrated network of signal transduction pathways. The major stimuli that activate JNK and p38 MAP kinases are pro-inflammatory cytokines and environmental stress. In contrast, the ERK group of MAP kinases are activated in cells treated with EGF or phorbol ester. This difference is accounted for, in part, by the substrate specificities of the MAP kinase kinases MEK1(53) , MEK2(53) , MKK3(49) , and MKK4 (49, 50) which activate ERK, p38, and JNK. These pathways are illustrated schematically.


The original identification of p38 demonstrated that this protein is tyrosine phosphorylated in LPS-treated cells(19, 51) . This study demonstrates that p38 is a member of the MAP kinase group that is activated by dual phosphorylation on Tyr and Thr by a stress-induced signal transduction pathway. Endotoxic LPS, an activator of the p38 MAP kinase pathway, can therefore be considered to be a form of environmental stress that elicits septic shock(52) .

FOOTNOTES

*
This work was supported by Grant CA58396 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Worcester, MA 01605.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; ATF2, activating transcription factor 2; cPLA(2), cytoplasmic phospholipase A(2); EGF, epidermal growth factor; EGF-R, EGF receptor; GST, glutathione S-transferase; JNK, c-Jun NH(2)-terminal kinase; LPS, lipopolysaccharide; UV, ultraviolet; PAGE, polyacrylamide gel electrophoresis.

(^2)
Control experiments demonstrated that JNK, but not p38 MAP kinase, bound to the activation domain of c-Jun.

(^3)
A caveat that must be placed on the interpretation of the immunofluorescence experiments is that the images obtained represent the cellular distribution of over-expressed p38 MAP kinase. The distribution of endogenous p38 MAP kinase may be different.

ACKNOWLEDGEMENTS

We thank Dr. D. J. Schmidt for assistance with immunofluorescence microscopy performed in the UMMC Biomedical Imaging Facility (directed by Dr. F. S. Fay). Dr. D. Baltimore and R. Cerione provided the GST-IkappaB and GST-EGF-R, respectively. The purified cPLA(2) was obtained from Dr. L-L. Lin. DNA sequence analysis was performed by T. Barrett. The technical assistance of I-H. Wu is greatly appreciated. The excellent secretarial assistance of Margaret Shepard is acknowledged.

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