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Big Mitogen-activated Protein Kinase 1 (BMK1) Is a Redox-sensitive Kinase

Big Mitogen-activated Protein Kinase 1 (BMK1) Is a Redox-sensitive Kinase THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 28, Issue of July 12, pp. 16586–16590, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Big Mitogen-activated Protein Kinase 1 (BMK1) Is a Redox-sensitive Kinase* (Received for publication, January 19, 1996, and in revised form, April 16, 1996) Jun-ichi Abe‡, Masatoshi Kusuhara‡, Richard J. Ulevitch§, Bradford C. Berk , and Jiing-Dwan Lee§ From the Department of Medicine, Cardiology Division, University of Washington, Seattle, Washington 98195 and the §Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 Mitogen-activated protein (MAP) kinases are a multi- major signaling system by which cells transduce extracellular gene family activated by many extracellular stimuli. stimuli into intracellular responses (1). Extracellular signal- There are three groups of MAP kinases based on their regulated protein kinases (ERKs) 1 and 2 were the first of the dual phosphorylation motifs, TEY, TPY, and TGY, which ERK/MAP kinase subfamily to be cloned, and these kinases are are termed extracellular signal-regulated protein ki- activated by diverse extracellular stimuli and by intracellular nases (ERK1/2), c-Jun N-terminal kinases, and p38, re- protooncogene products that induce proliferation or differenti- spectively. A new MAP kinase family member termed ation (2). Other related mammalian MAP kinases have been Big MAP kinase 1 (BMK1) or ERK5 was recently cloned. identified including two ERK3 isoforms (3, 4), ERK4 (5), Jun BMK1 has a TEY sequence similar to ERK1/2 but has N-terminal kinases/stress-activated protein kinases (JNK/ unique COOH-terminal and loop-12 domains. To define SAPK) (6, 7), p38 kinase (8), and p57 MAP kinase (9). MAP BMK1 regulation, its activation in cultured rat vascular kinases are activated by phosphorylation on T and Y residues smooth muscle cells was characterized. Angiotensin II, within a TXY phosphorylation motif, where X can be Glu (E), phorbol ester, platelet-derived growth factor, and tumor Pro (P), or Gly (G). Three classes of dual specificity MAP necrosis factor-a were the strongest stimuli for ERK1/2 kinases may be defined based on their motifs (TEY, TPY, and but were weak activators of BMK1. In contrast, H O 2 2 TGY), which we will term ERK1/2, JNK/SAPK, and p38, caused concentration-dependent activation of BMK1 but not ERK1/2. Sorbitol activated both BMK1 and respectively. ERK1/2. BMK1 activation by H O was calcium-depend- Relative activation of the three classes of MAP kinases is 2 2 ent and appeared ubiquitous as shown by stimulation in characteristic for particular stimuli. For example, growth fac- human skin fibroblasts, human vascular smooth muscle tors such as phorbol myristate acetate (PMA) and epidermal cells, and human umbilical vein endothelial cells. These growth factor activate ERK1/2 strongly but JNK/SAPK and findings demonstrate that activation of BMK1 is differ- p38 weakly (10). Hyperosmolar stress is a strong stimulus for ent from ERK1/2 and suggest an important role for p38 (8), and this stimulus also activates ERK1/2 and JNK in BMK1 as a redox-sensitive kinase. rat ventricular myocytes (11). In VSMC we have shown that growth factors and angiotensin II (AngII) are powerful activa- tors of ERK1/2 (12). Recently we found that oxidative stress 1,2 The mitogen-activated protein (MAP) kinase cascade is a activated ERK1/2 when the oxidative stress was superoxide but not H O (13). In other systems, oxidative stress has been 2 2 * This study was supported by a grant from the Japanese Heart demonstrated to activate JNK strongly (14). The specificity for Foundation and Bayer Yakuhin Research Grant Abroad for 1995 (to J. MAP kinase activation is determined, in part, by members of A.), by Grants HL44721 and HL49192 (to B. C. B.), GM37694 (to R. J. the MAP kinase/ERK kinase (MEK) family, which exhibit U.), and GM53214 (to J. D. L.) from the National Institutes of Health. unique pairing with downstream MAP kinases. For example, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked MEK1 and MEK2 activate ERK1/2, MKK3 activates p38, and “advertisement” in accordance with 18 U.S.C. Section 1734 solely to MKK4 (i.e. SEK1 the murine homolog of MKK4) activates indicate this fact. JNK/SAPK. These data suggest that cell-specific and stimulus- ‡ These authors contributed equally to this manuscript. ¶ specific events regulate the activities of the three classes of Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiology Division, Box MAP kinases. 357710, University of Washington, Seattle, WA 98195. Tel.: 206-685- The specificity of activation of MAP kinases by individual 6960; Fax: 206-616-1580; E-mail: [email protected]. 1 stimuli is reiterated by specific substrates for each class. Com- The abbreviations used are: MAP kinase, mitogen-activated protein mon substrates for the MAP kinases are transcription factors kinase; AngII, angiotensin II; BMK1, big mitogen-activated protein kinase 1; ERK, extracellular signal-regulated kinase; JNK, c-Jun N- that upon phosphorylation may be activated and induce terminal protein kinase; MEK, MAP kinase/ERK kinase; MKK, MAP changes in gene expression. ERK1/2 phosphorylate ternary kinase kinase; MBP, myelin basic protein; PAGE, polyacrylamide gel complex factor/Elk-1 on sites essential for transactivation (15), electrophoresis; PDGF, platelet-derived growth factor; PMA, phor- bol 12-myristate 13-acetate; SAPK, stress-activated protein kinase; which regulates c-fos induction. JNK/SAPK phosphorylates N- TNF-a, tumor necrosis factor-a; VSMC, vascular smooth muscle cell; terminal c-Jun and increases its transcriptional activating po- HUVEC, human umbilical vein endothelial cells; HASM, human arte- tential (6, 7, 16–18). Activating transcription factor 2 is phos- rial smooth muscle cells; HSF, human skin fibroblasts; BAPTA- phorylated and activated by both JNK and p38 (19, 20). AM, 1,2-bis(o-aminophenoxy)ethane-N, N, N9, N9-tetraacetic acid tetra(acetoxymethyl)ester. A new human MAP kinase gene termed Big MAP kinase 1 We use the term mitogen-activated protein (MAP) kinase for the (BMK1) or ERK5 was recently cloned by Lee et al. (21) and family of kinases that includes the extracellular signal-regulated pro- Zhou et al. (22). Because the primary structure of this MAP tein kinase (ERK), the c-Jun N-terminal kinase (JNK), p38, and big kinase is quite unique from ERK1/2 (21), the name BMK1 will mitogen-activated protein kinase 1 (BMK1) subfamilies. ERK1 and MAPK MAPK ERK2 are also known as p44 and p42 , respectively. be used in this paper. BMK1 has a TEY sequence in its dual This is an open access article under the CC BY license. 