miyake et al. 1 1 activation of mtk1/mekk4 by gadd45 through
TRANSCRIPT
Miyake et al.
1
1
Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and 2
dimerization-mediated trans-autophosphorylation of the MTK1 kinase domain 3
4
Zenshi Miyake1, 2, 3
, Mutsuhiro Takekawa1, 2, 3
, Qingyuan Ge4, and Haruo Saito
1, 2, * 5
6
1Division of Molecular Cell Signaling, 7
Institute of Medical Sciences, University of Tokyo, 8
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan 9
2Department of Biophysics and Biochemistry, 10
Graduate School of Science, University of Tokyo, 11
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 12
4Cell Signaling Technology, Inc., 13
3 Trask Lane, Danvers, MA 01923, USA 14
15
Running title: Activation of the MTK1 MAPKKK by GADD45 16
17
Word count for Materials and Methods: 801 18
Combined word count: 5035 19
20
3These authors contributed equally to this work. 21
*Corresponding author 22
Institute of Medical Sciences, The University of Tokyo, 23
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan 24
E-mail: [email protected] 25
Phone: +1-81-3-5449-5505; FAX: +1-81-3-5449-5701 26
ACCEPTED
Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.01435-06 MCB Accepts, published online ahead of print on 22 January 2007
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Abstract 1
2
The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK 3
kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is 4
conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses 5
activate two major subfamilies of MAPKs, namely the Jun N-terminal kinases (JNKs) and the 6
p38/SAPK kinases. MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to 7
and activated by the stress-inducible GADD45 family proteins (GADD45α/β/γ). Here, we 8
dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 9
N-terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. 10
This N-C interaction was disrupted by binding of GADD45 to the MTK1 N-terminal 11
GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization 12
domain containing a coiled-coil motif, which is essential for trans-autophosphorylation of MTK1 13
at Thr-1493 in the kinase activation loop. An MTK1 alanine-substitution mutant at Thr-1493 has a 14
severely reduced activity. Thus, we conclude that GADD45-binding induces MTK1 N-C 15
dissociation, dimerization, and autophosphorylation at Thr-1493, leading to activation of the 16
kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the 17
absence of GADD45. 18 ACCEPTED
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Introduction 1
2
Living organisms are frequently exposed to cellular stresses, which are defined as 3
diverse environmental conditions that are detrimental to the normal growth and survival of the cells. 4
Typical cellular stresses include ultraviolet (UV), ionizing radiation (IR), genotoxins, 5
hyperosmolarity, oxidative stress, low oxygen supply (hypoxia), and inhibition of protein synthesis 6
by antibiotics and plant toxins. In coping with the barrage of these and other cellular stresses, 7
multi-cellular eukaryotic organisms have developed a strategy as to how damaged cells will 8
respond to stresses. In general, if the intensity of damage is moderate, the affected cell will seek to 9
repair the damage. If, however, the damage to a cell is too severe for a complete repair, the affected 10
cells are eliminated by apoptosis. This reduces the risk to the organism as a whole, such as 11
development of a cancer. Such a crucial decision-making between repair or death is, at least in part, 12
mediated by the Stress-activated MAP kinase (SAPK) pathways (for general reviews on MAPK 13
and SAPK, see (8, 20, 24, 29, 30)). 14
As the name implies, the SAPK pathways are homologous to and share many 15
characteristics with the classic (ERK1/2) MAPK pathway. Eukaryotic MAPK pathways are 16
conserved signaling modules that serve to transmit signals from the cell surface to the nucleus. The 17
core of any MAPK pathway is composed of three tiers of sequentially activating protein kinases, 18
namely, MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK. Activation of 19
MAPKs is achieved by phosphorylation of a threonine and a tyrosine residues within a conserved 20
Thr-Xaa-Tyr motif in the activation loop (also called the T-loop) catalyzed by MAPKKs. 21
MAPKKs, in turn, are activated by any of several MAPKKKs, via phosphorylation of serine and/or 22
threonine residues within their activation loop. 23
All eukaryotic cells possess multiple MAPK pathways, each of which is activated by 24
