(Received for publication, May 29, 1997)
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
Big mitogen-activated kinase 1 (BMK1) or extracellular signal-regulated kinase-5 (ERK5) has recently been identified as a new member of the mitogen-activated protein kinase family. We have shown that BMK1 is activated to a greater extent by H2O2 than growth factors, suggesting that in comparison with other mitogen-activated protein kinase family members, BMK1 is a redox-sensitive kinase. Previous investigations indicate that the tyrosine kinase c-Src mediates signal transduction by reactive oxygen species, including H2O2. Therefore, the role of Src kinase family members (c-Src and Fyn) in activation of the BMK1 by H2O2 in mouse fibroblasts was studied. An essential role for c-Src was suggested by four experiments. First, H2O2 stimulated c-Src activity rapidly in fibroblasts (peak at 5 min), which preceded peak activity of BMK1 (20 min). Second, specific Src family tyrosine kinase inhibitors (herbimycin A and CP-118,556) blocked BMK1 activation by H2O2 in a concentration-dependent manner. Third, BMK1 activation in the response to H2O2 was completely inhibited in cells derived from mice deficient in c-Src, but not Fyn. Finally, BMK1 activity was much greater in v-Src-transformed NIH-3T3 cells than wild type cells. These results demonstrate an essential role for c-Src in H2O2-mediated activation of BMK1 and suggest that redox-sensitive regulation of BMK1 is a new function for c-Src.
The MAP1 kinases are activated by diverse stimuli to transduce signals from the cell membrane to the nucleus (1). Three MAP kinase groups may be defined based on their dual phosphorylation motifs, TEY, TPY, and TGY, which we will term ERK1/2 (2), c-Jun N-terminal kinase (JNK/SAPK) (3, 4), and p38 (5), respectively. Each MAP kinase group has relatively distinct upstream activators and substrate specificities. For example, growth factors and phorbol esters readily activate ERK1/2, but have little effect on JNK/SAPK; while UV irradiation and anisomycin are much better activators of JNK/SAPK than of ERK1/2 or p38. Similarly, ERK1/2 phosphorylates ternary complex factor/Elk-1 (6), while the transcription factor c-Jun is a specific substrate for the JNK/SAPK signaling pathway (3, 4). Common pathways also exist as shown by the fact that both JNK/SAPK and p38 are activated by apoptosis (7), and both kinases phosphorylate and regulate activating transcription factor 2 (8, 9).
A new MAP kinase family member termed BMK1 or ERK5 was cloned recently
(10, 11). BMK1 has a TEY sequence in its dual phosphorylation site,
like ERK1/2, but it has unique carboxyl-terminal and loop-12 domains
compared with ERK1/2. We have shown that activation of BMK1 in rat
aortic smooth muscle cells is distinct from activation of ERK1/2. In
particular, BMK1 participates in a redox-sensitive pathway activated by
H2O2 but not by agonists such as phorbol ester,
angiotensin II, platelet-derived growth factor, and tumor necrosis
factor- (12). Specific activation of BMK1 by oxidative stress
suggests that BMK1 may represent a unique redox-sensitive kinase
compared with other MAP kinase family members.
Reactive oxygen species, including H2O2,
O2, and OH
, have been
implicated in the pathogenesis of cardiovascular disease, especially atherosclerosis and hypertension (13-15). Recently, it has been shown
that these molecules and growth factors stimulate similar intracellular
signal events including activating kinases such as c-Src and ERK1/2
(16, 17). We have demonstrated that BMK1, a member of the MAP kinase
family, is specifically activated by oxidative and osmotic stress (12).
