Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase.

Mitogen-activated protein (MAP) kinases are a multigene family activated by many extracellular stimuli. There are three groups of MAP kinases based on their dual phosphorylation motifs, TEY, TPY, and TGY, which are termed extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinases, and p38, respectively. A new MAP kinase family member termed Big MAP kinase 1 (BMK1) or ERK5 was recently cloned. BMK1 has a TEY sequence similar to ERK1/2 but has unique COOH-terminal and loop-12 domains. To define BMK1 regulation, its activation in cultured rat vascular smooth muscle cells was characterized. Angiotensin II, phorbol ester, platelet-derived growth factor, and tumor necrosis factor-α were the strongest stimuli for ERK1/2 but were weak activators of BMK1. In contrast, H2O2 caused concentration-dependent activation of BMK1 but not ERK1/2. Sorbitol activated both BMK1 and ERK1/2. BMK1 activation by H2O2 was calcium-dependent and appeared ubiquitous as shown by stimulation in human skin fibroblasts, human vascular smooth muscle cells, and human umbilical vein endothelial cells. These findings demonstrate that activation of BMK1 is different from ERK1/2 and suggest an important role for BMK1 as a redox-sensitive kinase.

* This study was supported by a grant from the Japanese Heart Foundation and Bayer Yakuhin Research Grant Abroad for 1995 (to J. A.), by Grants HL44721 and HL49192 (to B. C. B.), GM37694 (to R. J. U.), and GM53214 (to J. D. L.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ These authors contributed equally to this manuscript. ¶ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiology Division, Box 357710, University of Washington, Seattle, WA 98195. Tel.: 206-685-6960; Fax: 206-616-1580; E-mail: bcberk@u.washington.edu. 1 The abbreviations used are: MAP kinase, mitogen-activated protein kinase; AngII, angiotensin II; BMK1, big mitogen-activated protein kinase 1; ERK, extracellular signal-regulated kinase; JNK, c-Jun Nterminal protein kinase; MEK, MAP kinase/ERK kinase; MKK, MAP kinase kinase; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; PDGF, platelet-derived growth factor; PMA, phorbol 12-myristate 13-acetate; SAPK, stress-activated protein kinase; TNF-␣, tumor necrosis factor-␣; VSMC, vascular smooth muscle cell; HUVEC, human umbilical vein endothelial cells; HASM, human arterial smooth muscle cells; HSF, human skin fibroblasts; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N, N, NЈ, NЈ-tetraacetic acid tetra(acetoxymethyl)ester. 2 We use the term mitogen-activated protein (MAP) kinase for the family of kinases that includes the extracellular signal-regulated protein kinase (ERK), the c-Jun N-terminal kinase (JNK), p38, and big mitogen-activated protein kinase 1 (BMK1) subfamilies. ERK1 and ERK2 are also known as p44 MAPK and p42 MAPK , respectively. 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 characterized its activation in cultured rat VSMC, which we have previously 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 activation of ERK1/2. In particular, BMK1 appears to participate in a redox-sensitive pathway activated by H 2 O 2 but not by agonists such as PMA, AngII, PDGF, and TNF-␣.

EXPERIMENTAL PROCEDURES
Cell Culture-Vascular smooth muscle cells (VSMC) were isolated from 200 -250-g male Sprague-Dawley rats and maintained in 10% calf serum/Dulbecco's modified Eagle's medium as described previously (23). Passage 5-15 VSMC at 70 -80% confluence in 100-mm dishes were 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 veins as described previously (24). Cells at passage 3 were grown in RPMI 1640 medium supplemented with 20% fetal bovine serum and were deprived of growth factors by incubation in serum-free RPMI 1640 containing 0.4% bovine serum albumin for 24 h. Human arterial smooth muscle cells (HASM) and human skin fibroblasts (HSF) were a kind gift from Dr. R. Ross and Dr. J. F. Oram, respectively, and were maintained in subconfluent state as described (25,26). In brief, human newborn (13 days) arteries were obtained from the thoracic aortas of infants on accidental death and cultured. Normal human skin fibroblasts were grown from explants of punch biopsies of skin from the inner thighs of normal volunteers in plastic tissue flasks containing Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Both HASM and HSF were maintained in Dulbecco's modified Eagle's medium/0.4% calf serum for 2 days before experiments.
