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J. Biol. Chem., Vol. 279, Issue 10, 8787-8791, March 5, 2004
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Binds to Big Mitogen-activated Protein Kinase 1 (BMK1/ERK5) and Regulates BMK1 Function*
From the Department of Medicine and Center for Cardiovascular Research, University of Rochester School of Medicine and Dentistry, Aab Institute of Biomedical Sciences, Rochester, New York 14642
Received for publication, September 15, 2003 , and in revised form, December 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in vitro and in vivo as demonstrated by glutathione S-transferase (GST)-14-3-3 fusion protein pull-down assays and coimmunoprecipitation. Phosphorylation of BMK1 was most likely required for this interaction. GST-14-3-3 pull-down assays using truncated constructs of BMK1 and site-directed BMK1 mutants demonstrated that the interaction requires serine 486 within the C terminus of BMK1. BMK1 bound to 14-3-3 basally, and the interaction was greatly abrogated when BMK1 was activated. The interaction of 14-3-3 and BMK1 inhibited kinase activities stimulated by constitutively active (CA)-MEK5 and epidermal growth factor. Mutation of serine 486 (BMK1-S486A) prevented the interaction with 14-3-3 and enhanced BMK1 activity upon epidermal growth factor stimulation. These data demonstrate an inhibitory function for 14-3-3 binding to BMK1 and show that serine 486 phosphorylation represents a novel regulatory mechanism for BMK1. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BMK1 is important in promoting cell proliferation and inhibiting cell apoptosis (5). BMK1 null embryos display defects in vascular remodeling and organization, indicating that BMK1 may play an important role in angiogenesis and cardiovascular development (6, 7). For example, in ischemic myocardium, activation of BMK1 counteracts ischemic cell damage and contributes to cell survival pathway.2 Previously, our lab showed that BMK1 was activated by fluid shear stress in endothelial cells (9) and that BMK1 mediates anti-apoptotic signaling stimulated by flow (10). These data suggest that BMK1 may be an important mediator of flow-stimulated atheroprotective signals in endothelial cells.
To characterize regulatory mechanisms for BMK1 that mediate cell growth and anti-apoptosis, we identified BMK1-binding proteins. By yeast two-hybrid analysis, we identified 14-3-3 as a BMK1-interacting protein. 14-3-3 proteins are a family of conserved, ubiquitously expressed proteins with monomeric molecular mass around 30,000 (11). To date, seven highly conserved 14-3-3 isoforms (, 
, 
, 
, 
, 
, 
) encoded by seven distinct genes have been identified in mammalian systems (11). 14-3-3 mainly exists in the cytoplasmic compartment, forming homo- or hetero-dimers with other 14-3-3 isoforms (12). Most reports indicate that 14-3-3 isoforms are interchangeable with respect to protein binding (11).
In the present study, we found that BMK1 bound to 14-3-3 under basal conditions, and the interaction was greatly diminished upon activation of MEK5 and phosphorylation of BMK1. 14-3-3 overexpression inhibited EGF-stimulated BMK1 kinase activity, and a BMK1-S486A mutant deficient in 14-3-3 binding exhibited increased kinase activity upon EGF stimulation. These findings define a new regulatory mechanism for BMK1 mediated by 14-3-3 binding to phospho-serine 486.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-galactosidase activity, 120 positive clones were obtained. 80 of them specifically interacted with BMK1 in yeast mating tests. Sequence analysis and bioinformatics studies indicated that 8 out of 80 BMK1-interacting sequences represented 14-3-3.
DNA Constructs and Mutagenesis—Human 14-3-3 cDNA was a gift from Dr. A. J. Muslin (Washington University School of Medicine). Human BMK1 cDNA was a gift from Dr. J. D. Lee (The Scripps Research Institute). Mouse BMK1 cDNA was cloned by Dr. Chen Yan as reported (1). BMK1 point mutations were made by using the QuikChange site-directed mutagenesis kit (Strategene) and confirmed by DNA sequencing.