Thus the the upstream signal mechanisms by which
H2O2 activates BMK1 should provide valuable insights into pathways of redox-sensitive signal transduction. Previous
studies have shown that c-Src is involved in signal events stimulated
by reactive oxygen species (16). Several receptors that lack intrinsic
tyrosine kinase activity stimulate tyrosine phosphorylation through
association with Src family kinases such as Lck, Lyn, and Fyn (18). To
determine whether Src family kinases are involved in
H2O2-mediated activation of BMK1, we
investigated the role of Src kinases in cultured mouse fibroblasts
using both specific Src inhibitors and cells derived from animals
deficient in c-Src or Fyn. We show here that activation of BMK1 by
H2O2 is positively regulated by c-Src, but not
Fyn. Thus, the c-Src-BMK1 signaling pathway may represent a new
redox-sensitive mechanism in fibroblasts.
The Src-/-, and Fyn-/- fibroblasts were isolated from mouse embryos, which were homozygous for disruption in Src or Fyn gene, and were immortalized with large T antigen (19, 20). Cells were kindly provided by Sheila M. Thomas (Fred Hutchinson Cancer Center, Seattle). NIH-3T3 cells and v-src-transformed NIH-3T3 cells were kindly provided by David Shalloway (21). Fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum as described previously (19). Cells 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 prior to use.
Immunoprecipitation and Western Blot AnalysisAfter treatment, the cells were washed with PBS, harvested in 0.5 ml of lysis buffer (50 mM sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 µM Na3VO4, 10 mM HEPES, pH 7.4, 0.1% Triton X-100, 500 µM phenylmethanesulfonyl fluoride, and 10 µg/ml leupeptin), and flash-frozen on a dry ice/ethanol bath. After allowing the cells to thaw, cells were scraped off the dish and centrifuged at 14,000 × 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 µl) or preimmune serum for 3 h at 4 °C and then incubated with 20 µl 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, 1 mM DTT), and 2 times in 1 ml of washing buffer (20 mM HEPES, pH 7.2, 2 mM EGTA, 10 mM MgCl2, 1 mM DTT, 0.1% Triton X-100). For Western blot analysis, immunoprecipitates were subjected to SDS-PAGE, and proteins were transferred to nitrocellulose membranes (HybondTM-ECL (enhanced chemiluminescence), Amersham International) as described previously (12). The membrane was blocked for 1 h at room temperature with a commercial blocking buffer from Life Technologies, Inc. The blots were then 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 ECL (Amersham International).
BMK1 and c-Src Kinase AssaysBMK1 kinase activity was
measured by autophosphorylation and myelin basic phosphorylation as
described previously with slight modification (12). Comparison of BMK1
activity measured by 32P incorporation into soluble myelin
basic protein versus autophosphorylation of BMK1 showed a
good correlation between the two techniques. Because
autophosphorylation of immunoprecipitated BMK1 was more robust than
myelin basic protein phosphorylation, we report only results from
autophosphorylation assays. Cells were harvested in lysis buffer at
4 °C, then flash-frozen on a dry ice/ethanol bath. After allowing
the cells to thaw, cells were scraped off the dish and centrifuged at
14,000 × g (4 °C for 30 min), and protein
concentration were determined. BMK1 was immunoprecipitated by
incubating 400 µg of protein from each sample with 3 µl of the
rabbit polyclonal anti-BMK1 antibody for 3 h and adding 40 µl of
a 1:1 slurry of protein A-Sepharose (Pharmacia) beads to the
extract/antibody mixture and incubation for 1 h 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, 1 mM DTT), and two
times in 1 ml of modified Buffer A (20 mM HEPES, pH 7.2, 2 mM EGTA, 10 mM MgCl2, 1 mM DTT, 0.1%Triton X-100). BMK1 kinase activity of the immunoprecipitate was
measured at 30 °C for 20 min in a reaction mixture (40 µl) containing 15 µM ATP, 10 mM
MgCl2, 10 mM MnCl2, and 3 µCi of
[-32P]ATP. The reaction was terminated by adding 8 µl of 6 × electrophoresis sample buffer and boiling for 5 min.
c-Src activity was measured by autophosphorylation exactly as described
previously (22). Samples were analyzed on 9% SDS-PAGE, followed by
autoradiography. BMK1 and c-Src autophosphorylation were determined by
densitometry of bands at the correct molecular weights in the linear
range of film exposure using a scanner and NIH Image 1.54.
