domain autophosphorylation of the mtk1 kinase trans dimerization

Published Ahead of Print 22 January 2007. 10.1128/MCB.01435-06.

2007, 27(7):2765. DOI:Mol. Cell. Biol. Haruo SaitoZenshi Miyake, Mutsuhiro Takekawa, Qingyuan Ge and Domain Autophosphorylation of the MTK1 Kinase

transDimerization-Mediated through Induced N-C Dissociation and Activation of MTK1/MEKK4 by GADD45

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MOLECULAR AND CELLULAR BIOLOGY, Apr. 2007, p. 2765–2776 Vol. 27, No. 70270-7306/07/$08.00�0 doi:10.1128/MCB.01435-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Activation of MTK1/MEKK4 by GADD45 through Induced N-CDissociation and Dimerization-Mediated trans Autophosphorylation

of the MTK1 Kinase Domain�†Zenshi Miyake,1,2‡ Mutsuhiro Takekawa,1,2‡* Qingyuan Ge,3 and Haruo Saito1,2*

Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, 4-6-1 Shirokanedai,Minato-ku, Tokyo 108-8639, Japan1; Department of Biophysics and Biochemistry, Graduate School of

Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan2;and Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, Massachusetts 019233

Received 4 August 2006/Returned for modification 3 October 2006/Accepted 9 January 2007

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK),and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryoticworld. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely,the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is astress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins(GADD45�/�/�). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. TheMTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. ThisN-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site.GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif,which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. AnMTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude thatGADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, lead-ing to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the sameevents, but in the absence of GADD45.

Living organisms are frequently exposed to cellular stresses,which are defined as diverse environmental conditions that aredetrimental to the normal growth and survival of the cells.Typical cellular stresses include UV, ionizing radiation, geno-toxins, hyperosmolarity, oxidative stress, low-oxygen supply(hypoxia), and inhibition of protein synthesis by antibiotics andplant toxins. In coping with the barrage of these and othercellular stresses, multicellular eukaryotic organisms have devel-oped strategies for how damaged cells will respond to stresses. Ingeneral, if the intensity of damage is moderate, the affected cellwill seek to repair the damage. If, however, the damage to a cellis too severe for a complete repair, the affected cells are elimi-nated by apoptosis. This reduces the risk to the organism as awhole, such as the development of a cancer. Such crucial decisionmaking between repair and death is, at least in part, mediated bythe stress-activated mitogen-activated protein kinase (SAPK)pathways (for general reviews on mitogen-activated protein ki-nase [MAPK] and SAPK, see references 8, 20, 24, 29, and 30).

As the name implies, the SAPK pathways are homologous toand share many characteristics with the classic (extracellularsignal-regulated kinase 1/2 [ERK1/2]) MAPK pathway. Eu-

karyotic MAPK pathways are conserved signaling modules thattransmit signals from the cell surface to the nucleus. The coreof any MAPK pathway is composed of three tiers of sequen-tially activating protein kinases, namely, MAPK kinase kinase(MAPKKK), MAPK kinase (MAPKK), and MAPK. The acti-vation of MAPKs is achieved by the phosphorylation of threonineand tyrosine residues within a conserved Thr-Xaa-Tyr motif in theactivation loop (also called the T-loop) catalyzed by MAPKKs.MAPKKs, in turn, are activated by any of several MAPKKKsvia the phosphorylation of serine and/or threonine residueswithin their activation loops.

All eukaryotic cells possess multiple MAPK pathways, eachof which is activated by a distinct set of stimuli. In the buddingyeast Saccharomyces cerevisiae, for example, hyperosmoticstress activates the Hog1 MAPK pathway, whereas matingpheromones activate the Fus3/Kss1 MAPK pathway (13, 18).In mammalian cells, four different subfamilies of MAPKs arepresent, namely, ERK1/2, Jun N-terminal protein kinase 1/2/3(JNK1/2/3), p38�/�/�/�, and ERK5. The ERK1/2 MAPK path-way is preferentially activated in response to mitogenic stimuli,such as growth factors and phorbol esters, and plays a role in cellgrowth and cell survival. The ERK1/2 pathway is regulatedmainly by the monomeric GTPase Ras, which recruits MAPKKKs of the Raf family to activate the two downstreamMAPKKs: MEK1 and -2. These MAPKKs, in turn, activate theERK1/2 MAPKs. The JNK and p38 MAPKs (collectivelycalled SAPKs), in contrast, preferentially respond to variouscellular stresses and are thus called SAPK pathways. Besidescell stresses, the SAPK pathways are also activated by cyto-kines such as interleukin-1, tumor necrosis factor alpha, and

* Corresponding author. Mailing address: Institute of Medical Sci-ences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo108-8639, Japan. Phone: 1-81-3-5449-5505. Fax: 1-81-3-5449-5701. E-mailfor Mutsuhiro Takekawa: [email protected]. E-mail for HaruoSaito: [email protected].

† Supplemental material for this article may be found at http://mcb.asm.org/.

‡ These authors contributed equally to this work.� Published ahead of print on 22 January 2007.

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transforming growth factor � (TGF-�). The JNK subfamilyof MAPKs are activated mainly by the MKK4 and MKK7MAPKKs, while the p38 subfamily MAPKs are activated bythe MKK3 and MKK6 MAPKKs. In clear contrast to thislimited number of MAPKKs in the SAPK pathways, there arenumerous MAPKKKs that function upstream of the JNK andp38 MAPKs. These include MEKK1/2/3, MTK1 (also knownas MEKK4), TAK1, ASK1/2, TAO1/2/3, MLKs, and perhapsothers. This multiplicity at the level of MAPKKK surely re-flects the vastly diverse stress stimuli that can recruit theseSAPK pathways.

MTK1 is one of the human MAPKKKs belonging to theSAPK pathways, and the mouse ortholog is called MEKK4 (16,33). The kinase domain of MTK1 (MEKK4) is homologous toother MAPKKKs and especially similar to mammalianMEKK1/2/3 and ASK1/2 and yeast SSK2/SSK22, but its N-terminal noncatalytic domain (regulatory domain) is unique(33). Analyses using MEKK4-deficient mice have shown thatthe MEKK4 signaling pathway integrates signals from both theT-cell antigen receptor and interleukin-12/STAT4 in develop-ing Th1 cells and promotes STAT4-independent gamma inter-feron production (9), and MEKK4 is essential for normal neu-ral and skeletal development (2, 10).

In a yeast two-hybrid screening aimed at identifying MTK1activators, we found three growth arrest and DNA damage-inducible 45 (GADD45) family proteins to be strong bindingpartners of MTK1 (34). The GADD45 gene was originallyidentified as a UV-inducible gene in Chinese hamster cells (14).The human genome encodes three GADD45-like proteins,GADD45� (the original GADD45), GADD45� (MyD118),and GADD45� (CR6 or OIG37) (25, 44). These will be re-ferred to collectively as the GADD45 proteins. The GADD45proteins share 55 to 58% sequence identity. The threeGADD45 genes are all inducible by cellular stresses, althoughoptimal stimuli for each gene appear to be different (25). Theexpression profiles of the three GADD45 genes are also dis-tinct in various tissues (34). The GADD45 proteins interactwith various intracellular molecules, such as proliferating cellnuclear antigen, Cdc2-cyclinB1 complex, p21Waf1/Cip1, andcore histones, and play important roles in stress-adaptive pro-cesses, including growth control, maintenance of genomic sta-bility, DNA repair, and apoptosis (3, 17, 25, 38). In otherwords, the GADD45 proteins are emergency calls in damagedcells.