In Vitro Transcription/Translation—By using the TNT T7-coupled reticulocyte lysate system (Promega), mouse BMK1 in pcDNA 3.1/Xpr-His was transcribed and translated in vitro. Briefly, 40 µl of TNT quick master mix, 1 µl of 1 mM methionine, 1 µg of DNA template, 1 µl of biotin-lysyl-tRNA were mixed in a final volume of 50 µl, incubated for 30 min at 30 °C. The nascent protein was labeled with Transcend biotin-lysyl-tRNA (Promega) and used for binding assay immediately.
Cell Culture and Transient Expression—293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, and 10% fetal calf serum in a 5% CO2, 95% O2 incubator at 37 °C. 293T cells were seeded onto 60-mm dishes 24 h prior to transfection and then transiently transfected at 60–70% confluence using LipofectAMINE reagent (Invitrogen). After 4 h of transfection, the medium was changed to 10% fetal calf serum for 24 h. Cells were growth-arrested (0% fetal calf serum) overnight prior to the experiment.
Cell Lysate Preparation—Cell monolayers were rinsed with ice-cold phosphate-buffered saline (150 mM NaCl, 20mMNa2PO4, pH7.4) and then harvested in 300 µl of cell lysis buffer (50 mM Tris-Cl, 2 mM EGTA, 1% Triton-X 100, 0.4 mMNa3VO4, 50 mM NaCl, plus 1:1000 protease inhibitor mixture (Sigma)) on ice. Cell lysates were then centrifuged at 14,000 x g at 4 °C for 15 min, and the supernatants were used immediately. For some experiments with phosphatase treatment, cell lysates were collected in calf intestinal alkaline phosphatase buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol, 0.5% Triton plus protease inhibitor mixture). Lysates were then incubated with or without 200 units of calf intestinal alkaline phosphatase (Promega) at 37 °C for 1 h.
GST Pull-downs and Coimmunoprecipitations—For the pull-down assays, cell lysates containing 1 mg of protein were incubated overnight with 10 µg of GST or GST-14-3-3 bound to glutathione-Sepharose. For coimmunoprecipitations, cell lysates from each sample were precleared by incubation with 10 µl of protein A/G agarose for 30 min at 4 °C and then incubated with 2 µg of specific antibody and 30 µl of protein A/G agarose overnight at 4 °C on a roller system. The immune complex beads were washed four times with 1 ml of cell lysate buffer before the addition of Laemmli buffer.
Western Analysis—Proteins were resolved on SDS-PAGE and transferred to nitrocellulose membranes (Hybond TM-ECL). Immunoblotting was performed with anti-Xpress antibody (Invitrogen), M2 anti-FLAG antibody (Sigma), anti-HA antibody (Sigma), anti-14-3-3 antibody (Santa Cruz Biotechnology), anti-BMK1 antibody (gift from Dr. J. D. Lee), or anti-phosphoBMK1 (Thr-218/Tyr-220) antibody (Cell Signaling). Immunoreactive bands were visualized using enhanced chemiluminescence with ECL reagent (Amersham Biosciences).
Reporter Gene Expression—A MEF2C reporter gene expression assay was performed as described previously (1). Briefly, 293T cells were seeded on 24-well plates the day before the transfection. Cells were transfected with the indicated cDNA constructs, the reporter plasmid pG5E1bLuc (containing five Gal4 sites and a luciferase gene), MEF2C fusion activator vectors (containing a GAL4-binding domain and MEF2C activation domain), and Renilla luciferase control vector for 5 h followed by incubation in 10% fetal calf serum for 24 h. Cell lysates were collected for luciferase assay performed with a dual reporter assay (Promega). To correct for the transfection efficiency, the firefly luciferase activities were divided by the Renilla luciferase activities.
BMK1 Kinase Assay—BMK1 kinase activity was measured by autophosphorylation as described previously (9). Briefly, immune complex kinase assay was performed in a reaction mixture (30 µl) containing 15 µM ATP, 10mM MgCl2, 10 mM MnCl2, and 3 µCi of [-32P]ATP at 30 °C for 20 min. The reaction was then terminated with 8 µl of 6x electrophoresis sample buffer and boiled for 5 min. Samples were analyzed on 7.5% SDS-polyacrylamide gel and then transferred to nitrocellulose membrane and autoradiographed.