distinct sets of stimuli. In the budding yeast Saccharomyces cerevisiae, for example, hyperosmotic 25
stress activates the Hog1 MAPK pathway, whereas mating pheromones activate the Fus3/Kss1 26
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MAPK pathway (13, 18). In mammalian cells, four different subfamilies of MAPKs are present, 1
namely, ERK1/2, JNK1/2/3, p38α/β/γ/δ, and ERK5. The ERK1/2 MAPK pathway is 2
preferentially activated in response to mitogenic stimuli, such as growth factors and phorbol esters, 3
and plays a role in cell growth and cell survival. The ERK1/2 pathway is mainly regulated by the 4
monomeric GTPase Ras, which recruits MAPKKKs of the Raf family to activate the two 5
downstream MAPKKs: MEK1/2. These MAPKKs, in turn, activate the ERK 1/2 MAPKs. The 6
JNK and p38 MAPKs (collectively called SAPKs), in contrast, preferentially respond to various 7
cellular stresses, and are thus called SAPK pathways. Besides cell stresses, the SAPK pathways 8
are also activated by cytokines such as IL-1, TNFα, and TGF-β. The JNK subfamily of MAPKs 9
are activated mainly by the MKK4 and MKK7 MAPKKs, while the p38 subfamily MAPKs are 10
activated by the MKK3 and MKK6 MAPKKs. In clear contrast to this limited number of 11
MAPKKs in the SAPK pathways, there are numerous MAPKKKs that function upstream of the 12
JNK and p38 MAPKs. These include MEKK1/2/3, MTK1 (also known as MEKK4), TAK1, 13
ASK1/2, TAO1/2/3, MLKs, and perhaps others. This multiplicity at the level of MAPKKK surely 14
reflects the vastly diverse stress stimuli that can recruit these SAPK pathways. 15
MTK1 is one of the human MAPKKKs belonging to the SAPK pathways, and the mouse 16
ortholog is called MEKK4 (16, 33). The kinase domain of MTK1 (MEKK4) is homologous to 17
other MAPKKKs, especially similar to mammalian MEKK1/2/3 and ASK1/2 and yeast 18
SSK2/SSK22, but its N-terminal non-catalytic domain (regulatory domain) is unique (33). 19
Analyses using MEKK4-deficient mice have shown that the MEKK4 signaling pathway integrates 20
signals from both the T cell antigen receptor and IL-12/STAT4 in developing Th1 cells, and 21
promotes STAT4-independent IFNγ production (9), and that MEKK4 is essential for normal neural 22
and skeletal development (2, 10). 23
In a yeast two-hybrid screening aimed to identify MTK1 activator(s), we found three 24
Growth-Arrest and DNA Damage-inducible 45 (GADD45) family proteins to be strong binding 25
partners of MTK1 (34). The GADD45 gene was originally identified as a UV-inducible gene in 26
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Chinese hamster cells (14). The human genome encodes three GADD45-like proteins, GADD45α 1
(the original GADD45), GADD45β (MyD118), and GADD45γ (CR6 or OIG37) (25, 44). These 2
will be referred to collectively as the GADD45 proteins. The GADD45 proteins share 55 to 58 % 3
sequence identity. The three GADD45 genes are all inducible by cellular stresses, although 4
optimal stimuli for each gene appear to be different (25). The expression profiles of the three 5
GADD45 genes are also distinct in various tissues (34). The GADD45 proteins interact with 6
various intracellular molecules, such as proliferating cell nuclear antigen (PCNA), Cdc2-CyclinB1 7
complex, p21Waf1/Cip1
, and core histones, and play important roles in stress-adaptive processes 8
including growth control, maintenance of genomic stability, DNA repair, and apoptosis (3, 17, 25, 9
38). In other words, the GADD45 proteins are emergency calls in damaged cells. 10
Expression of transfected GADD45 genes strongly activates co-expressed MTK1 kinase 11
and induces apoptosis in mammalian cells (34). TGF-β-induced GADD45β expression also 12
activates p38 MAPK through MTK1 activation (35). MEKK4-deficient mice have lost 13
GADD45-induced IFNγ production (9). Activation of the SAPK pathway by MTK1 and GADD45 14
is temporally a slow process, because it requires induction of GADD45 gene expression prior to 15
activation of MTK1. Thus, the activation of MTK1 by GADD45 differs from other modes of 16
SAPK activation that occur within minutes. GADD45/MTK1 mediated SAPK activation may 17
therefore serve as a more long-term adaptive mechanism for stressed cells. 18
We previously proposed that binding of GADD45 to the N-terminal region of MTK1 19
counters the autoinhibitory effect of the MTK1 N-terminal segment on the kinase domain, and at 20
the same time enables the MTK1 kinase domain to bind its cognate MAPKKs (MKK3 and MKK6) 21
via the latter’s DVD docking sites (28, 36). The details of MTK1 activation by GADD45, however, 22