Expression of transfected GADD45 genes strongly activatescoexpressed MTK1 kinase and induces apoptosis in mamma-lian cells (34). TGF-�-induced GADD45� expression also ac-tivates p38 MAPK through MTK1 activation (35). MEKK4-deficient mice have lost GADD45-induced gamma interferonproduction (9). The activation of the SAPK pathway by MTK1and GADD45 is temporally a slow process because it requiresthe induction of GADD45 gene expression prior to activationof MTK1. Thus, the activation of MTK1 by GADD45 differsfrom other modes of SAPK activation that occur within min-utes. GADD45/MTK1-mediated SAPK activation may there-fore serve as a more long-term adaptive mechanism forstressed cells.

We previously proposed that the binding of GADD45 to theN-terminal region of MTK1 counters the autoinhibitory effectof the MTK1 N-terminal segment on the kinase domain and, at

the same time, enables the MTK1 kinase domain to bind itscognate MAPKKs (MKK3 and MKK6) via the latter’s DVDdocking sites (28, 36). The details of MTK1 activation byGADD45, however, remained obscure. In this report, we in-vestigated the molecular mechanism by which GADD45 reg-ulates MTK1 kinase activity.

MATERIALS AND METHODS

Media and buffers. Lysis buffer contains 20 mM Tris-HCl (pH 7.5), 1% TritonX-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 50 mM �-glycerophosphate,10 mM NaF, 1 mM sodium vanadate, 1 mM dithiothreitol, 1 mM phenylmeth-ylsulfonyl fluoride, 10 �g/ml leupeptin, and 10 �g/ml aprotinin. Sodium dodecylsulfate (SDS) loading buffer is 65 mM Tris-HCl (pH 6.8), 5% (vol/vol) 2-mer-captoethanol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol. Kinase buffercontains 25 mM Tris-HCl (pH 7.5), 25 mM MgCl2, 0.5 mM sodium vanadate, 25mM �-glycerophosphate, 2 mM dithiothreitol, and 2 mM EGTA.

Plasmids. The mammalian expression plasmids pFlag-MTK1, pFlag-MTK1-K/R, pcDNA3-GADD45�, pFlag-MTK1(L534Q), pFlag-MTK1(Q637P), pFlag-MTK1(V1300F), pFlag-MTK1(I1360M), and pGST-MKK6(K/A) were de-scribed previously (28, 32, 34). GADD45� deletion mutants and MTK1 mutantswere generated by PCR mutagenesis. pEGFP-C1 (Clontech), pcDNA4Myc, andpcDNA3 vectors were used to generate green fluorescent protein (GFP)-taggedGADD45�, Myc-tagged MTK1, and GADD45�/GADD45� expression plas-mids, respectively. pcDNA3FKBP vector was used to generate FKBP-fused,hemagglutinin (HA)-tagged MTK1 expression plasmids. MTK1-N containsMet-22 through Ala-994, MTK1-C contains Ser-1247 through Glu-1607 (theC-terminal amino acid [aa]), and MTK1-N�BD contains Ser-253 through Ala-994.

Expression and purification of epitope-tagged proteins. Glutathione S-trans-ferase (GST)–MKK6(K/A) was expressed in Escherichia coli DH5 and purifiedusing glutathione-Sepharose beads as previously described (33).

Tissue culture and transient transfection. COS-7 cells were maintained inDulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum,L-glutamate, penicillin, and streptomycin. For transient transfection assays, cellsgrown in 60-mm dishes were transfected with the appropriate expression plas-mids using the Effectene transfection reagent (QIAGEN). The total amount ofplasmid DNA was adjusted to 1 �g per plate with vector DNA (pcDNA3).

Immunoblotting analyses. Immunoblotting analyses were carried out as de-scribed previously (36). The following antibodies were used: anti-Flag monoclo-nal antibody (MAb) M2 (Sigma), anti-HA MAb 3F10 (Roche), anti-GFP MAbB-2 (Santa Cruz), polyclonal anti-Myc (Santa Cruz), anti-GADD45� MAb 4T-27(Santa Cruz), and goat polyclonal antisera to GADD45� and GADD45� (SantaCruz). An anti-MTK1 antiserum has been described previously (9). An anti-phospho-T1493 rabbit antiserum (�P-T1493; lot no. A2340PE) was made inhouse.

In vitro binding assay for MTK1-N and C segments. Two plasmids that encodea Flag-tagged MTK1 N-terminal segment (residues 22 to 994) and a Myc-taggedMTK1 C-terminal segment (residues 1247 to 1607) were constructed (Fig. 1A).Flag-MTK1-N and Myc-MTK1-C were individually expressed in COS-7 cells,and cell lysates were prepared 24 h after transfection. The lysates were mixed invitro and incubated for 4 h at 4°C. Flag-MTK1-N was precipitated from themixture by an anti-Flag antibody conjugated to protein G-Sepharose, and co-precipitated Myc-MTK1-C was detected by immunoblotting using an anti-Mycantibody.

Detection of MTK1 Thr-1493 phosphorylation. Cell lysates, prepared in lysisbuffer with 0.5% deoxycholate, were incubated with an appropriate antibody for2 h at 4°C to precipitate epitope-tagged MTK1. Endogenous MTK1 was precip-itated using anti-MTK1 antibody. Immune complexes were recovered with theaid of protein G-Sepharose beads, washed three times with lysis buffer containing500 mM NaCl and 0.5% deoxycholate and twice with lysis buffer only, resus-pended in SDS loading buffer, and separated by SDS-polyacrylamide gel elec-trophoresis (PAGE) for immunoblotting analyses using �P-T1493.

Coimmunoprecipitation assay for protein binding. Cell lysates were incubatedwith protein G-Sepharose beads at 4°C for about 20 h. Then, precleared lysateswere incubated with anti-Flag MAb M2 bound to protein G-Sepharose beads at4°C for 4 h with gentle rotation. Immunoprecipitates were collected by centrif-ugation, washed six times with lysis buffer containing 500 mM NaCl and 0.5%deoxycholate, and subjected to SDS-PAGE.

In vitro kinase assay. Transfected cells were lysed in lysis buffer. Cell lysateswere incubated with the appropriate antibody for 2 h at 4°C. Immune complexeswere recovered with the aid of protein G-Sepharose beads, and washed twice

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with lysis buffer containing 500 mM NaCl and 0.5% deoxycholate, twice with lysisbuffer, and twice again with kinase buffer. Immunoprecipitates were resuspendedin 30 �l of kinase buffer containing 4 �g of GST-MKK6(K/A). The kinasereaction was initiated by the addition of 6 �l of [�-32P]ATP (3.3 �Ci/�l, 20 �MATP). Following a 30-min incubation at 30°C, reactions were terminated by theaddition of SDS loading buffer. Samples were boiled, separated by SDS-PAGE,dried, and visualized by autoradiography. The amounts of phospho-GST-MKK6(K/A) were quantified using the imaging analyzer FLA-3000.