16586 BMK1 Is a Redox-sensitive Kinase 16587 phosphorylation site like ERK1/2 but has unique C-terminal and loop-12 domains compared with ERK1/2, suggesting that its regulation and function may be different from those of ERK1/2. To define the regulation of BMK1, we have character- ized its activation in cultured rat VSMC, which we have pre- viously shown to have robust ERK activity in response to several stimuli. We show here that activation of BMK1 by hormonal and chemical stimuli is clearly distinct from activa- tion of ERK1/2. In particular, BMK1 appears to participate in a redox-sensitive pathway activated by H O but not by ago- 2 2 nists such as PMA, AngII, PDGF, and TNF-a. FIG.1. BMK1 is present in VSMC. Growth arrested VSMC were harvested and immunoprecipitated with BMK1 antiserum (3 ml) (A) EXPERIMENTAL PROCEDURES and preimmune serum (B). Samples were then analyzed by 10% SDS- Cell Culture—Vascular smooth muscle cells (VSMC) were isolated PAGE and Western blot analysis of immunoprecipitates using BMK1 from 200–250-g male Sprague-Dawley rats and maintained in 10% calf antibodies. A single band of ;110 kDa is present. Analysis of cell serum/Dulbecco’s modified Eagle’s medium as described previously lysates after immunoprecipitation demonstrated that $90% of BMK1 (23). Passage 5–15 VSMC at 70–80% confluence in 100-mm dishes were immunoreactive protein was precipitated. growth arrested by incubation in 0.4% calf serum/Dulbecco’s modified Eagle’s medium for 48 h to use. HUVEC were obtained from umbilical above. Equal amounts of protein (5–10 mg) were separated by SDS- veins as described previously (24). Cells at passage 3 were grown in PAGE through a gel containing 0.4 mg/ml myelin basic protein (MBP). RPMI 1640 medium supplemented with 20% fetal bovine serum and The gel was then incubated twice in buffer A (50 mM HEPES, pH 7.4, were deprived of growth factors by incubation in serum-free RPMI 1640 and5mM b-mercaptoethanol) containing 20% isopropyl alcohol for 30 containing 0.4% bovine serum albumin for 24 h. Human arterial smooth min, once in buffer A for 1 h, twice in buffer A containing 0.04% Tween muscle cells (HASM) and human skin fibroblasts (HSF) were a kind gift 20 at 4 °C for 16 h and for 2 h, and once in buffer A containing 100 mM from Dr. R. Ross and Dr. J. F. Oram, respectively, and were maintained 32 Na VO ,10mM MgCl ,50 mM ATP, and 50 mCi of [g- P]ATP for1hat 3 4 2 in subconfluent state as described (25, 26). In brief, human newborn (13 30 °C. The reaction was terminated by washing the gel five to eight days) arteries were obtained from the thoracic aortas of infants on times in fixative solution containing 10 mM sodium pyrophosphate and accidental death and cultured. Normal human skin fibroblasts were 5% trichloroacetic acid for 15 min. The gel was dried and subjected to grown from explants of punch biopsies of skin from the inner thighs of autoradiography, and ERK1/2 kinase activity was measured by densi- normal volunteers in plastic tissue flasks containing Dulbecco’s modi- tometry of autoradiogram (in the linear range of film exposure) using fied Eagle’s medium plus 10% fetal bovine serum. Both HASM and HSF NIH Image 1.49. We have previously shown that results for in gel were maintained in Dulbecco’s modified Eagle’s medium/0.4% calf se- kinase assay and immune complex kinase assay for ERK1/2 are highly rum for 2 days before experiments. 2 correlated (R 5 0.92) (28). The in gel kinase assay is more reproducible BMK1 Antibody—The peptide sequence used to generate rabbit anti- and less expensive, so it was used for ERK1/2 kinase assay. human BMK1 antibody was keyhole limpet hemocyanin-EGHGMN- BMK1 Kinase Assay—BMK1 kinase activity was assayed by MBP PADIESLQREIQMDSPML. The keyhole limpet hemocyanin-peptide phosphorylation as the substrate for BMK1 as described previously immunogen was emulsified by mixing with an equal volume of Freund’s (29). Cells were lysed as described above and centrifuged at 14,000 3 g adjuvant and injected into three to four subcutaneous dorsal sites, for a (4 °C for 30 min), and protein concentration was determined. BMK1 total volume of 1 ml (0.1 mg of peptide) per immunization. Two weeks was immunoprecipitated, and BMK1 kinase activity was measured at after the third boost, blood was allowed to clot, and serum was collected 30 °C for 20 min in the reaction mixture (40 ml) containing 0.1 mg/ml of by centrifugation. The anti-peptide antibody titer was determined with MBP, 15 mM of ATP, 10 mM MgCl ,10mM MnCl , and 3 mCi of 2 2 an enzyme linked immunosorbent assay with free peptide on the solid 32 [g- P]ATP. The reaction was terminated by adding 8 mlof6 3 electro- phase (1 mg/well). The results are expressed as the reciprocal of the phoresis sample buffer and boiling for 5 min. Samples were analyzed on serum dilution that resulted in an absorbance at 492 nm of 0.2 (detec- 15% SDS-PAGE followed by autoradiography. The radioactivity in the tion with goat anti-rabbit IgG-horseradish peroxidase conjugate and band corresponding to BMK1 was determined by densitometry of auto- peroxidase). The titer for the anti-serum was 1:29,900, and preimmune radiogram (in the linear range of film exposure) using NIH Image 1.49. serum was less than 1:50. Materials—All materials were from Sigma except where indicated. Immunoprecipitation and Western Blot Analysis—After treatment, Recombinant PDGF-BB was from Boehringer Mannheim, H O was 2 2 the cells were washed with PBS and 0.5 ml of lysis buffer (50 mM from Fisher, and BAPTA-AM was from Molecular Probes. sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 mM Na VO ,10mM HEPES, pH 7.4, 0.1% Triton X-100, 500 3 4 RESULTS mM phenylmethanesulfonyl fluoride, and 10 mg/ml leupeptin) and flash- Immunodetection of BMK1—An antibody was prepared frozen on a dry ice/ethanol bath. After allowing the cells to thaw, cells against the recently cloned MAP kinase, BMK1, as described were scraped off the dish and centrifuged at 14,000 3 g (4 °C for 30 min), and protein concentration was determined using the Bradford under “Experimental Procedures” (21). As shown in Fig. 1, protein assay (Bio-Rad). For immunoprecipitation, cell lysates were immunoprecipitation and Western blot analysis with BMK1 incubated with rabbit anti-BMK1 antibody (3 ml) or preimmune serum antibody revealed a prominent 110-kDa