Statistical Analysis—Data are shown as mean ± S.E. Differences were analyzed with Student's t test. Values of p < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Binds to BMK1, Identification by Yeast Two-hybrid Screen—To elucidate BMK1 regulatory mechanisms, we identified BMK1 binding partners by using full-length BMK1 as bait in a yeast two-hybrid library screen. We analyzed 80 positive clones that potentially interact with BMK1. Among these clones, we found proteins involved in multiple cellular processes, such as cell metabolism, growth regulation, and cytoskeleton maintenance. One prominent binding partner for BMK1 was 14-3-3. Since 14-3-3 isoforms are functionally similar, we investigated the interaction between BMK1 and 14-3-3 because cDNA constructs of this isoform were available for study.
14-3-3 Binds to BMK1 Both in Vitro and in Vivo —The interaction between 14-3-3 and BMK1 was confirmed in 293T cells. 293T cells were transfected with FLAG-BMK1, and cell lysates were pulled down by GST or GST-14-3-3 (Fig. 1A). BMK1 was pulled down by GST-14-3-3 but not by GST alone. The interaction was also demonstrated in coimmunoprecipitation experiments. Cell lysates from Xpress-14-3-3 and FLAG-BMK1 cotransfected 293T cells were immunoprecipitated with anti-FLAG antibody for BMK1, and anti-Xpress antibody was used to detect 14-3-3. We found that 14-3-3 coprecipitated with BMK1 in 293T cells (Fig. 1B, upper panel), and the reverse immunoprecipitation showed that BMK1 coprecipitated with 14-3-3 (Fig. 1B, lower panel). The fact that 14-3-3 interacts with BMK1 in vivo was further addressed in endogenous coimmunoprecipitation, which showed that endogenous BMK1 coprecipitated with 14-3-3. (Fig. 1C).
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Interacts with BMK1 Directly—To investigate whether the interaction between 14-3-3 and BMK1 is direct, BMK1 protein was synthesized in vitro by transcription/translation. As shown in Fig. 2, GST-14-3-3 pulled down BMK1 in the absence of other cellular proteins, demonstrating that these two proteins interact with each other directly in vitro.
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and BMK1—It has been well recognized that phosphorylation on serine and threonine is important for proteins to interact with 14-3-3 (13, 14). To investigate whether phosphorylation is required for BMK1 interaction, lysates from 293T cells transfected with BMK1 were treated with alkaline phosphatase for 1 h at 37 °C followed by GST-14-3-3 pull-down. As shown in Fig. 3A, in vitro treatment with calf intestinal alkaline phosphatase diminished the interaction between 14-3-3 and BMK1. This result was further supported by an in vivo experiment in which cells were treated with staurosporine, a general serine and threonine protein kinase inhibitor. The association between 14-3-3 and BMK1 was abolished by staurosporine (Fig. 3B). These results suggest that the interaction between 14-3-3 and BMK1 basally requires phosphorylation of BMK1.
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and BMK1 Occurs via Serine 486 within the C Terminus of BMK1—To identify the binding region for 14-3-3 on BMK1, a GST-14-3-3 pull-down assay was performed for several truncated constructs of BMK1. The design of the truncated BMK1 constructs was based on the BMK1 structure, which has a unique C terminus containing two proline-rich regions (P1 and P2) and one nuclear localization sequence (Fig. 4A) (1). GST-14-3-3 pulled down BMK1(aa 1–494), BMK1(aa 1–577), and wild type BMK1, but not BM-K(aa 1–418) (Fig. 4B). This result suggests that the interaction of 14-3-3 with BMK1 occurs within the C terminus of BMK1(aa 419–494). We further localized the binding region as aa 451–494 by GST-14-3-3 pull-down assay using BMK1 internal deletion mutants (data not shown). Although there is no obvious 14-3-3 recognition motif (RSXpSXP, where pS denotes phosphorylated serine) in this region, we focused on the serines within this region because phosphorylation was found to be required for the interaction with 14-3-3 (Fig. 3). We made four serine-to-alanine mutations, S460A, S472A, S486A and S489A, to investigate which serine may be important for the interaction. Only the BMK1-S486A mutant was not pulled down by GST-14-3-3, whereas the other serine-to-alanine mutants and wild type BMK1 were pulled down (Fig. 4C). Similarly, in 293T cells cotransfected with 14-3-3 and wild type BMK1 or BMK1-S486A mutant, 14-3-3 coimmunoprecipitated with wild type BMK1 but not BMK1-S486A (Fig. 4D). These data suggest that serine 486 within the motif LKpSLRS (where pS denotes phosphorylated serine) mediates the interaction between 14-3-3 and BMK1.