Two-hybrid analysis. Yeast two-hybrid analysis was performed using the yeastL40 strain as described previously (37). �-Galactosidase activity is expressed inMiller units (27).

Regulated protein homodimerization using AP20187. The ARGENT regu-lated homodimerization kit (version 2.0) and the ARGENT regulated transcrip-tion retrovirus kit (version 2.0) were obtained from ARIAD Pharmaceuticals,Inc. COS-7 cells were transfected with pFKBP-HA-MTK1, and 36 h later, themedium was replaced with fresh medium containing the appropriate amount ofAP20187 and the cells were incubated for 2 h at 37°C prior to lysis and assay.

RESULTS

GADD45 binds to and activates MTK1. Previously, we haveshown that the GADD45 family proteins bind to a GADD45-binding domain (BD) (Fig. 1A) in the N-terminal regulatory

region of MTK1 (34). An MTK1 mutant protein that lacks theGADD45 BD cannot be activated by GADD45 proteins. In thefollowing work, we used GADD45� as a representative mem-ber of the GADD45 family because GADD45� is moststrongly induced by methyl methanesulfonate (MMS) stressand by TGF-�. Whenever we compared the three GADD45proteins, however, there were no qualitative differences amongthem.

GADD45� is a small protein of 160 amino acid residues(Fig. 1B). To determine the region of GADD45� proteins thatis directly involved in MTK1 activation, we constructed a seriesof deletion mutants, each of which has a deletion of �10 aminoacids. COS-7 cells were cotransfected with a plasmid encodingGFP-tagged GADD45� (GFP-GADD45�) or one of its dele-tion derivatives and another plasmid encoding full-length Flag-tagged MTK1 (Flag-MTK1). After transfection for 36 h, kinaseactivity of Flag-MTK1 was assayed in vitro. Coexpression ofthe full-length GADD45� increased Flag-MTK1 kinase activ-ity more than 10-fold (Fig. 1C). Deletions at the N terminus

FIG. 1. GADD45 binds MTK1 and enhances MTK1 kinase activity. (A) Domain structure of MTK1. The top cartoon schematically indicatesthe positions of the GADD45 binding domain, the autoinhibitory domain, the dimerization domain (DD), and the kinase domain (KD) in MTK1.The lower bars represent the MTK1-N and MTK1-C constructs used for Fig. 1E. (B) Structure of GADD45. The position of the MTK1 bindingdomain in GADD45� is shown. (C) GADD45 region important for MTK1 activation. COS-7 cells were transiently cotransfected with an expressionplasmid for Flag-MTK1 and another plasmid encoding either full-length (FL) GFP-GADD45� or one of its deletion derivatives as indicated.Flag-MTK1 was immunoprecipitated from cell extracts, and its kinase activity was assayed in vitro using bacterially produced GST-MKK6(K/A)as a specific substrate. An autoradiogram of SDS-PAGE was digitally obtained using a PhosphorImager (top panel). Relative activity, calculatedversus the activity of mock-stimulated (by GFP) MTK1, is shown below the top panel (Fold activation). The average of three independentexperiments is shown. Expression levels of Flag-MTK1 and GFP-GADD45� derivatives were monitored by immunoblotting (middle and bottompanels). (D) Binding of GADD45 to MTK1. Flag-MTK1 and either GADD45� or GADD45��53-62 were transiently expressed in COS-7 cells.Flag-MTK1 was immunoprecipitated from the cell extracts, and the coprecipitation of GADD45� or GADD45��53-62 was assayed by immu-noblot analysis (top panel). Expression levels of GADD45� and Flag-MTK1were monitored by immunoblotting (middle and bottom panels). Thestar indicates the expected position of GADD45��53-62. (E) Disruption of MTK1 N-C interaction by GADD45. Flag-MTK1-N, Flag-MTK1-N�BD, Myc-MTK1-C, GADD45�, and GADD45��53-62 were individually expressed in COS-7 cells. Cell lysates were prepared and mixed invitro, and Flag-MTK1 or Flag-MTK1-N�BD was immunoprecipitated. Coprecipitated Myc-MTK1-C was detected by immunoblotting (top panel).Lower panels show the levels of protein expression. �, anti; IB, immunoblot; IP, immunoprecipitate.

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(residues 1 to 12) or C terminus (residues 133 to 160) hadrelatively little effect on MTK1 activation. In contrast, theseries of 10-aa deletion mutants between residue 13 and resi-due 132 was nonactivating, indicating that this region ofGADD45� is necessary for MTK1 activation.

To determine whether the GADD45 mutants’ failure toactivate MTK1 reflected an inability to bind MTK1, bindingwas assessed by two-hybrid analysis (see Fig. S1 in the supple-mental material) and coprecipitation assays (Fig. 1D and datanot shown). Whereas the full-length GADD45� and the acti-vating GADD45� mutants could bind to MTK1, the nonacti-vating GADD45� mutants failed to do so. Neither the expres-sion levels of the GADD45� deletion mutants (Fig. 1C) northeir subcellular localizations (data not shown) differed signif-icantly from those of wild-type (WT) GADD45�. These resultsthus support our hypothesis that a direct binding of GADD45to MTK1 is necessary to activate the MTK1 kinase domain.

GADD45 disrupts the MTK1 N-C association. The deletionof the entire N-terminal regulatory segment of MTK1 consti-tutively activates its C-terminal kinase domain (33), implyingthat the N-terminal segment contains an autoinhibitory do-main (AID) that binds to and inhibits the kinase domain. If so,

a possible mechanism by which GADD45 might activateMTK1 is by disruption of the N-C interaction in an MTK1molecule. As a first step in testing this model, we examinedwhether the N-terminal and C-terminal segments of MTK1 canassociate stably with each other in an in vitro binding assay (seeMaterials and Methods for details). The results in Fig. 1E (lane2) clearly demonstrate that Flag-MTK1-N (residues 22 to 994)bound to Myc-MTK1-C (residues 1247 to 1607). The N-Cinteraction, however, was completely disrupted when anothercell lysate containing GADD45� was included in the incuba-tion mixture (Fig. 1E, lane 3). The addition of a lysate con-taining GADD45��(53-62) (lacking residues 53 through 62),which cannot bind to MTK1, did not inhibit the interaction(Fig. 1E, lane 4). Flag-MTK1-N�BD, which lacks theGADD45 binding domain (residues 147 to 250), could bindMyc-MTK1-C (Fig. 1E, lane 5), but the interaction was nolonger susceptible to interference by GADD45� (lane 6). Insummary, it is concluded that the binding of GADD45� to theN-terminal segment of MTK1 disrupts MTK1 N-C association.