protein band in cul- for3hat4 °Cand then incubated with 20 ml of protein A-Sepharose tured rat VSMC. Preimmune serum showed no band except CL-4B (Pharmacia Biotech Inc.) for1hona roller system at 4 °C. The IgG. beads were washed two times with 1 ml of lysis buffer, 2 times with 1 BMK1 Is Poorly Activated by the VSMC Agonists AngII, ml of LiCl wash buffer (500 mM LiCl, 100 mM Tris-Cl, pH 7.6, 0.1% Triton X-100, and 1 mM dithiothreitol), and two times in 1 ml of washing PDGF, PMA, and TNF-a—To determine which known VSMC buffer (20 mM HEPES, pH 7.2, 2 mM EGTA, 10 mM MgCl ,1mM agonists activated BMK1, we stimulated growth arrested dithiothreitol, and 0.1% Triton X-100). For Western blot analysis, 15 mg VSMC with AngII, PDGF-BB, PMA, and TNF-a. We have of protein or immunoprecipitates were subjected to SDS-PAGE, and previously shown that these agonists are potent stimuli for proteins were transferred to nitrocellulose membrane (Hybondy-ECL, activation of ERK1/2 (12, 13), which contain a TEY dual phos- Amersham Corp.) as described previously (27). The membrane was phorylation site identical to that present in BMK1. The results blocked for1hat room temperature with a commercial blocking buffer from Life Technologies, Inc. The blots were incubated for4hat room presented below indicate that activation of BMK1 by these temperature with the BMK1 antibody, followed by incubation for 1 h agonists is very different from activation of ERK1/2. with secondary antibody (horseradish peroxidase-conjugated). Immu- As shown in Fig. 2A, AngII (100 nM) caused only a small noreactive bands were visualized using enhanced chemiluminescence activation of BMK1, approximately 2-fold greater than control (ECL, Amersham Corp.). at 5 min. In contrast, AngII caused a potent activation of ERK1/2 Kinase Activity Assay—An in gel kinase assay to measure ERK1/2 with a 10-fold increase in activity at 5 min. PDGF-BB ERK1/2 phosphotransferase activity was performed on cell lysates as described previously (27). In brief, cells were harvested as described (10 ng/ml) caused only a small increase in BMK1 (Fig. 2B), 16588 BMK1 Is a Redox-sensitive Kinase FIG.3. Phorbol ester and TNF-a weakly stimulate BMK1 activ- ity. Growth arrested VSMC were stimulated with 200 nM PMA (A) and FIG.2. Angiotensin II and PDGF-BB weakly stimulate BMK1. 10 ng/ml TNF-a (B) for the indicated times, cells were harvested, and Growth arrested VSMC were stimulated with 100 nM AngII (A) and 10 ERK1/2 and BMK1 activities were determined as described in the ng/ml PDGF-BB (B) for the indicated times, cells were harvested, and legend to Fig. 2. ERK1/2 and BMK1 activities were determined. Top, ERK1/2 were measured by an in gel kinase assay using MBP as substrate. MBP phosphorylation was detected after SDS-PAGE by autoradiography. Middle, BMK1 activity was measured by an immune complex protein kinase assay using MBP as substrate. MBP phosphorylation was de- tected after SDS-PAGE by autoradiography. Bottom, the results were quantified by densitometry of autoradiograms using NIH Image 1.49. The relative protein kinase activity was determined by setting the densitometric absorbance of cells at time 0 to 1.0. approximately 1.7-fold at 20 min. In contrast, PDGF-BB was a potent activator of ERK1/2, stimulating an 9-fold increase in activity at 20 min. PMA (200 nM) failed to stimulate BMK1 (Fig. 3A) but caused a 4.5-fold increase in ERK1/2 at 5 min. Finally, TNF-a failed to stimulate BMK1 (Fig. 3B) but caused an 11-fold increase in ERK1/2 activity at 20 min. Thus, these four hormonal agonists, which are potent stimuli for ERK1/2 in VSMC, caused minimal or no activation of BMK1. BMK1 Is Stimulated by H O and Sorbitol in VSMC—Be- 2 2 cause agonists known to activate ERK1/2 strongly were weak agonists for BMK1, we determined whether agonists known to FIG.4. H O and osmotic stress stimulate BMK1 activity. 2 2 activate JNK and p38 kinase could activate BMK1. Oxidative Growth arrested VSMC were stimulated with 200 mM H O (A) and 0.4 2 2 M sorbitol (B) for the indicated times, cells were harvested, and ERK1/2 stress has previously been shown to activate JNK (14), and and BMK1 activities were determined as described in the legend to hyperosmolar stress (e.g. 0.4 M sorbitol) has been shown to Fig. 2. activate p38 (8). Using the same experimental protocol de- scribed for Figs. 2 and 3, we assayed BMK1 activity in response to 200 mM H O and 0.4 M sorbitol. As shown in Fig. 4A,H O mined whether BMK1 activation by H O was calcium-depend- 2 2 2 2 2 2 was a potent stimulus for BMK1, causing a 3.8-fold increase in ent. To deplete intracellular calcium, we used thapsigargin (10 activity at 5 min that was sustained for 60 min. In contrast, mM for 10 min). Following thapsigargin treatment, H O was no 2 2 ERK1/2 was not significantly activated by H O . Of interest, longer able to stimulate BMK1 (Fig. 6). We also used 2 2 sorbitol was a potent stimulus for both BMK1 and ERK1/2 BAPTA-AM to chelate intracellular calcium as previously re- stimulating 10- and 5.7-fold increases in activity at peak time, ported (28). However, BAPTA-AM treatment caused a signifi- respectively (Fig. 4B) These data suggest that the regulation of cant increase in BMK1 activity in unstimulated cells, con- BMK1 may be more similar to JNK and p38 than ERK1/2. founding analysis of the results (not shown). Based on these H O and Sorbitol Stimulate BMK1 in a Concentration-de- findings it appears that calcium-dependent mechanisms are 2 2 pendent Manner—We determined the concentration depend- likely to be involved in regulation of BMK1 in VSMC. ence for BMK1 activation by H O and sorbitol. As shown in BMK1 Is Activated by H O in Several Cell Types—To deter- 2 2 2 2 Fig. 5A,H O stimulation of BMK1 was maximum at 200 mM mine whether activation of BMK1 by H O was a ubiquitous 2 2 2 2 with half-maximal effect at 50–100 mM. These concentrations characteristic, we determined the response to H O in several 2 2 are similar to those previously reported for H O -mediated different cell types. Cell lysates were prepared from HUVEC, 2 2 stimulation of c-fos mRNA and DNA synthesis in VSMC (30, HASM, HSF, and RASM cells, and Western blot analysis was 31). Sorbitol also stimulated a concentration-dependent in- performed. As shown in Fig. 7A, a band of 110 kDa was present crease in BMK1 activity, which was maximal at 0.8 M (Fig. 5B). in all cell types studied with the greatest relative expression in H O Stimulation of BMK1 Kinase Activity Is Calcium-de- HSF and RASM. In addition, a band of 112 kDa was present in 2 2 pendent in VSMC—We previously found