remained obscure. In this report, we investigated the molecular mechanism by which GADD45 23
regulates MTK1 kinase activity. 24
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Materials and Methods 1
2
Media and buffers 3
Lysis buffer contains 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 4
mM EDTA, 50 mM β-glycerophosphate, 10 mM NaF, 1 mM sodium vanadate, 1 mM dithiothreitol, 5
1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Kinase buffer contains 25 mM Tris-HCl 6
(pH 7.5), 20 mM MgCl2, 0.5 mM sodium vanadate, 25 mM β-glycerophosphate, 2 mM 7
dithiothreitol, 1 mM EGTA. SDS loading buffer is 65 mM Tris-HCl (pH 6.8), 5% (v/v) 8
2-mercaptoethanol, 2% SDS, 0.1% Bromophenol Blue, and 10% glycerol. Kinase buffer contains 9
25 mM Tris-HCl (pH 7.5), 25 mM MgCl2, 0.5 mM sodium vanadate, 25 mM β-glycerophosphate, 10
2 mM dithiothreitol, and 2 mM EGTA. 11
12
Plasmids 13
The mammalian expression plasmids pFlag-MTK1, pFlag-MTK1-K/R, pcDNA3-GADD45β, 14
pFlag-MTK1(L534Q), pFlag-MTK1(Q637P), pFlag-MTK1(V1300F), pFlag-MTK1(I1360M), 15
and pGST-MKK6(K/A) were described previously (28, 32, 34). GADD45β deletion mutants and 16
MTK1 mutants were generated by PCR mutagenesis. pEGFP-C1 (Clontech), pcDNA4Myc, and 17
pcDNA3 vectors were used to generate GFP-tagged GADD45β, Myc-tagged MTK1, and 18
GADD45α/GADD45γ expression plasmids, respectively. pcDNA3FKBP vector was used to 19
generate FKBP-fused, HA-tagged MTK1 expression plasmids. MTK1-N contains Met-22 through 20
Ala-994, MTK1-C contains Ser-1247 through Glu-1607 (the C-terminal amino acid), and 21
MTK1-N∆BD contains Ser-253 through Ala-994. 22
23
Expression and purification of epitope-tagged proteins 24
GST-MKK6 (K/A) was expressed in E. coli DH5 and purified using glutathione-Sepharose beads 25
as described (33). 26
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1
Tissue culture and transient transfection 2
COS-7 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 3
10 % fetal bovine serum, L-glutamate, penicillin, and streptomycin. For transient transfection 4
assays, cells grown in 60 mm dishes were transfected with the appropriate expression plasmids 5
using the Effectene Transfection Reagent (Qiagen). The total amount of plasmid DNA was 6
adjusted to 1µg per plate with vector DNA (pcDNA3). 7
8
Immunoblotting analyses 9
Immunoblotting analyses were carried out as described previously (36). The following antibodies 10
were used: anti-Flag mAb M2 (Sigma); anti-HA mAb 3F10 (Roche); anti-GFP mAb B-2 (Santa 11
Cruz); polyclonal anti-Myc (Santa Cruz); anti-GADD45α mAb 4T-27 (Santa Cruz); goat 12
polyclonal antisera to GADD45β and GADD45γ (Santa Cruz). An anti-MTK1 antiserum has been 13
described (9). An anti-phospho-T1493 rabbit antiserum (αP-T1493, lot #A2340PE) was made 14
in-house. 15
16
In vitro binding assay for MTK1-N and C segments 17
Two plasmids that encode, respectively, Flag-tagged MTK1 N-terminal segment (residues 22-994) 18
and a Myc-tagged MTK1 C-terminal segment (residues 1247-1607) were constructed (see Figure 19
1A). Flag-MTK1-N and Myc-MTK1-C were individually expressed in COS-7 cells, and cell 20
lysates were prepared 24 h after transfection. The lysates were mixed in vitro and incubated for 4 h 21
at 4°C. Flag-MTK1-N was precipitated from the mixture by an anti-Flag antibody conjugated to 22
Protein G-Sepharose, and co-precipitated Myc-MTK1-C was detected by immunoblotting using an 23
anti-Myc antibody. 24
25
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Detection of MTK1 Thr-1493 phosphorylation 1
Cell lysates, prepared in Lysis buffer with 0.5% deoxycholate, were incubated with an appropriate 2
antibody for 2 h at 4°C to precipitate epitope-tagged MTK1. Endogenous MTK1 was precipitated 3
using anti-MTK1 antibody. Immune-complexes were recovered with the aid of Protein 4
G-Sepharose beads, washed three times with Lysis buffer containing 500 mM NaCl and 0.5 % 5
deoxycholate, twice with Lysis buffer only, resuspended in SDS Loading buffer, and separated by 6
SDS-PAGE for immunoblotting analyses using αP-T1493. 7
8
Coimmunoprecipitation assay for protein binding 9
Cell lysates were incubated with Protein G-Sepharose beads at 4 °C for about 20 hr. Then, 10
precleared lysates were incubated with anti-Flag mAb M2 bound to Protein G-Sepharose beads at 11
4°C for 4 hr with gentle rotation. Immunoprecipitates were collected by centrifugation, washed six 12
times with Lysis buffer containing 500 mM NaCl and 0.5 % deoxycholate, and subjected to 13
SDS-PAGE. 14
15
In vitro kinase assay 16