Phosphorylation in the activation loop of the MTK1 kinasedomain. The activation of many protein kinases entails phos-phorylation at a specific residue(s) in the activation loop (T-

FIG. 2. Phosphorylation of specific amino acid residues in the MTK1 kinase activation loop. (A) Amino acid sequence of the activation loopof the MTK1 kinase. The black dot indicates the activating phosphorylation site. (B) Effects of mutations at potential phosphorylation sites in theMTK1 activation loop. Wild-type Flag-MTK1 (or MTK1 with an Ala substitution at the potential phosphorylation site) was coexpressed withGADD45� in COS-7 cells. Flag-MTK1 was immunoprecipitated from cell lysate and subjected to an in vitro kinase assay using GST-MKK6(K/A)as a substrate. Data were collected as described for Fig. 1C. Relative activity was calculated using the activity of the wild-type MTK1 as 100%. Theaverage of three independent experiments is shown below the top panel (% activity). (C) COS-7 cells were transfected with the WT Flag-MTK1expression construct or the T1493A phosphorylation site mutant, together with either a GADD45� expression plasmid (�) or the empty vectorpcDNA3 (). Immunoprecipitation was performed with an anti-Flag antibody, and the phosphorylation at Thr-1493 in MTK1 was probed byimmunoblot analysis using the antiserum �P-T1493. (D) COS-7 cells were transfected with the WT Flag-MTK1 expression construct or itskinase-dead derivative, K1371R (K/R), together with either a GADD45� expression plasmid (�) or an empty vector (). The phosphorylation atThr-1493 was probed as for panel C. (B through D) Expression levels of Flag-MTK1 and GADD45� were monitored by immunoblot analysis(middle and bottom panels). �, anti; IB, immunoblot; IP, immunoprecipitate.



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loop) between subdomains VII and VIII (21). There are fivepotential phosphorylation sites in the MTK1 activation loopregion (Ser-1484, Thr-1493, Ser-1500, Thr-1501, and Thr-1504)(Fig. 2A). To examine whether phosphorylation at any of theseresidues is required for MTK1 activation, we first individuallymutated each of these residues by substitution with Ala. TheseFlag-MTK1 Ala substitution mutants were coexpressed withGADD45�, and their kinase activities were measured in vitro(Fig. 2B). Kinase activities of MTK1-S1484A, S1500A, andT1501A were only moderately lower than that of wild-typeMTK1. In contrast, MTK1-T1504A was completely inactive,and MTK1-T1493A had only very weak activity.

To examine whether Thr-1493 and/or Thr-1504 is phosphor-ylated in vivo, we developed specific antisera to the MTK1T-loop peptides phosphorylated at Thr-1493 or at Thr-1504.To date, however, by using two different antisera, we haveobtained no evidence that Thr-1504 is phosphorylated. It ispossible that the mutation of Thr-1504 disturbs an essentialsecondary structure element unrelated to phosphorylation(45). In contrast, we could detect stimulation-dependent phos-phorylation at Thr-1493 using an anti-phospho-T1493 (�P-T1493) rabbit antibody. The �P-T1493 antibody reacted withMTK1 only when the latter was activated by GADD45� coex-pression; unstimulated MTK1 was unreactive (Fig. 2C, lanes 1and 2). Furthermore, the antibody did not react with theMTK1-T1493A mutant protein whether it was stimulated byGADD45� coexpression or not (Fig. 2C, lanes 4 and 3, respec-tively). These results indicate, on the one hand, that the �P-T1493 antibody is specific to phospho-T1493 and, on the otherhand, that the activation of MTK1 by GADD45� induces Thr-1493 phosphorylation. Occasionally, but not always, a substi-tution of Thr or Ser by an acidic amino acid (Asp or Glu)mimics the effect of phosphorylation. Thus, we tested the sub-stitution of Thr-1493 by Asp or Glu, but both T1493D andT1493E were inactive.

Next, we asked whether the phosphorylation of MTK1 atThr-1493 is affected by the autophosphorylation of MTK1 orby another kinase using the catalytically inactive pFlag-MTK1-K/R construct. As shown in Fig. 2D, no phosphorylation atThr-1493 was observed for MTK1-K/R under the same condi-tions in which wild-type MTK1 was strongly phosphorylated.This result indicates that Thr-1493 phosphorylation is depen-dent on MTK1 kinase activity. Because Thr-1493 phosphory-lation is also dependent on MTK1 homodimerization (see be-low), we conclude that the phosphorylation at Thr-1493 ismediated by the MTK1 kinase itself.

To test whether Thr-1493 phosphorylation plays a role in theactivation of MTK1 in response to stress, we analyzed Thr-1493 phosphorylation following the addition of MMS to thehuman embryonic kidney HEK293 cells. MMS is a well-knowncell stressor and a potent inducer of the GADD45 genes (34).Strong Thr-1493 phosphorylation was observed after 3 h ofexposure of the cells to MMS (Fig. 3A and see Fig. 8). Fur-thermore, this phosphorylation was dependent on MTK1 ki-nase activity because no Thr-1493 phosphorylation occurredwhen the kinase-defective Myc-MTK1-K/R mutant was usedinstead of wild-type Myc-MTK1. We also tested whether theendogenous MTK1 molecule is phosphorylated at Thr-1493when cells are stimulated by stress. This is a difficult experi-ment because the amount of the endogenous MTK1 molecules

is small, and the affinity of the available anti-MTK1 antibody isweak. Nonetheless, we could detect the MMS-induced phos-phorylation of Thr-1493 (Fig. 3B). Thus, the phosphorylationat Thr-1493 is a physiologically relevant reaction to externalstress stimuli.

Phosphorylation at Thr-1493 occurs in trans. We next de-termined whether MTK1 phosphorylates Thr-1493 by an in-tramolecular (cis) reaction or by an intermolecular (trans) re-action. We transfected COS-7 cells simultaneously with twodifferent MTK1 constructs, namely, Flag-tagged, wild-typeMTK1 and Myc-tagged, kinase-dead MTK1 (Myc-MTK1-K/R), together with pGADD45� or the empty vector (Fig. 4A,lanes 1 and 2). Myc-MTK1-K/R contains five repeats of theMyc tag and is easily distinguishable from Flag-MTK1 by SDSgel electrophoresis. If the Thr-1493 phosphorylation occursonly in cis, Myc-MTK1-K/R will not be phosphorylated, evenin the presence of the activated Flag-MTK1 protein. Thr-1493phosphorylation was detected, however, both in Myc-MTK1-K/R(Fig. 4A, lane 2) and in Flag-MTK1. When the tags werereversed and Myc-MTK1 and Flag-MTK1-K/R were used (Fig.4A, lanes 3 and 4), an essentially identical result was obtained.These data indicate that Thr-1493 phosphorylation is likely tobe an intermolecular (trans) reaction.

GADD45 enhances MTK1 oligomerization. We tested whetherMTK1 can form a stable homo-oligomer by coexpression ofMyc-MTK1 and Flag-MTK1. Flag-MTK1 was immunoprecipi-tated from cell lysates, and the presence of coprecipitatedMyc-MTK1 was probed by immunoblotting. As shown in Fig.4B, lane 2, Flag-MTK1 and Myc-MTK1 coprecipitated onlyvery weakly from unstimulated cells. However, when GADD45�/�/�was also expressed in the same cells, interaction between Myc-MTK1 and Flag-MTK1 was much enhanced (Fig. 4B, lanes 3

FIG. 3. Induction of MTK1 Thr-1493 phosphorylation by MMS.(A) HEK293 cells stably expressing either WT Myc-MTK1 or kinase-dead Myc-MTK1-K/R were stimulated with (�) or without () 100�g/ml MMS for 180 min, and cell lysates were prepared. The expressedMyc-MTK1 was affinity purified, and its phosphorylation at Thr-1493was probed using the antiserum �P-T1493. (B) HEK293 cells werestimulated with (�) or without () 100 �g/ml MMS for 180 min, andcell lysates were prepared. Endogenous MTK1 was immunoprecipi-tated by an affinity-purified anti-MTK1 antibody (or by a control poly-clonal anti-HA antibody), and its phosphorylation at Thr-1493 wasprobed as described for panel A. �, anti; Ab, antibody; IB, immuno-blot; IP, immunoprecipitate.