that H O -mediated the HASM. Next the response to H O was determined. As 2 2 2 2 c-fos expression was dependent on both calcium and protein shown in Fig. 7B, exposure to 200 mM H O for 5 min stimu- 2 2 kinase C (30). Because BMK1 appeared not to be activated by lated increases in BMK1 activity in both HSF and RASM. protein kinase C-dependent mechanisms (Fig. 3A), we deter- Smaller responses were observed in HUVEC and HASM. Thus BMK1 Is a Redox-sensitive Kinase 16589 FIG.5. H O and sorbitol stimulate a dose-dependent increase 2 2 FIG.7. BMK1 is activated by H O in multiple cell types. A, 2 2 in BMK1 activity. Quiescent cells were incubated with the indicated Western blot analysis. HUVEC, HASM, HSF, and RASM were obtained concentrations of H O (A) for 5 min and sorbitol (B) for 20 min. BMK1 2 2 and grown as described under “Experimental Procedures.” Western blot activity was measured by an immune complex protein kinase assay analysis was performed on whole cell lysates using BMK1 antibodies. A using MBP as substrate. MBP phosphorylation was detected after SDS- single band of ;110 kDa is present in HUVEC, HSF, and RASM. In PAGE by autoradiography. The results were quantified by densitome- HASM a predominant 110-kDa band as well as a less well expressed try of autoradiograms using NIH Image 1.49. The relative protein 112-kDa band were present. B, BMK1 activation by H O . The indi- 2 2 kinase activity was determined by setting the densitometric absorbance cated cells were growth arrested for 24 h as described under “Experi- of cells at time 0 to 1.0. mental Procedures” and then exposed to 200 mM H O for 5 min. BMK1 2 2 activity was measured by an immune complex protein kinase assay using MBP as substrate. MBP phosphorylation was detected after SDS- PAGE by autoradiography. upstream kinases that regulate MKK3 and MKK4 are likely different from those that regulate MEK5. The fact that H O was able to activate BMK1 but not 2 2 FIG.6. BMK1 activity in VSMC is inhibited by calcium deple- ERK1/2 is of particular interest. We have previously demon- tion. Intracellular calcium was depleted by exposure to thapsigargin (1 strated that oxidative stress, generated by xanthine and xan- mM) for 10 min prior to treatment with 200 mM H O for 5 min. Equal 2 2 thine oxidase, stimulates VSMC DNA synthesis (32). We also amounts of protein were then used to assay BMK1 kinase activity by showed that H O was able to stimulate c-fos expression (33). MBP phosphorylation as described in the legend to Fig. 2. 2 2 However, H O was unable to activate ERK1/2 (13), suggesting 2 2 that another kinase pathway was responsible for H O -medi- activation of BMK1 by H O appears to be a characteristic 2 2 2 2 ated gene expression. BMK1 appears a likely mediator based feature in multiple cell types. on its rapid activation by H O (peak at 5 min), its concentra- 2 2 DISCUSSION tion dependence (peak at 200 mM H O , similar to the peak 2 2 The major finding of this paper is that H O and osmotic effect on c-fos induction), and its ability to stimulate BMK1 in 2 2 stress activate BMK1 in VSMC. Although BMK1 has the same multiple cell types. Thus BMK1 is a new candidate as a redox- dual phosphorylation site (TEY) that is present in ERK1/2, sensitive kinase. regulation of BMK1 by hormonal and chemical stimuli is quite We previously showed in VSMC that H O and superoxide 2 2 different from ERK1/2. The most potent stimuli for BMK1 were induced proto-oncogene mRNA expression in a protein kinase H O and hyperosmolar stress (sorbitol). Although sorbitol also C-dependent manner (32, 33). In addition, activation of ERK1/2 2 2 activated ERK1/2, H O activated BMK1 but not ERK1/2 (sim- by superoxide is protein kinase C-dependent. In contrast, 2 2 ilar to results we previously reported (13)). In contrast, the BMK1 was not activated by PMA, suggesting that a protein most potent stimuli for ERK1/2 were AngII, PMA, PDGF, and kinase C-independent pathway may be involved. Other inves- TNF-a. These agonists failed to activate BMK1 significantly. tigators have reported that H O and superoxide cause myo- 2 2 These data indicate that the mechanism of activation of BMK1 cardial injury with intracellular calcium overload (34). Deple- is not identical to the ERK group of MAP kinases but is more tion of intracellular calcium stores by thapsigargin treatment similar to p38 and JNK/SAPK, which are activated by environ- caused nearly complete inhibition of BMK1 activation by H O . 2 2 mental stress. Thus calcium-dependent tyrosine kinases, such as the recently Zhou et al. (22) cloned a new member of the MAPK/ERK described PYK2 (35), may be important upstream activators of kinase family termed MEK5. This kinase interacted with a BMK1. Finally, previous investigators have suggested that Src kinase identical to BMK1 in the yeast two hybrid screen, which may be an upstream mediator of redox-sensitive signal trans- was termed ERK5 by these authors. They showed that MEK5 duction (14). Future work will be necessary to identify up- specifically interacted with BMK1 and that MEK1 was unable stream mediators of H O -stimulated BMK1 activity. 2 2 to interact with BMK1, suggesting that the MEK1/ERK1 path- In summary, we have shown that BMK1 is present in VSMC way and the MEK5/BMK1(ERK5) pathways have different and activated by both H O and hyperosmolar stress. The 2 2 functions. The results of the present study support their con- hormonal and chemical mediators that activate BMK1 clearly cept. There also appear to be important differences in the differ from the mediators that activate ERK1/2, suggesting activation of BMK1 compared with JNK and p38. For example, that these two classes of MAP kinases serve different intracel- Raingeaud et al. (20) showed that TNF-a was a powerful stim- lular functions. The exciting finding that BMK1 is activated by ulus for both JNK and p38 in HeLa cells, whereas we observed H O , whereas ERK1/2 are not suggests that BMK1 may rep- 2 2 no activation of BMK1 in VSMC treated with TNF-a. Thus the resent a new class of redox-sensitive kinases. 16590 BMK1 Is a Redox-sensitive Kinase 14, 8376–8384 Acknowledgments—We thank members of the Berk laboratory, espe- 18. Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes Dev. 7, cially Drs. A. S. Baas, M. Ishida, T. Ishida, M. Takahashi, and T. E. 2135–2148 Peterson, for help. 19. Gupta, S., Campbell, D., D’Erijard, B., and Davis, R. J. (1995) Science 267, 389–393 REFERENCES 20. 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Big Mitogen-activated Protein Kinase 1 (BMK1) Is a Redox-sensitive Kinase