BMK1 Antibody-The peptide sequence used to generate rabbit antihuman BMK1 antibody was keyhole limpet hemocyanin-EGHGMN-PADIESLQREIQMDSPML. The keyhole limpet hemocyanin-peptide immunogen was emulsified by mixing with an equal volume of Freund's adjuvant and injected into three to four subcutaneous dorsal sites, for a total volume of 1 ml (0.1 mg of peptide) per immunization. Two weeks after the third boost, blood was allowed to clot, and serum was collected by centrifugation. The anti-peptide antibody titer was determined with an enzyme linked immunosorbent assay with free peptide on the solid phase (1 g/well). The results are expressed as the reciprocal of the serum dilution that resulted in an absorbance at 492 nm of 0.2 (detection with goat anti-rabbit IgG-horseradish peroxidase conjugate and peroxidase). The titer for the anti-serum was 1:29,900, and preimmune serum was less than 1:50.
Immunoprecipitation and Western Blot Analysis-After treatment, the cells were washed with PBS and 0.5 ml of lysis buffer (50 mM sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 M Na 3 VO 4 , 10 mM HEPES, pH 7.4, 0.1% Triton X-100, 500 M phenylmethanesulfonyl fluoride, and 10 g/ml leupeptin) and flashfrozen on a dry ice/ethanol bath. After allowing the cells to thaw, cells were scraped off the dish and centrifuged at 14,000 ϫ g (4°C for 30 min), and protein concentration was determined using the Bradford protein assay (Bio-Rad). For immunoprecipitation, cell lysates were incubated with rabbit anti-BMK1 antibody (3 l) or preimmune serum for 3 h at 4°C and then incubated with 20 l of protein A-Sepharose CL-4B (Pharmacia Biotech Inc.) for 1 h on a roller system at 4°C. The beads were washed two times with 1 ml of lysis buffer, 2 times with 1 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 buffer (20 mM HEPES, pH 7.2, 2 mM EGTA, 10 mM MgCl 2 , 1 mM dithiothreitol, and 0.1% Triton X-100). For Western blot analysis, 15 g of protein or immunoprecipitates were subjected to SDS-PAGE, and proteins were transferred to nitrocellulose membrane (Hybond-ECL, Amersham Corp.) as described previously (27). The membrane was blocked for 1 h at room temperature with a commercial blocking buffer from Life Technologies, Inc. The blots were incubated for 4 h at room temperature with the BMK1 antibody, followed by incubation for 1 h with secondary antibody (horseradish peroxidase-conjugated). Immunoreactive bands were visualized using enhanced chemiluminescence (ECL, Amersham Corp.). ERK1/2 Kinase Activity Assay-An in gel kinase assay to measure ERK1/2 phosphotransferase activity was performed on cell lysates as described previously (27). In brief, cells were harvested as described above. Equal amounts of protein (5-10 g) were separated by SDS-PAGE through a gel containing 0.4 mg/ml myelin basic protein (MBP). The gel was then incubated twice in buffer A (50 mM HEPES, pH 7.4, and 5 mM ␤-mercaptoethanol) containing 20% isopropyl alcohol for 30 min, once in buffer A for 1 h, twice in buffer A containing 0.04% Tween 20 at 4°C for 16 h and for 2 h, and once in buffer A containing 100 M Na 3 VO 4 , 10 mM MgCl 2 , 50 M ATP, and 50 Ci of [␥-32 P]ATP for 1 h at 30°C. The reaction was terminated by washing the gel five to eight times in fixative solution containing 10 mM sodium pyrophosphate and 5% trichloroacetic acid for 15 min. The gel was dried and subjected to autoradiography, and ERK1/2 kinase activity was measured by densitometry of autoradiogram (in the linear range of film exposure) using NIH Image 1.49. We have previously shown that results for in gel kinase assay and immune complex kinase assay for ERK1/2 are highly correlated (R 2 ϭ 0.92) (28). The in gel kinase assay is more reproducible and less expensive, so it was used for ERK1/2 kinase assay.
BMK1 Kinase Assay-BMK1 kinase activity was assayed by MBP phosphorylation as the substrate for BMK1 as described previously (29). Cells were lysed as described above and centrifuged at 14,000 ϫ g (4°C for 30 min), and protein concentration was determined. BMK1 was immunoprecipitated, and BMK1 kinase activity was measured at 30°C for 20 min in the reaction mixture (40 l) containing 0.1 mg/ml of MBP, 15 M of ATP, 10 mM MgCl 2 , 10 mM MnCl 2 , and 3 Ci of [␥-32 P]ATP. The reaction was terminated by adding 8 l of 6 ϫ electrophoresis sample buffer and boiling for 5 min. Samples were analyzed on 15% SDS-PAGE followed by autoradiography. The radioactivity in the band corresponding to BMK1 was determined by densitometry of autoradiogram (in the linear range of film exposure) using NIH Image 1.49.