Transfected cells were lysed in Lysis buffer. Cell lysates were incubated with the appropriate 17
antibody for 2 h at 4°C. Immune-complexes were recovered with the aid of protein G-Sepharose 18
beads, washed twice with Lysis buffer containing 500 mM NaCl and 0.5 % deoxycholate, twice 19
with Lysis buffer, and twice again with kinase buffer. Immunoprecipitates were resuspended in 30 20
µl of kinase buffer containing 4 µg of GST-MKK6 (K/A). The kinase reaction was initiated by the 21
addition of 6 µl of [γ-32
P]ATP (3.3 µCi/µl, 20 µM ATP). Following a 30 min incubation at 30°C, 22
reactions were terminated by the addition of SDS loading buffer. Samples were boiled, separated 23
by SDS-PAGE, dried and visualized by autoradiography. The amounts of phospho-GST-MKK6 24
(K/A) were quantified using the imaging analyzer FLA-3000. 25
26
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Two-hybrid analysis 1
Yeast two-hybrid analysis was performed using the yeast L40 strain, as described previously (37). 2
β-galactosidase activity is expressed as Miller units (27). 3
4
Regulated protein homo-dimerization using AP20187 5
The ARGENT Regulated Homodimerization Kit [v2.0] and the ARGENT Regulated Transcription 6
Retrovirus Kit [v2.0] were obtained from ARIAD Pharmaceuticals, Inc. 7
(www/ariad.com/regulationkits). COS-7 cells were transfected with pFKBP-HA-MTK1, and 36 8
hr later, the medium was replaced with a fresh medium containing the appropriate amount of 9
AP20187, and the cells were incubated for 2 hr at 37°C prior to lysis and assay. 10
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Results 1
2
GADD45 binds to and activates MTK1 3
Previously, we have shown that the GADD45 family proteins bind to a 4
GADD45-binding domain (BD; see Figure 1A) in the N-terminal regulatory region of MTK1 (34). 5
An MTK1 mutant protein that lacks the GADD45 BD cannot be activated by GADD45 proteins. 6
In the following work, we used GADD45β as a representative member of the GADD45 family, 7
because GADD45β is most strongly induced by MMS stress and by TGF-β. Whenever we 8
compared the three GADD45 proteins, however, there were no qualitative differences among them. 9
GADD45β is a small protein of 160 amino acid residues (Figure 1B). To determine the 10
region of GADD45β proteins that is directly involved in MTK1 activation, we constructed a series 11
of deletion mutants, each of which has an ~10-amino-acid deletion. COS-7 cells were 12
co-transfected with a plasmid encoding GFP-tagged GADD45β (GFP-GADD45β), or one of its 13
deletion derivatives, and another plasmid encoding Flag-tagged full-length MTK1 (Flag-MTK1). 14
After transfection for 36 hr, kinase activity of Flag-MTK1 was assayed in vitro. Co-expression of 15
the full-length (FL) GADD45β increased Flag-MTK1 kinase activity more than 10-fold (Figure 16
1C). Deletions at the N-terminus (residues 1-12) or the C-terminus (residues 133-160) had 17
relatively little effect on MTK1 activation. In contrast, the series of 10-aa deletion mutants 18
between residue 13 and residue 132 were non-activating, indicating that this region of GADD45β 19
is necessary for MTK1 activation. 20
To determine if the GADD45 mutants’ failure to activate MTK1 reflected an inability to 21
bind MTK1, binding was assessed by 2-hybrid analysis (Supplementary Figure S1) and by 22
co-precipitation assays (Figure 1D, and data not shown). Whereas the full-length GADD45β, and 23
the activating GADD45β mutants could bind to MTK1, the non-activating GADD45β mutants 24
failed to do so. Neither the expression levels of the GADD45β deletion mutants (Figure 1C), nor 25
their subcellular localizations (data not shown), differed significantly from those of the wild-type 26
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GADD45β. These results thus support our hypothesis that a direct binding of GADD45 to MTK1 1
is necessary to activate the MTK1 kinase domain. 2
3
GADD45 disrupts the MTK1 N-C association 4
Deletion of the entire N-terminal regulatory segment of MTK1 constitutively activates 5
its C-terminal kinase domain (33), implying that the N-terminal segment contains an autoinhibitory 6
domain (AID) that binds to and inhibits the kinase domain. If so, a possible mechanism by which 7
GADD45 might activate MTK1 is by disruption of the N-C interaction in an MTK1 molecule. As 8
a first step in testing this model, we examined if the N-terminal and C-terminal segments of MTK1 9
can associate stably with each other, in an in vitro binding assay (see Materials and Methods for 10
details). The results in Figure 1E (lane 2) clearly demonstrate that Flag-MTK1-N (residues 11