The adhesion assay was performed as described previously (23). Briefly, cells were incubated at 37 °C in 2 mM EDTA in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3) for 5 min and detached from dishes by gentle pipetting. The cells were washed three times with RPMI 1640, collected by low speed centrifugation, and resuspended in RPMI 1640 with 0.1% bovine serum albumin. 1 × 106 cells were placed onto 60-mm bacteriologic plastic dishes coated with fibronectin and incubated at 37 °C. The bacteriological plastic dishes and plates were coated with human fibronectin (10 µg/ml) for 16 h at 4 °C, and nonspecific binding sites were blocked with 1% heat-denatured bovine serum albumin in PBS for 1 h at room temperature. Prior to use the dishes and plates were rinsed three times with PBS.
MaterialsAll materials were from Sigma except where indicated. H2O2 was from Fisher, and CP-118,556 was from Pfizer.
Statistical AnalysisData are reported as mean ± S.D. Differences were analyzed with unpaired two-tailed Student's t test, or Welch's t test as appropriate.
As reported previously (10), BMK1 resembles other members
of the MAP kinase family in that it has a consensus TXY
phosphorylation motif (specifically TEY) that
upon dual phosphorylation is associated with stimulation of kinase
activity. However, BMK1 differs from other MAP kinase family members by
virture of its larger size as shown by Western blot analysis with BMK1
carboxyl-terminal antibody, which revealed a prominent 110-kDa protein
band in cultured fibroblasts (Fig. 1).
H2O2 caused a retardation in electrophoretic mobility of immunoreactive BMK1 that was readily detected on Western blot as two distinct bands of higher apparent molecular weight. We
interpret these bands to represent phosphorylated BMK1, although the
precise sites of phosphorylation are unknown. However, the highest
molecular weight band has the same apparent molecular weight as the
autophosphorylated protein band shown in subsequent figures, suggesting
that these BMK1 proteins have undergone TEY phosphorylation. A similar
band shift has been reported for the p42 and p44 MAP kinases when they
undergo phosphorylation on T and Y (24).
H2O2 Stimulates BMK1 Kinase Activity
Stimulation of BMK1 kinase activity by
H2O2 was measured by the change in
electrophoretic mobility on Western blot (Fig.
2A), by autophosphorylation
(Fig. 2B), and by myelin basic protein phosphorylation (not
shown). In serum-deprived quiescent cells, BMK1 was almost exclusively
in the unphosphorylated, lower molecular weight form (Fig.
2A). Stimulation with 200 µM
H2O2 for 20-40 min markedly increased the
higher molecular weight forms of BMK1.
Because it was difficult to quantitate BMK1 kinase activity by Western blot analysis, we performed an in vitro kinase assay based on BMK1 autophosphorylation activity. The time course for BMK1 autophosphorylation (Fig. 2, B and C) was similar to that observed for changes in electrophoretic mobility (Fig. 2A) with peak activation at 20 min. The H2O2-induced increase in BMK1 autophosphorylation was not due to changes in BMK1 protein expression. There was a good correlation between the magnitude and time course of BMK1 autophosphorylation and in vitro phosphorylation of myelin basic protein. Because the autophosphorylation assay was more reproducible and of greater magnitude it was chosen for subsequent experiments. The role of autophosphorylation of BMK1 in functional activation is unknown at this time.
H2O2 Activates c-Src in FibroblastsPrevious investigators have suggested that c-Src may
be an upstream mediator of redox-sensitive signal transduction (16). To
explore the role of c-Src in H2O2-mediated
signal transduction in fibroblasts, we measured c-Src kinase activity
by an immune complex assay with c-Src autophosphorylation. c-Src
activity increased within 2 min in response to 200 µM
H2O2 (Fig. 3)
with a maximum 3.1 ± 1.0-fold increase 5 min after
H2O2 stimulation. No difference in c-Src
protein expression was observed in lysates from control and
H2O2-stimulated cells as determined by
immunoprecipitation and Western blot analysis with anti-Src
antibody.