Abe, Jun-ichi; Kusuhara, Masatoshi; Ulevitch, Richard J.; Berk, Bradford C.; Lee, Jiing-Dwan
Journal of Biological Chemistry – Jul 1, 1996
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10.1074/jbc.271.28.16586
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 28, Issue of July 12, pp. 16586–16590, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Big Mitogen-activated Protein Kinase 1 (BMK1) Is a Redox-sensitive Kinase* (Received for publication, January 19, 1996, and in revised form, April 16, 1996) Jun-ichi Abe‡, Masatoshi Kusuhara‡, Richard J. Ulevitch§, Bradford C. Berk , and Jiing-Dwan Lee§ From the Department of Medicine, Cardiology Division, University of Washington, Seattle, Washington 98195 and the §Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 Mitogen-activated protein (MAP) kinases are a multi- major signaling system by which cells transduce extracellular gene family activated by many extracellular stimuli. stimuli into intracellular responses (1). Extracellular signal- There are three groups of MAP kinases based on their regulated protein kinases (ERKs) 1 and 2 were the first of the dual phosphorylation motifs, TEY, TPY, and TGY, which ERK/MAP kinase subfamily to be cloned, and these kinases are are termed extracellular signal-regulated protein ki- activated by diverse extracellular stimuli and by intracellular nases (ERK1/2), c-Jun N-terminal kinases, and p38, re- protooncogene products that induce proliferation or differenti- spectively. A new MAP kinase family member termed ation (2). Other related mammalian MAP kinases have been Big MAP kinase 1 (BMK1) or ERK5 was recently cloned. identified including two ERK3 isoforms (3, 4), ERK4 (5), Jun BMK1 has a TEY sequence similar to ERK1/2 but has N-terminal kinases/stress-activated protein kinases (JNK/ unique COOH-terminal and loop-12 domains. To define SAPK) (6, 7), p38 kinase (8), and p57 MAP kinase (9). MAP BMK1 regulation, its activation in cultured rat vascular kinases are activated by phosphorylation on T and Y residues smooth muscle cells was characterized. Angiotensin II, within a TXY phosphorylation motif, where X can be Glu (E), phorbol ester, platelet-derived growth factor, and tumor Pro (P), or Gly (G). Three classes of dual specificity MAP necrosis factor-a were the strongest stimuli for ERK1/2 kinases may be defined based on their motifs (TEY, TPY, and but were weak activators of BMK1. In contrast, H O 2 2 TGY), which we will term ERK1/2, JNK/SAPK, and p38, caused concentration-dependent activation of BMK1 but not ERK1/2. Sorbitol activated both BMK1 and respectively. ERK1/2. BMK1 activation by H O was calcium-depend- Relative activation of the three classes of MAP kinases is 2 2 ent and appeared ubiquitous as shown by stimulation in characteristic for particular stimuli. For example, growth fac- human skin fibroblasts, human vascular smooth muscle tors such as phorbol myristate acetate (PMA) and epidermal cells, and human umbilical vein endothelial cells. These growth factor activate ERK1/2 strongly but JNK/SAPK and findings demonstrate that activation of BMK1 is differ- p38 weakly (10). Hyperosmolar stress is a strong stimulus for ent from ERK1/2 and suggest an important role for p38 (8), and this stimulus also activates ERK1/2 and JNK in BMK1 as a redox-sensitive kinase. rat ventricular myocytes (11). In VSMC we have shown that growth factors and angiotensin II (AngII) are powerful activa- tors of ERK1/2 (12). Recently we found that oxidative stress 1,2 The mitogen-activated protein (MAP) kinase cascade is a activated ERK1/2 when the oxidative stress was superoxide but not H O (13). In other systems, oxidative stress has been 2 2 * This study was supported by a grant from the Japanese Heart demonstrated to activate JNK strongly (14). The specificity for Foundation and Bayer Yakuhin Research Grant Abroad for 1995 (to J. MAP kinase activation is determined, in part, by members of A.), by Grants HL44721 and HL49192 (to B. C. B.), GM37694 (to R. J. the MAP kinase/ERK kinase (MEK) family, which exhibit U.), and GM53214 (to J. D. L.) from the National Institutes of Health. unique pairing with downstream MAP kinases. For example, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked MEK1 and MEK2 activate ERK1/2, MKK3 activates p38, and “advertisement” in accordance with 18 U.S.C. Section 1734 solely to MKK4 (i.e. SEK1 the murine homolog of MKK4) activates indicate this fact. JNK/SAPK. These data suggest that cell-specific and stimulus- ‡ These authors contributed equally to this manuscript. ¶ specific events regulate the activities of the three classes of Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiology Division, Box MAP kinases. 357710, University of Washington, Seattle, WA 98195. Tel.: 206-685- The specificity of activation of MAP kinases by individual 6960; Fax: 206-616-1580; E-mail: [email protected]. 1 stimuli is reiterated by specific substrates for each class. Com- The abbreviations used are: MAP kinase, mitogen-activated protein mon substrates for the MAP kinases are transcription factors kinase; AngII, angiotensin II; BMK1, big mitogen-activated protein kinase 1; ERK, extracellular signal-regulated kinase; JNK, c-Jun N- that upon phosphorylation may be activated and induce terminal protein kinase; MEK, MAP kinase/ERK kinase; MKK, MAP changes in gene expression. ERK1/2 phosphorylate ternary kinase kinase; MBP, myelin basic protein; PAGE, polyacrylamide gel complex factor/Elk-1 on sites essential for transactivation (15), electrophoresis; PDGF, platelet-derived growth factor; PMA, phor- bol 12-myristate 13-acetate; SAPK, stress-activated protein kinase; which regulates c-fos induction. JNK/SAPK phosphorylates N- TNF-a, tumor necrosis factor-a; VSMC, vascular smooth muscle cell; terminal c-Jun and increases its transcriptional activating po- HUVEC, human umbilical vein endothelial cells; HASM, human arte- tential (6, 7, 16–18). Activating transcription factor 2 is phos- rial smooth muscle cells; HSF, human skin fibroblasts; BAPTA- phorylated and activated by both JNK and p38 (19, 20). AM, 1,2-bis(o-aminophenoxy)ethane-N, N, N9, N9-tetraacetic acid tetra(acetoxymethyl)ester. A new human MAP kinase gene termed Big MAP kinase 1 We use the term mitogen-activated protein (MAP) kinase for the (BMK1) or ERK5 was recently cloned by Lee et al. (21) and family of kinases that includes the extracellular signal-regulated pro- Zhou et al. (22). Because the primary structure of this MAP tein kinase (ERK), the c-Jun N-terminal kinase (JNK), p38, and big kinase is quite unique from ERK1/2 (21), the name BMK1 will mitogen-activated protein kinase 1 (BMK1) subfamilies. ERK1 and MAPK MAPK ERK2 are also known as p44 and p42 , respectively. be used in this paper. BMK1 has a TEY sequence in its dual This is an open access article under the CC BY license. 