22-994) bound to Myc-MTK1-C (residues 1247-1607). The N-C interaction, however, was 12
completely disrupted when another cell lysate containing GADD45β was included into the 13
incubation mixture (lane 3). The addition of a lysate containing GADD45β∆(53-62), which cannot 14
bind to MTK1, did not inhibit the interaction (lane 4). Flag-MTK1-N∆BD, which lacks the 15
GADD45 binding domain (residues 147-250), could bind Myc-MTK1-C (lane 5), but the 16
interaction was no longer susceptible to interference by GADD45β (lane 6). In summary, it is 17
concluded that binding of GADD45β to the N-terminal segment of MTK1 disrupts MTK1 N-C 18
association. 19
20
Phosphorylation in the activation loop of the MTK1 kinase domain 21
Activation of many protein kinases entails phosphorylation at (a) specific residue(s) in 22
the activation loop (T-loop) between subdomains VII and VIII (21). There are five potential 23
phosphorylation sites in the MTK1 activation loop region (Ser-1484, Thr-1493, Ser-1500, 24
Thr-1501, and Thr-1504; see Figure 2A). To examine whether phosphorylation at any of these 25
residues is required for MTK1 activation, we first individually mutated each of these residues by 26
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substitution with Ala. These Flag-MTK1 Ala substitution mutants were co-expressed with 1
GADD45β, and their kinase activities were measured in vitro (Figure 2B). Kinase activity of 2
MTK1-S1484A, S1500A, and T1501A, was only moderately lower than that of the wild-type 3
MTK1. In contrast, MTK1-T1504A was completely inactive, and MTK1-T1493A had only very 4
weak activity. 5
To examine whether Thr-1493 and/or Thr-1504 is phosphorylated in vivo, we developed 6
specific antisera to the MTK1 T-loop peptides phosphorylated at Thr-1493 or at Thr-1504. To date, 7
however, using two different antisera we have obtained no evidence that Thr-1504 is 8
phosphorylated. It is possible that mutation of Thr-1504 disturbs an essential secondary structure 9
element unrelated to phosphorylation (45). In contrast, we could detect stimulation-dependent 10
phosphorylation at Thr-1493 using an anti-phospho-T1493 (αP-T1493) rabbit antibody. The 11
αP-T1493 antibody reacted with MTK1 only when the latter was activated by GADD45β 12
co-expression; unstimulated MTK1 was un-reactive (Figure 2C; lanes 1 and 2). Furthermore, the 13
antibody did not react with the MTK1-T1493A mutant protein, whether it was stimulated or not by 14
GADD45β co-expression (Figure 2C; lanes 4 and 3, respectively). These results indicate, on the 15
one hand, that the α-pT1493 antibody is specific to phospho-T1493, and, on the other hand, that 16
activation of MTK1 by GADD45β induces Thr-1493 phosphorylation. Occasionally, but not 17
always, a substitution of Thr or Ser by an acidic amino acid (Asp or Glu) mimics the effect of 18
phosphorylation. Thus, we tested the substitution of Thr-1493 by Asp or Glu, but both T1493D 19
and T1493E were inactive. 20
Next, we asked whether the phosphorylation of MTK1 at Thr-1493 is effected by 21
autophosphorylation of MTK1, or by another kinase, using the catalytically inactive 22
pFlag-MTK1-K/R construct. As shown in Figure 2D, no phosphorylation at Thr-1493 was 23
observed for MTK1-K/R, under the same conditions in which wild-type MTK1 was strongly 24
phosphorylated. This result indicates that Thr-1493 phosphorylation is dependent on MTK1 25
kinase activity. Because Thr-1493 phosphorylation is also dependent on MTK1 26
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homo-dimerization (see below), we conclude that the phosphorylation at Thr-1493 is mediated by 1
the MTK1 kinase itself. 2
To test if Thr-1493 phosphorylation plays a role in the activation of MTK1 in response to 3
stress, we analyzed Thr-1493 phosphorylation following addition of MMS to the human 4
embryonic kidney HEK293 cells. MMS is a well-known cell stressor and a potent inducer of the 5
GADD45 genes (34). Strong Thr-1493 phosphorylation was observed after 3 hr exposure of the 6
cells to MMS (Figures 3A and 8). Furthermore, this phosphorylation was dependent on MTK1 7
kinase activity, because no Thr-1493 phosphorylation occurred when the kinase-defective 8
Myc-MTK1-K/R mutant was used instead of the wild-type Myc-MTK1. We also tested if the 9
endogenous MTK1 molecule is phosphorylated at Thr-1493 when cells are stimulated by a stress. 10
This is a difficult experiment because the amount of the endogenous MTK1 molecules is low and 11
the affinity of the available anti-MTK1 antibody is weak. Nonetheless, we could detect the 12