Both Herbimycin A and CP-118,556 Inhibit BMK1 Activation by H2O2
To determine whether c-Src is an
upstream signaling mediator of BMK1, we studied the effect of two
different inhibitors, herbimycin A and CP-118,556, on
H2O2-mediated BMK1 activity (Fig.
4). These two reagents have been shown to
exhibit specificity for inhibition of c-Src (25, 26). Herbimycin A, a
benzoquinone ansamycin antibiotic, inhibits Src family kinases by
covalent interactions with sulfhydryl groups (25) and by disrupting Src
interactions with heat shock proteins (especially HSP90) (27).
CP-118,556, a pyrazolopyrimidine, interacts specifically with Src
family kinases and is a competitive inhibitor of ATP (25). CP-118,556
inhibits Src family kinases preferentially compared with ZAP-70, JAK2, and the epidermal growth factor receptor (25), although the relative
specificity for inhibition of individual Src family kinases is not
established. Herbimycin A and CP-118,556 caused a
concentration-dependent inhibition of
H2O2-mediated BMK1 activation with approximate
IC50 values of 0.3 and 1 µM,
respectively.
Activation of BMK1 in Response to H2O2 Is Dependent on c-Src, but Not Fyn
The ability of herbimycin A and
CP-118,556 to inhibit BMK1 activation by H2O2
suggested an important role for c-Src. To verify that activation of
BMK1 occurred by a c-Src-dependent mechanism, we utilized
cells derived from mice deficient in c-Src kinase family members (19).
There was no immunoreactive c-Src in Src/
cells, while
immunoreactive Fyn was expressed equally (or to a greater extent) than
in wild type cells (Fig. 5,
top). Likewise, there was no immunoreactive Fyn in
(Fyn
/
) cells, while there was no change in expression of c-Src in
Fyn
/
cells compared with the wild type cells (Fig. 5,
top). H2O2 stimulated BMK1 activity in wild type fibroblasts that was maximal at 20 min (Fig.
6, right). In contrast, in
Src
/
fibroblasts, H2O2 failed to stimulate
BMK1 activity at any time (Fig. 6, left). Interestingly, in
Fyn
/
fibroblasts, H2O2 enhanced BMK1
activity by 69 ± 19% compared with the wild type cells (Fig.
7). These results indicate that H2O2 activation of BMK1 is dependent on c-Src,
but not Fyn. To confirm that Src
/
cells are sufficiently healthy to
mediate a Src-independent responses, we investigated protein tyrosine phosphorylation stimulated by adhesion in Src
/
and wild type fibroblasts. Anti-phosphotyrosine Western blot analysis of total cell
lysates showed that adhesion to fibronectin (a ligand for
3,4,5,v
1,
v
3,
v
5, and
v
6 integrins) stimulated tyrosine phosphorylation of 72-75-, and 110-kDa proteins, and there was no
significant difference in the tyrosine phosphorylation of these proteins in Src
/
cells compared with the wild type cells (Fig. 8). To investigate the role of Src
further we studied NIH-3T3 wild type (WT-3T3) and v-Src-transformed
(v-Src-3T3) cells. There was significantly more immunoreactive Src in
the v-Src cells (Fig. 5, bottom). In addition, there was
significantly greater BMK1 activation (7.1 ± 1.4-fold increase)
in the v-Src cells under basal conditions than in wild type-3T3 cells
(Fig. 9).
The major finding of this paper is that
H2O2-mediated BMK1 activation requires c-Src.
Redox-sensitive regulation of BMK1 is thus a new function for c-Src.