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to 5). GADD45��53-62, which cannot bind to MTK1 (Fig.1B), could not enhance the Myc-MTK1/Flag-MTK1 interac-tion (Fig. 4C, lane 4). These results prove that MTK1 oligo-merizes when stimulated by GADD45. Because full-lengthMTK1 is a very large molecule, it is difficult to measure theextent of oligomerization with any accuracy by gel filtration orother methods. Gel filtration analyses of a shorter fragmentthat contains the oligomerization domain (see below) wereconsistent with a dimer formation, but a trimer formationcould not be excluded (data not shown). With this caveat, weconclude that MTK1 dimerizes when stimulated by GADD45.

MTK1 contains a dimerization domain. GADD45 proteinsare known to form homo- and hetero-oligomeric complexes(23). In principle, therefore, MTK1 dimerization could be aconsequence of dimerization of the associated GADD45. Al-ternatively, a GADD45-induced conformational change mightunmask a latent dimerization domain in MTK1. To distinguishthese possibilities, we undertook the experiments describedbelow.

Initially, using a full-length Flag-MTK1 and a series of Myc-MTK1 truncation constructs, we mapped the region in MTK1that is responsible for dimerization. Full-length Myc-MTK1coprecipitated with full-length Flag-MTK1 in the presence ofGADD45� (see Fig. S2 [lane 2] in the supplemental material).Most notably, Myc-MTK1(853-1341), in which both the C-terminal kinase catalytic domain (amino acids 1342 to 1607)and the N-terminal 852 amino acids are absent, could also bindto full-length Flag-MTK1 in the presence of GADD45 (see

Fig. S2 [lane 10] in the supplemental material). Because Myc-MTK1(853-1341) lacks the entire GADD45-binding domain(amino acids 147 to 250), this result disproves the hypothesisthat MTK1 dimerization is an indirect consequence of theGADD45 dimerization.

Mapping of the dimerization domain in MTK1. The MTK1dimerization domain was further mapped using additional de-letion constructs. The progressive deletion of MTK1(853-1341) from its N-terminal side revealed that MTK1(941-1341)can bind full-length MTK1, but MTK1(953-1341) cannot, in-dicating that the sequence between amino acid residues 941and 953 is essential for dimerization (Fig. 5A, left panel). Thedeletion of MTK1(941-1341) from its C-terminal side showedthat the shortest segment that can bind full-length MTK1 isMTK1(941-1272) (Fig. 5A, right panel). Thus, we concludethat the minimal region required for MTK1 dimerization is theregion between amino acid residues 941 and 1272, although aslightly longer segment (residues 941 to 1321) binds full-lengthMTK1 more efficiently.

Because the dimerization assay in the above mapping wasperformed entirely in vitro, we verified that MTK1(941-1321)and full-length MTK1 interact in vivo, by coexpressing Myc-MTK1(941-1321) and full-length Flag-MTK1 in COS-7 cells ineither the presence or the absence of GADD45� (Fig. 5B). Inthis in vivo experiment, as in the in vitro experiments, Myc-MTK1(941-1321) and full-length Flag-MTK1 dimerized, butonly in the presence of GADD45�.

FIG. 4. trans phosphorylation of Thr-1493 and dimerization of MTK1 are induced by GADD45. (A) trans phosphorylation of MTK1 Thr-1493induced by GADD45�. COS-7 cells were cotransfected with expression vectors for Myc-MTK1-K/R and Flag-MTK1 (or for Myc-MTK1 andFlag-MTK1-K/R), together with pGADD45� (�) or the empty vector pcDNA3 (). Flag-MTK1 and Myc-MTK1 were simultaneously immuno-precipitated using both anti-Flag and anti-Myc antibodies. Phosphorylation of precipitated Flag-MTK1 and Myc-MTK1 at Thr-1493 was probedwith �P-T1493 antibody. The stars indicate the positions of kinase-dead MTK1 proteins. (B) Dimerization of MTK1 induced by GADD45.Flag-MTK1 and Myc-MTK1 were coexpressed in COS-7 cells, together with GADD45�, GADD45�, or GADD45�. Flag-MTK1 was immuno-precipitated from cell lysates, and coprecipitated Myc-MTK1 was probed by immunoblotting using an anti-Myc antibody (top panel). (C) GADD45binding is necessary for MTK1 dimerization. Flag-MTK1 and Myc-MTK1 were coexpressed in COS-7 cells, together with either GADD45�, orGADD45��53-62. Flag-MTK1 was immunoprecipitated from cell lysates, and the coprecipitated Myc-MTK1 was probed by immunoblotting usingan anti-Myc antibody (top panel). For panels B and C, the expression levels of Myc-MTK1, Flag-MTK1, and GADD45 proteins are shown in thelower portions. �, anti; IB, immunoblot; IP, immunoprecipitate.



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Coiled-coil motif in the dimerization domain. A databasesearch revealed no significant candidates that are structurallysimilar to the MTK1 dimerization domain (residues 941 to1272), except the corresponding regions in MTK1 orthologs ofvertebrates, insects, and nematodes. The COILS program (26),however, predicted that the short region of residues 982 to1012 may form a coiled-coil structure (Fig. 6A). A coiled-coilis made of two �-helices that wrap around each other to form

a twisted supercoil structure and is often involved in protein-protein assemblage. The MTK1 coiled-coil motif is conservedin the MTK1 orthologs of evolutionarily diverse organisms (seeFig. S3 in the supplemental material).

To examine whether the coiled-coil motif plays a role inMTK1 dimerization and/or activation, we constructed in vivocoimmunoprecipitation assays using four MTK1 mutants in theputative coiled-coil motif. The first mutant, MTK1-�CC, lacksthe entire coiled-coil motif (amino acids 982 to 1012). In theother three mutants, one or more positions (Leu-997, Ile-1001,and Val-1008) are substituted by a helix-disrupting proline; theL997P/I1001P double mutant is abbreviated as PP, and theL997P/I1001P/V1008P triple mutant is abbreviated as PPP.When the entire coiled-coil region was deleted (MTK1-�CC)or two critical amino acid residues were replaced by proline(MTK1-PP), very little interaction occurred between the Flag-and Myc-tagged constructs, even in the presence of GADD45�(Fig. 6B).

From these results, we conclude that the coiled-coil forma-tion is critical for GADD45-induced MTK1 dimerization. Wealso conclude that the dimerization domain in full-lengthMTK1 is usually masked and is uncovered only in the presenceof the GADD45 proteins.