MMS-induced phosphorylation of Thr-1493 (Figure 3B). Thus, the phosphorylation at Thr-1493 is 13
a physiologically relevant reaction to external stress stimuli. 14
15
Phosphorylation at Thr-1493 occurs in trans 16
We next determined whether MTK1 phosphorylates Thr-1493 by an intramolecular (cis) 17
reaction, or by an intermolecular (trans) reaction. We transfected COS-7 cells simultaneously with 18
two different MTK1 constructs, namely Flag-tagged wild-type MTK1 (Flag-MTK1) and 19
Myc-tagged kinase-dead MTK1 (Myc-MTK1(K/R)), together with pGADD45β or the empty 20
vector (Figure 4A, lanes 1 and 2). Myc-MTK1(K/R) contains 5 repeats of the Myc-tag, and is 21
easily distinguishable from Flag-MTK1 by SDS gel. If the Thr-1493 phosphorylation occurs only 22
in cis, Myc-MTK1(K/R) will not be phosphorylated even in the presence of the activated 23
Flag-MTK1 protein. Thr-1493 phosphorylation was detected, however, both in Myc-MTK1(K/R) 24
(Figure 4A, lane 2, asterisk) and in Flag-MTK1. When the tags were reversed, and Myc-MTK1 25
and Flag-MTK1(K/R) were used (Figure 4A, lanes 3 and 4), an essentially identical result was 26
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obtained. These data indicate that Thr-1493 phosphorylation is likely to be an intermolecular 1
(trans) reaction. 2
3
GADD45 enhances MTK1 oligomerization 4
We tested if MTK1 can form a stable homo-oligomer, by co-expression of Myc-MTK1 5
and Flag-MTK1. Flag-MTK1 was immunoprecipitated from cell lysates, and the presence of 6
co-precipitated Myc-MTK1 was probed by immunoblotting. As shown in Figure 4B, lane 2, 7
Flag-MTK1 and Myc-MTK1 co-precipitated only very weakly from unstimulated cells. However, 8
when either GADD45α, β, or γ was also expressed in the same cells, interaction between 9
Myc-MTK1 and Flag-MTK1 was much enhanced (Figure 4B, lanes 3 – 5). GADD45β∆53-62, 10
which cannot bind to MTK1 (see Figure 1B), could not enhance Myc-MTK1/Flag-MTK1 11
interaction (Figure 4C, lane 4). These results prove that MTK1 oligomerizes when stimulated by 12
GADD45. Because full-length MTK1 is a very large molecule, it is difficult to measure the extent 13
of oligomerization with any accuracy by gel filtration or other methods. Gel filtration analyses of a 14
shorter fragment that contains the oligomerization domain (see below) were consistent with a 15
dimer formation, but a trimer formation could not be excluded (data not shown). With this caveat, 16
we conclude that MTK1 dimerizes when stimulated by GADD45. 17
18
MTK1 contains a dimerization domain 19
GADD45 proteins are known to form homo- and hetero-oligomeric complexes (23). In 20
principle, therefore, MTK1 dimerization could be a consequence of dimerization of the associated 21
GADD45. Alternatively, a GADD45-induced conformational change might unmask a latent 22
dimerization domain in MTK1. To distinguish these possibilities, we undertook the following 23
experiments. 24
Initially, we mapped the region in MTK1 that is responsible for dimerization, using a 25
full-length Flag-MTK1 and a series of Myc-MTK1 truncation constructs. Full-length Myc-MTK1 26
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co-precipitated with full-length Flag-MTK1, in the presence of GADD45β (Supplementary Figure 1
S2, lane 2). Most notably, Myc-MTK1(853-1341), in which both the C-terminal kinase catalytic 2
domain (1342-1607) and the N-terminal 852 amino acids are absent, could also bind to full-length 3
Flag-MTK1, in the presence of GADD45 (Figure S2, lane 10). Because Myc-MTK1(853-1341) 4
lacks the entire GADD45-binding domain (147-250), this result disproves the hypothesis that 5
MTK1 dimerization is an indirect consequence of the GADD45 dimerization. 6
7
Mapping of the dimerization domain in MTK1 8
The MTK1 dimerization domain was further mapped using additional deletion 9
constructs. Progressive deletion of MTK1(853-1341) from its N-terminal side revealed that 10
MTK1(941-1341) can bind MTK1-FL, but MTK1(953-1341) cannot, indicating that the sequence 11
between aa residues 941 and 953 is essential for dimerization (Figure 5A, left panel). Deletion of 12
MTK1(941-1341) from its C-terminal side showed that the shortest segment that can bind 13
MTK1-FL is MTK1(941-1272) (Figure 5A, right panel). Thus, we conclude that the minimal 14
region required for MTK1 dimerization is the region between aa residues 941 and 1272, although a 15
slightly longer segment (residues 941-1321) binds MTK1-FL more efficiently. 16
Because the dimerization assay in the above mapping was done entirely in vitro, we 17