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and BMK1 Is Reduced upon BMK1 Activation—To address whether the interaction between 14-3-3 and BMK1 is altered by BMK1 activation, constitutively active MEK5 (CA-MEK5) was used to activate BMK1. As shown in Fig. 5A, GST-14-3-3 bound to BMK1 under basal conditions, whereas the interaction was significantly reduced when BMK1 was activated by CA-MEK5. This result was further supported in a coimmunoprecipitation experiment, as shown in Fig. 5B. 293T cells were cotransfected with FLAG-BMK1 and CA-MEK5. Cell lysates were immunoprecipitated with anti-FLAG antibody for BMK1 and immunoblotted with anti-14-3-3 antibody. Compared with the basal condition, the interaction between BMK1 and 14-3-3 was significantly reduced when BMK1 was activated by CA-MEK5 (Fig. 5B).
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Inhibits BMK1 Activity through Interaction at Serine 486 —The fact that BMK1 dissociates from 14-3-3 upon BMK1 activation suggests that 14-3-3 binding may modulate BMK1 kinase activity. To investigate this possibility, 293T cells were cotransfected with Xpress-14-3-3 and CA-MEK5. Endogenous BMK1 was immunoprecipitated, and its activity was measured by in vitro kinase assay based on BMK1 autophosphorylation (Fig. 6A). BMK1 activation was inhibited significantly by 14-3-3 expression. Based on this result, we hypothesized that wild type BMK1 and BMK1-S486A may have different activities since BMK1-S486A does not bind to 14-3-3. To test this hypothesis, we used EGF to stimulate BMK1. 293T cells transfected with BMK1 or BMK1-S486A were stimulated with EGF (Fig. 6B). The BMK1-S486A mutant exhibited greater activity (
3-fold-increase at 20 min) than wild type BMK1 as measured by anti-phosphoBMK1 antibody. Consistent with this result, in 14-3-3-overexpressing 293T cells, EGF-stimulated endogenous BMK1 activity was significantly inhibited (by 70%) (Fig. 6C).