MTK1 dimerization is required for Thr-1493 autophosphor-ylation. Both dimerization and Thr-1493 autophosphorylationof MTK1 are induced by the coexpression of GADD45, sug-gesting that these events might be causally related. More spe-cifically, we hypothesized that MTK1 dimerization is a prerequi-site for autophosphorylation at Thr-1493. To test this hypothesis,we examined whether dimerization-defective MTK1 mutants canautophosphorylate at Thr-1493. GADD45-stimulated phos-phorylation of Thr-1493 was undetectable in MTK1-�CC,compared to the robust Thr-1493 phosphorylation of the wild-type MTK1 molecule (Fig. 7A). PP and PPP substitution mu-tants are also completely defective in autophosphorylation,although the L997P single mutant could weakly autophosphor-ylate at Thr-1493 (Fig. 7B). Perhaps consistent with these find-

FIG. 5. Mapping of the dimerization domain in MTK1. (A) In vitro MTK1 dimerization assays. COS-7 cells were separately transfected withfull-length Flag-MTK1, one of Myc-MTK1 deletion mutants, or GADD45�. Cell lysates were prepared, and extracts containing full-lengthFlag-MTK1, GADD45�, and one of the Myc-MTK1 deletions were mixed in vitro and subjected to immunoprecipitation with an anti-Flag antibody(�Flag). The presence or absence of coprecipitating Myc-MTK1 deletion mutant proteins in the Flag-MTK1 immunoprecipitates was determinedby immunoblot analysis using an anti-Myc antibody (top panel). Stars indicate the expected positions of the proteins. (B) In vivo dimerization ofMTK1. COS-7 cells were cotransfected with expression plasmids for Myc-MTK1(941-1321) and full-length Flag-MTK1, together with anotherplasmid encoding GADD45� (�) or the empty vector pcDNA3 (). Flag-MTK1 was immunoprecipitated from cell lysates, and coprecipitatedMyc-MTK1(941-1321) was probed with an anti-Myc antibody (top panel). The expression level of each protein was monitored by immunoblotanalysis of the whole extracts (lower panels). �, anti; IB, immunoblot; IP, immunoprecipitate.

FIG. 6. MTK1 dimerizes through the coiled-coil motif. (A) Aminoacid sequence of the putative coiled-coil region (black bar) in MTK1and flanking sequences. The three amino acid positions in which pro-line substitution mutations were made are indicated by large blackdots. The �CC mutant lacks the entire coiled-coil region from residues982 to 1012. (B) The coiled-coil region is necessary for MTK1 dimer-ization. COS-7 cells were cotransfected with expression plasmids en-coding the wild-type or coiled-coil mutant versions of Myc-MTK1 andFlag-MTK1 as indicated, together with a GADD45� expression plas-mid (�) or the empty vector pcDNA3 (). Flag-MTK1 was immuno-precipitated from cell lysates, and coprecipitated Myc-MTK1 wasprobed with an anti-Myc antibody (top panel). �, anti; IB, immunoblot;IP, immunoprecipitate.



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ings, the L997P single mutant did not inhibit MTK1 dimeriza-tion to an appreciable degree (data not shown). Thus, Thr-1493autophosphorylation requires MTK1 dimerization.

Earlier, we suggested that Thr-1493 autophosphorylation isan intermolecular, or trans, reaction between a pair of MTK1molecules in a dimer. To further examine whether Thr-1493phosphorylation is an intermolecular reaction, we transfectedCOS-7 cells with two MTK1 constructs: a Myc-tagged,catalytically active, full-length molecule (Myc-MTK1) and aFlag-tagged, catalytically defective molecule, without or with amutation in the coiled-coil motif (Flag-MTK1-K/R or Flag-MTK1-PP-K/R, respectively). Because Flag-MTK1-K/R hasno catalytic activity, the only way its Thr-1493 can be phosphor-ylated by the coexpressed Myc-MTK1 is in trans. When Myc-MTK1 and Flag-MTK1-K/R were coexpressed, immunopre-cipitated Flag-MTK1-K/R was phosphorylated at Thr-1493when GADD45� was also expressed (Fig. 7C, lanes 1 and 2).However, when Myc-MTK1 and the dimerization-defectiveFlag-MTK1-PP-K/R construct were coexpressed, Flag-MTK1-PP-K/R was not phosphorylated at all by the coexpressed Myc-MTK1 kinase, even in the presence of GADD45� (Fig. 7C,lane 4). From these results, we conclude that the MTK1 dimer-ization is required for Thr-1493 phosphorylation in trans be-tween two MTK1 molecules.

MTK1 dimerization is required for the activation of theMTK1 kinase domain. So far, we have shown separately thatMTK1 dimerization is required for Thr-1493 phosphorylationand that Thr-1493 phosphorylation precedes the full activationof the MTK1 kinase. Thus, we can predict that dimerization isrequired for the full activation of the MTK1 kinase catalyticdomain. This prediction was tested using an in vitro kinase

assay (Fig. 7D). MTK1-L997P, which weakly autophosphory-lates in the presence of GADD45� (Fig. 7B), also had aweaker kinase activity than wild-type MTK1 did (Fig. 7D, lanes4 and 2, respectively). MTK1-PP and MTK1-PPP, which areboth completely defective in dimerization (Fig. 6B and datanot shown) and in autophosphorylation at Thr-1493 (Fig. 7B),are completely defective in kinase activity (Fig. 7D, lanes 6 and8, respectively). Thus, there is a very strong correlation be-tween dimerization, autophosphorylation, and catalytic activa-tion of MTK1.

Next, we tested whether the dimerization of MTK1 by itselfis sufficient to achieve kinase domain activation by usingFK506-binding protein 12 (FKBP12)-mediated dimerization ofMTK1. An FKBP12 domain can be induced to dimerize byadding the dimeric FK506 reagent AP20187 to the medium(31, 40). MTK1 phosphorylation at Thr-1493 could not beinduced by AP20187 at any concentration between 0 and 100nM (see Fig. S4 in the supplemental material) The coexpres-sion of GADD45� induced Thr-1493 phosphorylation ofFKBP-MTK1, showing that the fusion protein is capable ofautophosphorylation, but only after binding of GADD45.Thus, dimerization is necessary but not sufficient for MTK1activation. Perhaps the AP20187-induced dimer has a less op-timal orientation of the coiled-coil segments for activation.

To test whether GADD45 binding and MTK1 dimerizationplay roles in the phosphorylation of MTK1 Thr-1493 in re-sponse to stress, we analyzed Thr-1493 phosphorylation fol-lowing the addition of MMS to the cells (Fig. 3A). HEK293cells were stably transfected with a wild type, GADD45-bind-ing site deletion (�BD) mutant, or coiled-coil defective (PP)mutant version of a Myc-MTK1 construct. MTK1 phosphory-

FIG. 7. MTK1 dimerization is essential for Thr-1493 autophosphorylation and for MTK1 activation. (A and B) Dimerization-defective MTK1mutants cannot autophosphorylate. COS-7 cells were cotransfected with either wild-type or mutant Flag-MTK1, as indicated, together with aGADD45� expression vector (�) or the empty control vector (), and cell extracts were prepared 36 h after transfection. Immunoprecipitationwas performed with an anti-Flag antibody, and Thr-1493 phosphorylation of the precipitated Flag-MTK1 was analyzed by immunoblotting using�P-T1493 antibody (top panel). (C) MTK1 autophosphorylation is an intermolecular reaction. COS-7 cells were cotransfected with expressionvectors for Myc-MTK1 and either Flag-MTK1-K/R or Flag-MTK1-PP-K/R, together with a GADD45� expression vector (�) or the empty vectorpcDNA3 (). Immunoprecipitation was performed with an anti-Flag antibody, and phosphorylation of the precipitated Flag-MTK1-K/R (orFlag-MTK1-PP-K/R) was detected by immunoblot analysis using �P-T1493 antibody (top panel). (D) Dimerization-defective MTK1 mutantscannot be activated by GADD45. COS-7 cells were transfected with either an expression plasmid for Flag-MTK1 or one of its coiled-coil motifmutants, together with another plasmid encoding GADD45� (�) or the empty vector pcDNA3 (). Cell lysates were subjected to an in vitro kinaseassay using GST-MKK6(K/A) as a specific substrate. An autoradiogram of SDS-PAGE was digitally obtained using a PhosphorImager (top panel).Relative activity (Fold activation) was calculated versus the activity of unstimulated MTK1 (the leftmost sample). The average of two independentexperiments is shown below the top panel. For panels A through D, the expression level of each protein was monitored by immunoblotting, andshown in the lower portions. �, anti; IB, immunoblot; IP, immunoprecipitate.