Materials-All materials were from Sigma except where indicated. Recombinant PDGF-BB was from Boehringer Mannheim, H 2 O 2 was from Fisher, and BAPTA-AM was from Molecular Probes.

RESULTS
Immunodetection of BMK1-An antibody was prepared against the recently cloned MAP kinase, BMK1, as described under "Experimental Procedures" (21). As shown in Fig. 1, immunoprecipitation and Western blot analysis with BMK1 antibody revealed a prominent 110-kDa protein band in cultured rat VSMC. Preimmune serum showed no band except IgG.
BMK1 Is Poorly Activated by the VSMC Agonists AngII, PDGF, PMA, and TNF-␣-To determine which known VSMC agonists activated BMK1, we stimulated growth arrested VSMC with AngII, PDGF-BB, PMA, and TNF-␣. We have previously shown that these agonists are potent stimuli for activation of ERK1/2 (12,13), which contain a TEY dual phosphorylation site identical to that present in BMK1. The results presented below indicate that activation of BMK1 by these agonists is very different from activation of ERK1/2.
As shown in Fig. 2A, AngII (100 nM) caused only a small activation of BMK1, approximately 2-fold greater than control at 5 min. In contrast, AngII caused a potent activation of ERK1/2 with a 10-fold increase in activity at 5 min. PDGF-BB (10 ng/ml) caused only a small increase in BMK1 (Fig. 2B),   FIG. 1. BMK1 is present in VSMC. Growth arrested VSMC were harvested and immunoprecipitated with BMK1 antiserum (3 l) (A) and preimmune serum (B). Samples were then analyzed by 10% SDS-PAGE and Western blot analysis of immunoprecipitates using BMK1 antibodies. A single band of ϳ110 kDa is present. Analysis of cell lysates after immunoprecipitation demonstrated that Ն90% of BMK1 immunoreactive protein was precipitated. 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-␣ 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 2 O 2 and Sorbitol in VSMC-
Because agonists known to activate ERK1/2 strongly were weak agonists for BMK1, we determined whether agonists known to activate JNK and p38 kinase could activate BMK1. Oxidative stress has previously been shown to activate JNK (14), and hyperosmolar stress (e.g. 0.4 M sorbitol) has been shown to activate p38 (8). Using the same experimental protocol described for Figs. 2 and 3, we assayed BMK1 activity in response to 200 M H 2 O 2 and 0.4 M sorbitol. As shown in Fig. 4A, H 2 O 2 was a potent stimulus for BMK1, causing a 3.8-fold increase in activity at 5 min that was sustained for 60 min. In contrast, ERK1/2 was not significantly activated by H 2 O 2 . Of interest, sorbitol was a potent stimulus for both BMK1 and ERK1/2 stimulating 10-and 5.7-fold increases in activity at peak time, respectively (Fig. 4B) These data suggest that the regulation of BMK1 may be more similar to JNK and p38 than ERK1/2.  (30,31). Sorbitol also stimulated a concentration-dependent increase in BMK1 activity, which was maximal at 0.8 M (Fig. 5B).
H 2 O 2 Stimulation of BMK1 Kinase Activity Is Calcium-dependent in VSMC-We previously found that H 2 O 2 -mediated c-fos expression was dependent on both calcium and protein kinase C (30). Because BMK1 appeared not to be activated by protein kinase C-dependent mechanisms (Fig. 3A), we deter-mined whether BMK1 activation by H 2 O 2 was calcium-dependent. To deplete intracellular calcium, we used thapsigargin (10 M for 10 min). Following thapsigargin treatment, H 2 O 2 was no longer able to stimulate BMK1 (Fig. 6). We also used BAPTA-AM to chelate intracellular calcium as previously reported (28). However, BAPTA-AM treatment caused a significant increase in BMK1 activity in unstimulated cells, confounding analysis of the results (not shown). Based on these findings it appears that calcium-dependent mechanisms are likely to be involved in regulation of BMK1 in VSMC.