16586 BMK1 Is a Redox-sensitive Kinase 16587 phosphorylation site like ERK1/2 but has unique C-terminal and loop-12 domains compared with ERK1/2, suggesting that its regulation and function may be different from those of ERK1/2. To define the regulation of BMK1, we have character- ized its activation in cultured rat VSMC, which we have pre- viously shown to have robust ERK activity in response to several stimuli. We show here that activation of BMK1 by hormonal and chemical stimuli is clearly distinct from activa- tion of ERK1/2. In particular, BMK1 appears to participate in a redox-sensitive pathway activated by H O but not by ago- 2 2 nists such as PMA, AngII, PDGF, and TNF-a. FIG.1. BMK1 is present in VSMC. Growth arrested VSMC were harvested and immunoprecipitated with BMK1 antiserum (3 ml) (A) EXPERIMENTAL PROCEDURES and preimmune serum (B). Samples were then analyzed by 10% SDS- Cell Culture—Vascular smooth muscle cells (VSMC) were isolated PAGE and Western blot analysis of immunoprecipitates using BMK1 from 200–250-g male Sprague-Dawley rats and maintained in 10% calf antibodies. A single band of ;110 kDa is present. Analysis of cell serum/Dulbecco’s modified Eagle’s medium as described previously lysates after immunoprecipitation demonstrated that $90% of BMK1 (23). Passage 5–15 VSMC at 70–80% confluence in 100-mm dishes were immunoreactive protein was precipitated. growth arrested by incubation in 0.4% calf serum/Dulbecco’s modified Eagle’s medium for 48 h to use. HUVEC were obtained from umbilical above. Equal amounts of protein (5–10 mg) were separated by SDS- veins as described previously (24). Cells at passage 3 were grown in PAGE through a gel containing 0.4 mg/ml myelin basic protein (MBP). RPMI 1640 medium supplemented with 20% fetal bovine serum and The gel was then incubated twice in buffer A (50 mM HEPES, pH 7.4, were deprived of growth factors by incubation in serum-free RPMI 1640 and5mM b-mercaptoethanol) containing 20% isopropyl alcohol for 30 containing 0.4% bovine serum albumin for 24 h. Human arterial smooth min, once in buffer A for 1 h, twice in buffer A containing 0.04% Tween muscle cells (HASM) and human skin fibroblasts (HSF) were a kind gift 20 at 4 °C for 16 h and for 2 h, and once in buffer A containing 100 mM from Dr. R. Ross and Dr. J. F. Oram, respectively, and were maintained 32 Na VO ,10mM MgCl ,50 mM ATP, and 50 mCi of [g- P]ATP for1hat 3 4 2 in subconfluent state as described (25, 26). In brief, human newborn (13 30 °C. The reaction was terminated by washing the gel five to eight days) arteries were obtained from the thoracic aortas of infants on times in fixative solution containing 10 mM sodium pyrophosphate and accidental death and cultured. Normal human skin fibroblasts were 5% trichloroacetic acid for 15 min. The gel was dried and subjected to grown from explants of punch biopsies of skin from the inner thighs of autoradiography, and ERK1/2 kinase activity was measured by densi- normal volunteers in plastic tissue flasks containing Dulbecco’s modi- tometry of autoradiogram (in the linear range of film exposure) using fied Eagle’s medium plus 10% fetal bovine serum. Both HASM and HSF NIH Image 1.49. We have previously shown that results for in gel were maintained in Dulbecco’s modified Eagle’s medium/0.4% calf se- kinase assay and immune complex kinase assay for ERK1/2 are highly rum for 2 days before experiments. 2 correlated (R 5 0.92) (28). The in gel kinase assay is more reproducible BMK1 Antibody—The peptide sequence used to generate rabbit anti- and less expensive, so it was used for ERK1/2 kinase assay. human BMK1 antibody was keyhole limpet hemocyanin-EGHGMN- BMK1 Kinase Assay—BMK1 kinase activity was assayed by MBP PADIESLQREIQMDSPML. The keyhole limpet hemocyanin-peptide phosphorylation as the substrate for BMK1 as described previously immunogen was emulsified by mixing with an equal volume of Freund’s (29). Cells were lysed as described above and centrifuged at 14,000 3 g adjuvant and injected into three to four subcutaneous dorsal sites, for a (4 °C for 30 min), and protein concentration was determined. BMK1 total volume of 1 ml (0.1 mg of peptide) per immunization. Two weeks was immunoprecipitated, and BMK1 kinase activity was measured at after the third boost, blood was allowed to clot, and serum was collected 30 °C for 20 min in the reaction mixture (40 ml) containing 0.1 mg/ml of by centrifugation. The anti-peptide antibody titer was determined with MBP, 15 mM of ATP, 10 mM MgCl ,10mM MnCl , and 3 mCi of 2 2 an enzyme linked immunosorbent assay with free peptide on the solid 32 [g- P]ATP. The reaction was terminated by adding 8 mlof6 3 electro- phase (1 mg/well). The results are expressed as the reciprocal of the phoresis sample buffer and boiling for 5 min. Samples were analyzed on serum dilution that resulted in an absorbance at 492 nm of 0.2 (detec- 15% SDS-PAGE followed by autoradiography. The radioactivity in the tion with goat anti-rabbit IgG-horseradish peroxidase conjugate and band corresponding to BMK1 was determined by densitometry of auto- peroxidase). The titer for the anti-serum was 1:29,900, and preimmune radiogram (in the linear range of film exposure) using NIH Image 1.49. serum was less than 1:50. Materials—All materials were from Sigma except where indicated. Immunoprecipitation and Western Blot Analysis—After treatment, Recombinant PDGF-BB was from Boehringer Mannheim, H O was 2 2 the cells were washed with PBS and 0.5 ml of lysis buffer (50 mM from Fisher, and BAPTA-AM was from Molecular Probes. sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 mM Na VO ,10mM HEPES, pH 7.4, 0.1% Triton X-100, 500 3 4 RESULTS mM phenylmethanesulfonyl fluoride, and 10 mg/ml leupeptin) and flash- Immunodetection of BMK1—An antibody was prepared frozen on a dry ice/ethanol bath. After allowing the cells to thaw, cells against the recently cloned MAP kinase, BMK1, as described were scraped off the dish and centrifuged at 14,000 3 g (4 °C for 30 min), and protein concentration was determined using the Bradford under “Experimental Procedures” (21). As shown in Fig. 1, protein assay (Bio-Rad). For immunoprecipitation, cell lysates were immunoprecipitation and Western blot analysis with BMK1 incubated with rabbit anti-BMK1 antibody (3 ml) or preimmune serum antibody revealed a prominent 110-kDa