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lation at Thr-1493 was monitored before and after the expo-sure of these cells to MMS (Fig. 8). Strong Thr-1493 phosphor-ylation of the wild-type MTK1 was observed after 3 h ofexposure to MMS. In contrast, Thr-1493 phosphorylation wascompletely absent in �BD and PP mutants, indicating that

both GADD45 binding and MTK1 dimerization are importantfor phosphorylation at Thr-1493 by extracellular stress stimuli.

Constitutively active MTK1 mutants revisited. Previously,we described constitutively active MTK1 mutants that can bindto and phosphorylate MKK6 in the absence of GADD45 (28).The mechanism of activation of these MTK1 mutants in theabsence of GADD45 binding should shed further light on howGADD45 activates the MTK1 kinase. Thus, we investigatedwhether these constitutively active mutations can mimic MTK1activation by GADD45 binding. Specifically, we tested threeconstitutively active mutants (L534Q, Q637P, and I1360M)that can phosphorylate the MKK6 substrate, both in vivo andin vitro, in the absence of GADD45 expression. The fourthmutant, V1300F, can bind but does not phosphorylate MKK6in the absence of GADD45.

First, we tested MTK1 Thr-1493 phosphorylation in thesemutants. As shown in Fig. 9A, MTK1-L534Q, MTK1-Q637P,and MTK1-I1360M were constitutively phosphorylated at Thr-1493 in the absence of any GADD45 protein (lanes 3, 4, and 6,respectively), whereas the wild-type MTK1 or MTK1-V1300Fwas not (lanes 1 and 5, respectively). Next, we tested whetherthese mutants were dimerized by coexpressing Myc-tagged andFlag-tagged versions of each mutant. The three constitutivelyactive mutants (L534Q, Q637P, and I1360M) were dimerized

FIG. 8. GADD45 binding and subsequent MTK1 dimerization isrequired for stress-induced MTK1 autophosphorylation. Human em-bryonic kidney HEK293 cells were stably transfected with expressionvectors for WT Myc-MTK1, GADD45 binding-defective MTK1-�BD,or dimerization-defective MTK1-PP. Transfected cells were treatedwithout () or with (�) 100 �g/ml MMS for 180 min before prepa-ration of cell extracts. Myc-MTK1 was affinity purified, and its phos-phorylation status at Thr-1493 was probed using the �P-T1493 anti-body (upper panel). The same filter was reprobed with anti-Mycantibody (bottom panel). �, anti; IB, immunoblot; IP, immunoprecipi-tate.

FIG. 9. Constitutively active MTK1 mutants. (A) Constitutively active MTK1 mutants are phosphorylated at Thr-1493 in the absence ofGADD45 binding. COS-7 cells were transfected with an expression plasmid for wild-type Myc-MTK1 or its constitutively active mutant versions,together with a second plasmid encoding GADD45� (�) or the control empty vector pcDNA3 (). Immunoprecipitation was carried out with ananti-Myc antibody, and the phosphorylation status of Thr-1493 was probed with the �P-T1493 antibody (top panel). (B) Constitutively activeMTK1 mutants are dimerized in the absence of GADD45 binding. Wild-type or mutant versions of Flag-MTK1 and Myc-MTK1 and GADD45�were coexpressed in COS-7 cells as indicated. Cell lysates were prepared and subjected to immunoprecipitation with an anti-Flag antibody. Thepresence of Myc-MTK1 in the Flag-MTK1 precipitates was probed by an anti-Myc antibody (top panel). (C) The dimerization domain is essentialfor GADD45-independent Thr-1493 autophosphorylation in a constitutively active MTK1 mutant. COS-7 cells were transfected with an expressionplasmid for either wild-type Myc-MTK1, constitutively active Myc-MTK1 L534Q, or the dimerization-defective Myc-MTK1 L534Q-PP, togetherwith a second plasmid encoding GADD45� (�) or the control pcDNA3 empty vector (). Immunoprecipitation was performed with an anti-Mycantibody, and Thr-1493 phosphorylation of the precipitates was detected by immunoblot analysis with the �P-T1493 antibody. (D) A constitutivelyactive MTK1 mutation disrupts the MTK1 N-C interaction independent of GADD45 binding. Flag-MTK1-N, Flag-MTK1-N-L534Q, Myc-MTK1-C, or GADD45� was separately expressed in COS-7 cells. Cell lysates were prepared and mixed in vitro as indicated. Flag-MTK1 orFlag-MTK1-N-L534Q was immunoprecipitated, and coprecipitated Myc-MTK1-C was detected by immunoblotting. For panels A through D,protein expression levels are shown in the lower portions. �, anti; IB, immunoblot; IP, immunoprecipitate.



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in the absence of GADD45 (Fig. 9B, lanes 3, 4, and 6), whereasMTK1-V1300F was dimerized only at the level of the wild-typeMTK1 protein without GADD45 (lane 5). Thr-1493 phosphor-ylation of the constitutively active mutants was dependent onthe MTK1 dimerization domain because the dimerization-de-fective PP mutation completely abolished Thr-1493 phosphor-ylation of the L534Q mutant (Fig. 9C). Lastly, we tested theeffects of constitutively active mutations on MTK1 N-C inter-action. As we showed earlier in this paper, N- and C-terminalsegments of MTK1 bind to each other and this association isdisrupted by GADD45 binding to the N-terminal fragment(Fig. 9D, lanes 2 and 3). The constitutively active mutationL534Q in the MTK1 N-terminal fragment inhibited the N-Cinteraction, and this inhibition does not require GADD45 (Fig.9D, lane 4). Thus, the constitutively active MTK1 mutationsmimic the effects of GADD45 binding, including the Thr-1493autophosphorylation, MTK1 dimerization, and disruption ofthe N-C interaction, suggesting that they are key residues inGADD45-mediated MTK1 activation.