BMK1 Is Activated by H 2 O 2 in Several Cell
Types-To determine whether activation of BMK1 by H 2 O 2 was a ubiquitous characteristic, we determined the response to H 2 O 2 in several different cell types. Cell lysates were prepared from HUVEC, HASM, HSF, and RASM cells, and Western blot analysis was performed. As shown in Fig. 7A, a band of 110 kDa was present in all cell types studied with the greatest relative expression in HSF and RASM. In addition, a band of 112 kDa was present in the HASM. Next the response to H 2 O 2 was determined. As shown in Fig. 7B  Growth arrested VSMC were stimulated with 100 nM AngII (A) and 10 ng/ml PDGF-BB (B) for the indicated times, cells were harvested, and 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 detected 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. activation of BMK1 by H 2 O 2 appears to be a characteristic feature in multiple cell types. DISCUSSION The major finding of this paper is that H 2 O 2 and osmotic stress activate BMK1 in VSMC. Although BMK1 has the same dual phosphorylation site (TEY) that is present in ERK1/2, regulation of BMK1 by hormonal and chemical stimuli is quite different from ERK1/2. The most potent stimuli for BMK1 were H 2 O 2 and hyperosmolar stress (sorbitol). Although sorbitol also activated ERK1/2, H 2 O 2 activated BMK1 but not ERK1/2 (similar to results we previously reported (13)). In contrast, the most potent stimuli for ERK1/2 were AngII, PMA, PDGF, and TNF-␣. These agonists failed to activate BMK1 significantly. These data indicate that the mechanism of activation of BMK1 is not identical to the ERK group of MAP kinases but is more similar to p38 and JNK/SAPK, which are activated by environmental stress.
Zhou et al. (22) cloned a new member of the MAPK/ERK kinase family termed MEK5. This kinase interacted with a kinase identical to BMK1 in the yeast two hybrid screen, which was termed ERK5 by these authors. They showed that MEK5 specifically interacted with BMK1 and that MEK1 was unable to interact with BMK1, suggesting that the MEK1/ERK1 pathway and the MEK5/BMK1(ERK5) pathways have different functions. The results of the present study support their concept. There also appear to be important differences in the activation of BMK1 compared with JNK and p38. For example, Raingeaud et al. (20) showed that TNF-␣ was a powerful stimulus for both JNK and p38 in HeLa cells, whereas we observed no activation of BMK1 in VSMC treated with TNF-␣. Thus the upstream kinases that regulate MKK3 and MKK4 are likely different from those that regulate MEK5.
The fact that H 2 O 2 was able to activate BMK1 but not ERK1/2 is of particular interest. We have previously demonstrated that oxidative stress, generated by xanthine and xanthine oxidase, stimulates VSMC DNA synthesis (32). We also showed that H 2 O 2 was able to stimulate c-fos expression (33). However, H 2 O 2 was unable to activate ERK1/2 (13), suggesting that another kinase pathway was responsible for H 2 O 2 -mediated gene expression. BMK1 appears a likely mediator based on its rapid activation by H 2 O 2 (peak at 5 min), its concentration dependence (peak at 200 M H 2 O 2 , similar to the peak effect on c-fos induction), and its ability to stimulate BMK1 in multiple cell types. Thus BMK1 is a new candidate as a redoxsensitive kinase.
We previously showed in VSMC that H 2 O 2 and superoxide induced proto-oncogene mRNA expression in a protein kinase C-dependent manner (32,33). In addition, activation of ERK1/2 by superoxide is protein kinase C-dependent. In contrast, BMK1 was not activated by PMA, suggesting that a protein kinase C-independent pathway may be involved. Other investigators have reported that H 2 O 2 and superoxide cause myocardial injury with intracellular calcium overload (34). Depletion of intracellular calcium stores by thapsigargin treatment caused nearly complete inhibition of BMK1 activation by H 2 O 2 . Thus calcium-dependent tyrosine kinases, such as the recently described PYK2 (35), may be important upstream activators of BMK1. Finally, previous investigators have suggested that Src may be an upstream mediator of redox-sensitive signal transduction (14). Future work will be necessary to identify upstream mediators of H 2 O 2 -stimulated BMK1 activity.
In summary, we have shown that BMK1 is present in VSMC and activated by both H 2 O 2 and hyperosmolar stress. The hormonal and chemical mediators that activate BMK1 clearly differ from the mediators that activate ERK1/2, suggesting that these two classes of MAP kinases serve different intracellular functions. The exciting finding that BMK1 is activated by H 2 O 2 , whereas ERK1/2 are not suggests that BMK1 may represent a new class of redox-sensitive kinases. FIG. 7. BMK1 is activated by H 2 O 2 in multiple cell types. A, Western blot analysis. HUVEC, HASM, HSF, and RASM were obtained and grown as described under "Experimental Procedures." Western blot analysis was performed on whole cell lysates using BMK1 antibodies. A single band of ϳ110 kDa is present in HUVEC, HSF, and RASM. In HASM a predominant 110-kDa band as well as a less well expressed 112-kDa band were present. B, BMK1 activation by H 2 O 2 . The indicated cells were growth arrested for 24 h as described under "Experimental Procedures" and then exposed to 200 M H 2 O 2 for 5 min. BMK1 activity was measured by an immune complex protein kinase assay using MBP as substrate. MBP phosphorylation was detected after SDS-PAGE by autoradiography.