protein band in cul- for3hat4 °Cand then incubated with 20 ml of protein A-Sepharose tured rat VSMC. Preimmune serum showed no band except CL-4B (Pharmacia Biotech Inc.) for1hona roller system at 4 °C. The IgG. beads were washed two times with 1 ml of lysis buffer, 2 times with 1 BMK1 Is Poorly Activated by the VSMC Agonists AngII, ml of LiCl wash buffer (500 mM LiCl, 100 mM Tris-Cl, pH 7.6, 0.1% Triton X-100, and 1 mM dithiothreitol), and two times in 1 ml of washing PDGF, PMA, and TNF-a—To determine which known VSMC buffer (20 mM HEPES, pH 7.2, 2 mM EGTA, 10 mM MgCl ,1mM agonists activated BMK1, we stimulated growth arrested dithiothreitol, and 0.1% Triton X-100). For Western blot analysis, 15 mg VSMC with AngII, PDGF-BB, PMA, and TNF-a. We have of protein or immunoprecipitates were subjected to SDS-PAGE, and previously shown that these agonists are potent stimuli for proteins were transferred to nitrocellulose membrane (Hybondy-ECL, activation of ERK1/2 (12, 13), which contain a TEY dual phos- Amersham Corp.) as described previously (27). The membrane was phorylation site identical to that present in BMK1. The results blocked for1hat room temperature with a commercial blocking buffer from Life Technologies, Inc. The blots were incubated for4hat room presented below indicate that activation of BMK1 by these temperature with the BMK1 antibody, followed by incubation for 1 h agonists is very different from activation of ERK1/2. with secondary antibody (horseradish peroxidase-conjugated). Immu- As shown in Fig. 2A, AngII (100 nM) caused only a small noreactive bands were visualized using enhanced chemiluminescence activation of BMK1, approximately 2-fold greater than control (ECL, Amersham Corp.). at 5 min. In contrast, AngII caused a potent activation of ERK1/2 Kinase Activity Assay—An in gel kinase assay to measure ERK1/2 with a 10-fold increase in activity at 5 min. PDGF-BB ERK1/2 phosphotransferase activity was performed on cell lysates as described previously (27). In brief, cells were harvested as described (10 ng/ml) caused only a small increase in BMK1 (Fig. 2B), 16588 BMK1 Is a Redox-sensitive Kinase FIG.3. Phorbol ester and TNF-a weakly stimulate BMK1 activ- ity. Growth arrested VSMC were stimulated with 200 nM PMA (A) and FIG.2. Angiotensin II and PDGF-BB weakly stimulate BMK1. 10 ng/ml TNF-a (B) for the indicated times, cells were harvested, and Growth arrested VSMC were stimulated with 100 nM AngII (A) and 10 ERK1/2 and BMK1 activities were determined as described in the ng/ml PDGF-BB (B) for the indicated times, cells were harvested, and legend to Fig. 2. ERK1/2 and BMK1 activities were determined. Top, ERK1/2 were measured by an in gel kinase assay using MBP as substrate. MBP phosphorylation was detected after SDS-PAGE by autoradiography. Middle, BMK1 activity was measured by an immune complex protein kinase assay using MBP as substrate. MBP phosphorylation was de- tected after SDS-PAGE by autoradiography. Bottom, the results were quantified by densitometry of autoradiograms using NIH Image 1.49. The relative protein kinase activity was determined by setting the densitometric absorbance of cells at time 0 to 1.0. approximately 1.7-fold at 20 min. In contrast, PDGF-BB was a potent activator of ERK1/2, stimulating an 9-fold increase in activity at 20 min. PMA (200 nM) failed to stimulate BMK1 (Fig. 3A) but caused a 4.5-fold increase in ERK1/2 at 5 min. Finally, TNF-a failed to stimulate BMK1 (Fig. 3B) but caused an 11-fold increase in ERK1/2 activity at 20 min. Thus, these four hormonal agonists, which are potent stimuli for ERK1/2 in VSMC, caused minimal or no activation of BMK1. BMK1 Is Stimulated by H O and Sorbitol in VSMC—Be- 2 2 cause agonists known to activate ERK1/2 strongly were weak agonists for BMK1, we determined whether agonists known to FIG.4. H O and osmotic stress stimulate BMK1 activity. 2 2 activate JNK and p38 kinase could activate BMK1. Oxidative Growth arrested VSMC were stimulated with 200 mM H O (A) and 0.4 2 2 M sorbitol (B) for the indicated times, cells were harvested, and ERK1/2 stress has previously been shown to activate JNK (14), and and BMK1 activities were determined as described in the legend to hyperosmolar stress (e.g. 0.4 M sorbitol) has been shown to Fig. 2. activate p38 (8). Using the same experimental protocol de- scribed for Figs. 2 and 3, we assayed BMK1 activity in response to 200 mM H O and 0.4 M sorbitol. As shown in Fig. 4A,H O mined whether BMK1 activation by H O was calcium-depend- 2 2 2 2 2 2 was a potent stimulus for BMK1, causing a 3.8-fold increase in ent. To deplete intracellular calcium, we used thapsigargin (10 activity at 5 min that was sustained for 60 min. In contrast, mM for 10 min). Following thapsigargin treatment, H O was no 2 2 ERK1/2 was not significantly activated by H O . Of interest, longer able to stimulate BMK1 (Fig. 6). We also used 2 2 sorbitol was a potent stimulus for both BMK1 and ERK1/2 BAPTA-AM to chelate intracellular calcium as previously re- stimulating 10- and 5.7-fold increases in activity at peak time, ported (28). However, BAPTA-AM treatment caused a signifi- respectively (Fig. 4B) These data suggest that the regulation of cant increase in BMK1 activity in unstimulated cells, con- BMK1 may be more similar to JNK and p38 than ERK1/2. founding analysis of the results (not shown). Based on these H O and Sorbitol Stimulate BMK1 in a Concentration-de- findings it appears that calcium-dependent mechanisms are 2 2 pendent Manner—We determined the concentration depend- likely to be involved in regulation of BMK1 in VSMC. ence for BMK1 activation by H O and sorbitol. As shown in BMK1 Is Activated by H O in Several Cell Types—To deter- 2 2 2 2 Fig. 5A,H O stimulation of BMK1 was maximum at 200 mM mine whether activation of BMK1 by H O was a ubiquitous 2 2 2 2 with half-maximal effect at 50–100 mM. These concentrations characteristic, we determined the response to H O in several 2 2 are similar to those previously reported for H O -mediated different cell types. Cell lysates were prepared from HUVEC, 2 2 stimulation of c-fos mRNA and DNA synthesis in VSMC (30, HASM, HSF, and RASM cells, and Western blot analysis was 31). Sorbitol also stimulated a concentration-dependent in- performed. As shown in Fig. 7A, a band of 110 kDa was present crease in BMK1 activity, which was maximal at 0.8 M (Fig. 5B). in all cell types studied with the greatest relative expression in H O Stimulation of BMK1 Kinase Activity Is Calcium-de- HSF and RASM. In addition, a band of 112 kDa was present in 2 2 pendent in VSMC—We previously