Data that support an essential role for c-Src in
H2O2-mediated BMK1 activation include the
following. 1) The time course for c-Src activation was rapid (peak at 5 min) and preceded BMK1 activation (peak at 20 min). 2) c-Src
inhibitors, herbimycin A and CP-118,556, blocked BMK1 activation by
H2O2 at concentrations consistent with a
specific effect on c-Src. 3) In Src-/- fibroblasts, there was no BMK1
activation in response to H2O2. In contrast, in
Fyn/
fibroblasts, H2O2 stimulated BMK1 activation significantly. 4) In v-Src-transformed NIH-3T3 cells BMK1
activation was increased relatively to wild type fibroblasts. Our
results are the first to show that c-Src, but not Fyn, is involved
specifically in oxidative stress-mediated BMK1 activation.
Previous investigators have suggested that c-Src may be an upstream mediator of redox-sensitive signal transduction based on findings with UV irradiation (16). There are at least nine members of the Src family of cytoplasmic protein kinases. Three family members (c-Src, Fyn, and Yes) are expressed ubiquitously and studies suggest that their functions may be at least partially overlapping (28). In the present study we observed that c-Src, but not Fyn, was required for H2O2-mediated BMK1 activation. This result suggests that unique activators of c-Src, but not Fyn, are generated by H2O2 in fibroblasts. Future studies will be required to define the precise nature of these activators.
Several investigators (29-32), including our group (17, 33), have suggested that reactive oxygen species regulate cell function by stimulating many of the same signal transduction pathways utilized by growth factors. For example, both platelet-derived growth factor and superoxide activate ERK1/2 in vascular smooth muscle cells, stimulate c-fos and c-myc expression, and increase DNA synthesis (17, 33). Other investigators have shown that reactive oxygen species stimulate increases in intracellular calcium and raise intracellular pH (31). However, several results suggest that activation of BMK1 by reactive oxygen species does not occur via a pathway shared by growth factors. First, we showed previously that activation of BMK1 occurred in response to H2O2 and sorbitol but not to growth factors such as platelet-derived growth factor and phorbol esters (12). Second, growth factors stimulate ERK1/2 in rat aortic smooth muscle cells, while H2O2 does not (17). Finally, growth factors such as angiotensin II and platelet-derived growth factor stimulate paxillin phosphorylation in smooth muscle cells and fibroblasts (34), but H2O2 does not (data not shown). These findings suggest that H2O2 uses a unique signal pathway to activate BMK1 which is different from growth factors.
Activation of BMK1 by H2O2 likely reflects a balance between positive and negative regulators as shown for other members of the MAP kinase family. For example, ERK1/2 is positively regulated by MAP kinase/ERK kinase and negatively regulated by the MAP kinase phosphatase-1. To date the phosphatase responsible for inactivating BMK1 has not been identified. c-Src itself is positively and negatively regulated by dephosphorylation and phosphorylation. One of the residues that appears to be critical for regulation of c-Src is Tyr530, which is not present in v-Src. Phosphorylation of Tyr530 by Csk (C Src kinase) family members inhibits c-Src activity (35), whereas dephosphorylation of this residue appears to be an activating mechanism. Phosphorylation of Tyr419 in the catalytic domain may be an activating signal, although the kinase(s) responsible have not been identified. Future work, including analysis of Csk function and phosphorylation of specific c-Src residues, will be necessary to identify the mechanisms for c-Src activation in response to H2O2.
In summary, we have shown that c-Src and BMK1 are activated by oxidative stress in fibroblasts. The fact that BMK1 is not activated by growth factors indicates that c-Src-mediated activation of BMK1 represents a growth-independent function of c-Src. Furthermore, the demonstration that H2O2-mediated activation of BMK1 required Src, but not Fyn, suggests that these two Src family kinases serve different intracellular functions with respect to oxidative stress and that the c-Src-BMK1 signaling pathway may involve novel intracellular mediators.
We thank U. Schmitz from the Berk laboratory for critical reading of this manuscript.