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—To demonstrate the physiological significance of BMK1 and 14-3-3 interaction, we studied BMK1-mediated transactivation of MEF2C, an important transcription factor in cardiovascular development. To determine the effect of 14-3-3-BMK1 interaction on MEF2C activity, we used a reporter gene assay (Fig. 7). BMK1 overexpression increased MEF2C activity by 100% (Fig. 7, column 2), although 14-3-3 cotransfection inhibited MEF2C activity by 40% (Fig. 7, column 3). The increase in MEF2C activity (550%) upon cotransfection of BMK1 and CA-MEK5 (Fig. 7, column 4) was also significantly inhibited by 40% upon 14-3-3 cotransfection (Fig. 7, column 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The interaction between BMK1 and 14-3-3 inhibits BMK1 phosphorylation and activation of MEF2C. 14-3-3 binding to BMK1 at serine 486 occurs within the motif LKSLRS (s = site of phosphorylation), which is conserved in human, mouse, and rat BMK1 proteins (1, 15). As is typical for 14-3-3 binding, the interaction with BMK1 is most likely phosphorylation-dependent. Several motifs in 14-3-3 binding partners have been recognized. Among 14-3-3 motifs, one prominent binding motif is RX1–2pSX2–3S (where pS denotes phosphorylated serine and X represents any amino acid) (13, 14). The motif LK(SLRS) in BMK1 is similar to RX1–2[pSX2–3S], as shown by the bracketed amino acids. 14-3-3 proteins bind to several kinases, including ASK, protein kinase C-µ, and p90 ribosomal s6 kinase isoform-1 (RSK-1), and inhibit their activities (16–18). Here, we found that BMK1 binds to 14-3-3 basally through serine 486 site on the C terminus and dissociates from 14-3-3 upon BMK1 activation. The mechanism of BMK1 activation is not fully understood, although the upstream kinase that activates BMK1 via phosphorylation of TEY motif is MEK5 (2). Based on the mechanisms of 14-3-3 inhibiting other kinases, we propose a putative model for 14-3-3 in regulation of BMK1 signaling (Fig. 8). 14-3-3 binds to BMK1 directly and stabilizes its inactive form by blocking MEK5 interaction under basal conditions. Upon mitogen activation, a BMK1 activation intermediate forms that includes phosphorylation of TEY motif by MEK5 and dephosphorylation of serine 486 by an unknown phosphatase. It is possible that either action could be the initiation step facilitating the other. Fully activated BMK1 is characterized by phosphorylation at TEY and absence of 14-3-3 binding. The supporting evidence for this model is findings that overexpression of 14-3-3 inhibits BMK1 activity upon EGF stimulation, and the 14-3-3 binding-deficient BMK1 mutant has a higher kinase activity than wild type. Two unanswered questions are the identity of the kinase that phosphorylates BMK1 at serine 486 basally and the mechanism by which BMK1 and 14-3-3 dissociate from each other. We think it is unlikely that BMK1 autophosphorylates serine 486 since it is not surrounded by a MAP kinase phosphorylation motif. A protein kinase C isoform is more likely based on sequence motif. Based on the similarity to ASK1, we think the dissociation may be caused by recruitment of a phosphatase (phosphoserine phosphatase) that dephosphorylates serine 486. The protein identified as AIP-1 that binds to ASK1 and dissociates 14-3-3 from ASK1 is the first example of this mechanism (19, 20).
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To date, seven 14-3-3 isoforms have been identified, and they form homo- or hetero-dimers naturally. 14-3-3 proteins bind to their ligands through their conserved inner, concave surfaces within a cup-like shape as a whole. Most reports indicate that these 14-3-3 isoforms are non-selective regarding interaction with their ligands (11). Our findings that 14-3-3 bound to BMK1 in yeast two-hybrid analysis and the binding of 14-3-3 to BMK1 in the mammalian system shown here also indicate the non-selective characteristic of 14-3-3 isoforms.
In summary, we demonstrate for the first time that 14-3-3 is a BMK1-binding protein that regulates BMK1 function. An important extension of the present study is identification of serine 486 as a regulatory site for BMK1 activation. Further work will be required to elucidate the nature of the kinase that phosphorylates serine 486 and the phosphatase and/or competing protein that dissociates 14-3-3 from serine 486.
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FOOTNOTES |
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To whom correspondence should be addressed: Center for Cardiovascular Research, University of Rochester, 601 Elmwood Ave., Box 679, Rochester, NY 14642. Tel.: 585-273-1946; Fax: 585-273-1497; E-mail: bradford_berk{at}urmc.rochester.edu.
1 The abbreviations used are: BMK1, big mitogen-activated protein kinase 1; ASK, apoptosis signal-regulating kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; CA-MEK, constitutively active MEK; GST, glutathione S-transferase; EGF, epidermal growth factor; aa, amino acids; HA, hemagglutinin.
2 Y. Takeishi, Q. Huang, T. Wang, M. Glassman, M. Yoshizumi, J.-D. Lee, H. Kawakatsu, C. P. Baines, W. Che, S. Ohta, R. A. Walsh, B. B. Berk, and J. Abe, submitted.
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REFERENCES |
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