found that H O -mediated the HASM. Next the response to H O was determined. As 2 2 2 2 c-fos expression was dependent on both calcium and protein shown in Fig. 7B, exposure to 200 mM H O for 5 min stimu- 2 2 kinase C (30). Because BMK1 appeared not to be activated by lated increases in BMK1 activity in both HSF and RASM. protein kinase C-dependent mechanisms (Fig. 3A), we deter- Smaller responses were observed in HUVEC and HASM. Thus BMK1 Is a Redox-sensitive Kinase 16589 FIG.5. H O and sorbitol stimulate a dose-dependent increase 2 2 FIG.7. BMK1 is activated by H O in multiple cell types. A, 2 2 in BMK1 activity. Quiescent cells were incubated with the indicated Western blot analysis. HUVEC, HASM, HSF, and RASM were obtained concentrations of H O (A) for 5 min and sorbitol (B) for 20 min. BMK1 2 2 and grown as described under “Experimental Procedures.” Western blot activity was measured by an immune complex protein kinase assay analysis was performed on whole cell lysates using BMK1 antibodies. A using MBP as substrate. MBP phosphorylation was detected after SDS- single band of ;110 kDa is present in HUVEC, HSF, and RASM. In PAGE by autoradiography. The results were quantified by densitome- HASM a predominant 110-kDa band as well as a less well expressed try of autoradiograms using NIH Image 1.49. The relative protein 112-kDa band were present. B, BMK1 activation by H O . The indi- 2 2 kinase activity was determined by setting the densitometric absorbance cated cells were growth arrested for 24 h as described under “Experi- of cells at time 0 to 1.0. mental Procedures” and then exposed to 200 mM H O for 5 min. BMK1 2 2 activity was measured by an immune complex protein kinase assay using MBP as substrate. MBP phosphorylation was detected after SDS- PAGE by autoradiography. upstream kinases that regulate MKK3 and MKK4 are likely different from those that regulate MEK5. The fact that H O was able to activate BMK1 but not 2 2 FIG.6. BMK1 activity in VSMC is inhibited by calcium deple- ERK1/2 is of particular interest. We have previously demon- tion. Intracellular calcium was depleted by exposure to thapsigargin (1 strated that oxidative stress, generated by xanthine and xan- mM) for 10 min prior to treatment with 200 mM H O for 5 min. Equal 2 2 thine oxidase, stimulates VSMC DNA synthesis (32). We also amounts of protein were then used to assay BMK1 kinase activity by showed that H O was able to stimulate c-fos expression (33). MBP phosphorylation as described in the legend to Fig. 2. 2 2 However, H O was unable to activate ERK1/2 (13), suggesting 2 2 that another kinase pathway was responsible for H O -medi- activation of BMK1 by H O appears to be a characteristic 2 2 2 2 ated gene expression. BMK1 appears a likely mediator based feature in multiple cell types. on its rapid activation by H O (peak at 5 min), its concentra- 2 2 DISCUSSION tion dependence (peak at 200 mM H O , similar to the peak 2 2 The major finding of this paper is that H O and osmotic effect on c-fos induction), and its ability to stimulate BMK1 in 2 2 stress activate BMK1 in VSMC. Although BMK1 has the same multiple cell types. Thus BMK1 is a new candidate as a redox- dual phosphorylation site (TEY) that is present in ERK1/2, sensitive kinase. regulation of BMK1 by hormonal and chemical stimuli is quite We previously showed in VSMC that H O and superoxide 2 2 different from ERK1/2. The most potent stimuli for BMK1 were induced proto-oncogene mRNA expression in a protein kinase H O and hyperosmolar stress (sorbitol). Although sorbitol also C-dependent manner (32, 33). In addition, activation of ERK1/2 2 2 activated ERK1/2, H O activated BMK1 but not ERK1/2 (sim- by superoxide is protein kinase C-dependent. In contrast, 2 2 ilar to results we previously reported (13)). In contrast, the BMK1 was not activated by PMA, suggesting that a protein most potent stimuli for ERK1/2 were AngII, PMA, PDGF, and kinase C-independent pathway may be involved. Other inves- TNF-a. These agonists failed to activate BMK1 significantly. tigators have reported that H O and superoxide cause myo- 2 2 These data indicate that the mechanism of activation of BMK1 cardial injury with intracellular calcium overload (34). Deple- is not identical to the ERK group of MAP kinases but is more tion of intracellular calcium stores by thapsigargin treatment similar to p38 and JNK/SAPK, which are activated by environ- caused nearly complete inhibition of BMK1 activation by H O . 2 2 mental stress. Thus calcium-dependent tyrosine kinases, such as the recently Zhou et al. (22) cloned a new member of the MAPK/ERK described PYK2 (35), may be important upstream activators of kinase family termed MEK5. This kinase interacted with a BMK1. Finally, previous investigators have suggested that Src kinase identical to BMK1 in the yeast two hybrid screen, which may be an upstream mediator of redox-sensitive signal trans- was termed ERK5 by these authors. They showed that MEK5 duction (14). Future work will be necessary to identify up- specifically interacted with BMK1 and that MEK1 was unable stream mediators of H O -stimulated BMK1 activity. 2 2 to interact with BMK1, suggesting that the MEK1/ERK1 path- In summary, we have shown that BMK1 is present in VSMC way and the MEK5/BMK1(ERK5) pathways have different and activated by both H O and hyperosmolar stress. The 2 2 functions. The results of the present study support their con- hormonal and chemical mediators that activate BMK1 clearly cept. There also appear to be important differences in the differ from the mediators that activate ERK1/2, suggesting activation of BMK1 compared with JNK and p38. For example, that these two classes of MAP kinases serve different intracel- Raingeaud et al. (20) showed that TNF-a was a powerful stim- lular functions. The exciting finding that BMK1 is activated by ulus for both JNK and p38 in HeLa cells, whereas we observed H O , whereas ERK1/2 are not suggests that BMK1 may rep- 2 2 no activation of BMK1 in VSMC treated with TNF-a. Thus the resent a new class of redox-sensitive kinases. 16590 BMK1 Is a Redox-sensitive Kinase 14, 8376–8384 Acknowledgments—We thank members of the Berk laboratory, espe- 18. Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes Dev. 7, cially Drs. A. S. Baas, M. Ishida, T. Ishida, M. Takahashi, and T. E. 2135–2148 Peterson, for help. 19. Gupta, S., Campbell, D., D’Erijard, B., and Davis, R. J. (1995) Science 267, 389–393 REFERENCES 20. 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Published: Jul 1, 1996

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