verified that MTK1(941-1321) and MTK1-FL interact in vivo, by co-expressing 18
Myc-MTK1(941-1321) and Flag MTK1-FL in COS-7 cells, either in the presence or the absence of 19
GADD45β (Figure 5B). In this in vivo experiment, as in the in vitro experiments, 20
Myc-MTK1(941-1321) and Flag MTK1-FL dimerized, but only in the presence of GADD45β. 21
22
A coiled-coil motif in the dimerization domain 23
A database search revealed no significant candidates that are structurally similar to the 24
MTK1 dimerization domain (residues 941-1272), except the corresponding regions in MTK1 25
orthologs of the vertebrate, insect, and nematode. The COILS program (26), however, predicted 26
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that the short region 982-1012 may form a coiled-coil structure (Figure 6A). A coiled-coil is made 1
of two α-helices that wrap around each other to form a twisted supercoil structure, and is often 2
involved in protein-protein assemblage. The MTK1 coiled-coil motif is conserved in the MTK1 3
orthologs of evolutionarily diverse organisms (Supplementary Figure S3). 4
To examine if the coiled-coil motif plays a role in MTK1 dimerization and/or activation, 5
we did in vivo coimmunoprecipitation assays using four MTK1 mutants in the putative coiled-coil 6
motif. The first mutant, MTK1-∆CC, lacks the entire coiled-coil motif (982-1012). In the other 7
three mutants, one or more positions (Leu-997, Ile-1001, and Val-1008) are substituted by a 8
helix-disrupting proline: the L997P/I1001P double mutant is abbreviated as PP, and the 9
L997P/I1001P/V1008P triple mutant as PPP. When the entire coiled-coil region was deleted 10
(MTK1-∆CC), or two critical amino acid residues were replaced by proline (MTK1-PP), very little 11
interaction occurred between the Flag- and Myc-tagged constructs, even in the presence of 12
GADD45β (Figure 6B). 13
From these results, we conclude that coiled-coil formation is critical for 14
GADD45-induced MTK1 dimerization. We also conclude that the dimerization domain in 15
full-length MTK1 is usually masked, and is uncovered only in the presence of the GADD45 16
proteins. 17
18
MTK1 dimerization is required for Thr-1493 autophosphorylation 19
Both dimerization and Thr-1493 autophosphorylation of MTK1 are induced by 20
co-expression of GADD45, suggesting that these events might be causally related. More 21
specifically, we hypothesized that MTK1 dimerization is a prerequisite for autophosphorylation at 22
Thr-1493. To test this hypothesis, we examined whether dimerization-defective MTK1 mutants 23
can autophosphorylate at Thr-1493 or not. GADD45-stimulated phosphorylation of Thr-1493 was 24
undetectable in MTK1-∆CC, compared to the robust Thr-1493 phosphorylation of the wild-type 25
MTK1 molecule (Figure 7A). Double (PP) and triple (PPP) substitution mutants are also 26
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completely defective in autophosphorylation, although the L997P single mutant could weakly 1
autophosphorylate at Thr-1493 (Figure 7B). Perhaps consistent with these findings, the L997P 2
single mutant did not inhibit MTK1 dimerization to an appreciable degree (data not shown). Thus, 3
Thr-1493 autophosphorylation requires MTK1 dimerization. 4
Earlier, we suggested that Thr-1493 autophosphorylation is an intermolecular, or trans, 5
reaction between a pair of MTK1 molecules in a dimer. To further examine if Thr-1493 6
phosphorylation is an intermolecular reaction, we transfected COS-7 cells with two MTK1 7
constructs: one a Myc-tagged, catalytically active, full-length molecule (Myc-MTK1), and the 8
other a Flag-tagged, catalytically defective molecule, without or with a mutation in the coiled-coil 9
motif (Flag-MTK1-K/R or Flag-MTK1-PP-K/R, respectively). Because Flag-MTK1-K/R has no 10
catalytic activity, the only way its Thr-1493 can be phosphorylated by the co-expressed 11
Myc-MTK1 is in trans. When Myc-MTK1 and Flag-MTK1-K/R were co-expressed, 12
immunoprecipitated Flag-MTK1-K/R was phosphorylated at Thr-1493, when GADD45β was also 13
expressed (Figure 7C; lanes 1 and 2). However, when Myc-MTK1 and the dimerization-defective 14
Flag-MTK1-PP-K/R construct were co-expressed, Flag-MTK1-PP-K/R was not phosphorylated at 15
all by the co-expressed Myc-MTK1 kinase, even in the presence of GADD45β (Figure 7C; lane 4). 16
From these results, we conclude that the MTK1 dimerization is required for Thr-1493 17
phosphorylation in trans between two MTK1 molecules. 18
19
MTK1 dimerization is required for activation of the MTK1 kinase domain 20