DISCUSSION

The data from this study lead to the following model ofGADD45-mediated activation of the MTK1 MAPKKK. Theactivation of MTK1 by GADD45 occurs through a series ofmolecular steps I through VI, as summarized in Fig. 10. Inbrief, each step is as follows. For step I, in unstimulated cells,MTK1 is in a closed (inhibited) conformation in which theN-terminal AID blocks the C-terminal kinase catalytic domain.In step II, extracellular stimuli, such as MMS exposure, inducethe expression of stress-inducible GADD45 proteins, whichbind to the MTK1 N-terminal GADD45-BD. In step III,GADD45 binding to MTK1 dissociates the latter’s AID fromthe C-terminal kinase catalytic domain. In step IV, at the sametime, GADD45 binding unmasks the MTK1 dimerization do-main, inducing homodimer formation. At this stage, the MTK1kinase domain is in open conformation (i.e., not actively in-hibited) but not yet fully active as a kinase. In step V, dimer-ized MTK1 becomes fully activated when Thr-1493 is transautophosphorylated. In step VI, GADD45 binding also un-masks a site in the MTK1 kinase domain that interacts with theMAPKK DVD docking sites, allowing MTK1 to interact withand phosphorylate the cognate MAPKKs, namely, MKK3 andMKK6 (36).

Because the GADD45 proteins are highly conserved (34), itis likely that all three members of the GADD45 family activateMTK1 by the same mechanism. Indeed, in our investigation ofGADD45-mediated MTK1 activation, we did not find anyqualitative differences among the three GADD45 proteins. Wedid find, however, that there are quantitative differencesamong them; e.g., GADD45� does not stimulate MTK1 asefficiently as GADD45� or GADD45� does (34). It is possiblethat they have slightly different affinities for MTK1. In terms offunction, however, their varied expression patterns are perhapsmore important. The expression of each member of theGADD45 gene family is induced by a distinct set of environ-mental stresses and cytokines in different tissues. For example,the expression of GADD45�, but not GADD45� or -�, isinduced by TGF-� (35) and the expression of GADD45�, butnot GADD45� or -�, is modulated by p53 (25). Consistently,

the GADD45 proteins (GADD45�/�/�) serve overlapping butnonidentical functions in different apoptotic and growth inhib-itory pathways (25).

Full activation of MTK1 by GADD45 entails four differentmolecular mechanisms: removal of the autoinhibitory domain,dimerization, phosphorylation of the activation loop, and un-masking of the docking site for MAPKKs. Individually, thesemechanisms are used by other MAPKKKs. However, the de-tails are different for each MAPKKK, reflecting different phys-iological roles. Therefore, in order to appreciate its physiolog-ical function, it is important to analyze how the binding of oneprotein (GADD45) orchestrates these mechanisms, therebyconverting an inert enzyme (MTK1) to a fully active one.

The autoinhibition of the kinase catalytic domain by N-terminal (or sometimes C-terminal) regulatory sequences is amechanism employed by many MAPKKKs. Artificial deletionof such autoinhibitory domains converts the kinase into a con-stitutively active one (5, 6, 11, 43, 46). In physiologically rele-vant activation processes, however, release from autoinhibitionis effected by a different mechanism for each kinase. For ex-ample, MEKK1 is activated by caspase-3-mediated shedding ofits N-terminal inhibitory domain (41) and Ste11 is activated bySte20-mediated phosphorylation of the autoinhibitory domain(39). In the case of MTK1, the binding of GADD45 to MTK1seems to compete with the adjacent autoinhibitory domain forinteraction with the MTK1 catalytic domain.

Dimerization is also frequently utilized to activate proteinkinases, including MAPKKKs. For example, it has been re-ported that forced dimerization of full-length MEKK1 inducesits autophosphorylation and the subsequent phosphorylationof the MKK4 (SEK1) MAPKK (7). It was also reported re-cently that the dimerization of the MEKK4 (MTK1) kinasedomain (without the N-terminal autoinhibitory domain) byonly AP20187 increases MEKK4 kinase activity (1). Unlikethese cases, however, artificial dimerization of full-lengthMTK1 using FKBP and AP20187 did not enhance its in vitrocatalytic activity and did not lead to Thr-1493 phosphorylationin vivo. Therefore, it is likely that dimerization is necessary butnot sufficient for the activation of full-length MTK1. Perhaps it

FIG. 10. Schematic model of MTK1 activation. The activation ofMTK1 by GADD45 can be dissected into several stages, as indicatedby the roman numerals in the scheme (steps I through VI). See theDiscussion for details.



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is important for MTK1 activation that GADD45 dissociatesthe N-terminal autoinhibitory domain from the C-terminal cat-alytic domain. The specific configuration of the coiled-coil-mediated dimerization might also be important.

Since GADD45 proteins can themselves form homo- and het-erodimers (or oligomers) (23), one possibility was that MTK1indirectly dimerized through the dimerization of bound GADD45molecules. This is unlikely, however, for two reasons. First, MTK1fragments that do not contain the GADD45-BD can bind MTK1.Second, constitutively active MTK1 mutants dimerize in the ab-sence of GADD45 proteins (Fig. 9B). Nonetheless, it is possiblethat GADD45 dimerization might serve to concentrate MTK1molecules, hence enhancing the latter’s dimerization. Finally, itshould be noted that MTK1 is dimerized by other stimuli, such asTRAF4, presumably via a GADD45-independent mechanism (1).

The phosphorylation of the activation loop is perhaps themost common activation mechanism for diverse families ofprotein kinases (4, 15, 19, 42). However, different kinasesmight use different mechanisms to achieve phosphorylation ofthe activation loop. Many kinases are phosphorylated by theirupstream activating kinase in a manner similar to that for thephosphorylation of MAPKKs by MAPKKKs or the phosphor-ylation of MAPKs by MAPKKs. Other kinases autophosphor-ylate the activation loop (12, 22). Thr-1493 phosphorylation ofMTK1 is induced by the expression of the GADD45 proteinsor by extracellular stimuli, such as MMS, that are known toinduce GADD45 expression. Dimerization-defective MTK1mutants, however, cannot autophosphorylate Thr-1493. Thus,stable dimerization induced by GADD45 binding is responsi-ble for efficient intermolecular Thr-1493 phosphorylation ofMTK1.

Finally, the GADD45 proteins indirectly control the interac-tion between activated MTK1 and its substrates, i.e., MKK3 andMKK6. Both MKK3 and MKK6 contain an �20-amino-acid pep-tide, termed the DVD domain, at their C termini (36). Thedeletion and point mutations of the MKK3/MKK6 DVD se-quence inhibit stable interaction between MTK1 and MKK3/MKK6 and, consequently, inhibit the activation of MKK3/MKK6by MTK1. The DVD-mediated interaction between MKK6 andfull-length MTK1 does not take place unless GADD45 is alsopresent (28). It is likely that the MTK1 N-terminal autoinhibitorydomain prevents the MTK1 catalytic domain from binding theMKK3/MKK6 DVD docking site.

With the detailed activation mechanism of MTK1 now avail-able, it will be possible to study this important but underex-plored area of cellular signal transduction.

ACKNOWLEDGMENTS

We thank P. O’Grady for critical reading of the manuscript, M.Kitamura and N. Yoshida for excellent technical assistance, andARIAD Pharmaceuticals, Inc., for the gift of the ARGENT regulatedhomodimerization kit and the ARGENT transcription retrovirus kit.

This work was supported in part by several Grants-in-Aid from theMinistry of Education, Culture, Sports, Science and Technology ofJapan (to H.S. and M.T.) and a PRESTO program from the JapanScience and Technology Agency (to M.T.).

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domain autophosphorylation of the mtk1 kinase trans dimerization

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