So far, we have shown separately that MTK1 dimerization is required for Thr-1493 21
phosphorylation, and that Thr-1493 phosphorylation precedes the full activation of the MTK1 22
kinase. Thus, we can predict that dimerization is required for the full activation of the MTK1 23
kinase catalytic domain. This prediction was tested using an in vitro kinase assay (Figure 7D). 24
MTK1-L997P, which weakly autophosphorylates in the presence of GADD45β (see Figure 7B), 25
also had a weaker kinase activity than wild-type MTK1 (Figure 7D, lanes 4 and 2, respectively). 26
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MTK1-PP and MTK1-PPP, which are both completely defective in dimerization (Figure 6B, and 1
data not shown) and in autophosphorylation at Thr-1493 (Figure 7B), are completely defective in 2
kinase activity (Figure 7D, lanes 6 and 8). Thus, there is a very strong correlation between 3
dimerization, autophosphorylation, and catalytic activation of MTK1. 4
Next, we tested if dimerization of MTK1 by itself is sufficient to achieve kinase domain 5
activation, by using an FK506-binding protein 12 (FKBP12)-mediated dimerization of MTK1. An 6
FKBP12 domain can be induced to dimerize by adding the dimeric FK506 reagent, AP20187 to the 7
media (31, 40). As demonstrated in Supplementary Figure S4, MTK1 phosphorylation at Thr-1493 8
could not be induced by AP20187, at any concentration between 0 and 100 nM. Co-expression of 9
GADD45β induced Thr-1493 phosphorylation of FKBP-MTK1, showing that the fusion protein is 10
capable of autophosphorylation, but only after binding of GADD45. Thus, dimerization is 11
necessary, but not sufficient, for MTK1 activation. Perhaps, the AP20187-induced dimer has a less 12
optimal orientation of the coiled-coil segments for activation. 13
To test if GADD45 binding and MTK1 dimerization play roles in phosphorylation of 14
MTK1 Thr-1493 in response to stress, we analyzed Thr-1493 phosphorylation following addition 15
of MMS to the cells (see Figure 3A). HEK293 cells were stably transfected with either a wild-type 16
(WT), GADD45-binding site deletion (∆BD) mutant, or a coiled-coil defective (PP) mutant version 17
of a Myc-MTK1 construct. MTK1 phosphorylation at Thr-1493 was monitored before and after 18
exposure of these cells to MMS (Figure 8). Strong Thr-1493 phosphorylation of the wild-type 19
MTK1 was observed after 3 hr exposure to MMS. In contrast, Thr-1493 phosphorylation was 20
completely absent in ∆BD and PP mutants, indicating that both GADD45 binding and MTK1 21
dimerization are important for phosphorylation at Thr-1493 by extracellular stress stimuli. 22
23
Constitutively active MTK1 mutants revisited 24
Previously, we described constitutively active MTK1 mutants that can bind to and 25
phosphorylate MKK6 in the absence of GADD45 (28). The mechanism of activation of these 26
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MTK1 mutants in the absence of GADD45 binding should shed further light on how GADD45 1
activates the MTK1 kinase. Thus, we investigated whether these constitutively active mutations 2
can mimic MTK1 activation by GADD45 binding. Specifically, we tested three constitutively 3
active mutants (L534Q, Q637P, and I1360M), which can phosphorylate the MKK6 substrate, both 4
in vivo and in vitro, in the absence of GADD45 expression. The fourth mutant, V1300F, can bind, 5
but does not phosphorylate, MKK6, in the absence of GADD45. 6
First, we tested MTK1 Thr-1493 phosphorylation in these mutants. As shown in Figure 7
9A, MTK1-L534Q, MTK1-Q637P, and MTK1–I1360M were constitutively phosphorylated at 8
Thr-1493, in the absence of any GADD45 protein (lanes 3, 4, and 6), whereas the wild-type MTK1 9
or MTK1-V1300F was not (Figure 9A, lanes 1 and 5). Next, we tested if these mutants were 10
dimerized by co-expressing a Myc-tagged and a Flag-tagged versions of each mutant. The three 11
constitutively active mutants (L534Q, Q637P, and I1360M) were dimerized in the absence of 12
GADD45 (Figure 9B, lanes 3, 4, and 6), whereas MTK1-V1300F was dimerized only at the level 13
of the wild-type MTK1 protein without GADD45 (Figure 9B, lane 5). Thr-1493 phosphorylation 14
of the constitutively active mutants was dependent on the MTK1 dimerization domain, because the 15
dimerization-defective PP mutation completely abolished Thr-1493 phosphorylation of the L534Q 16
mutant (Figure 9C). Lastly, we tested the effects of constitutively active mutations on MTK1 N-C 17
interaction. As we showed earlier in this paper, N- and C-terminal segments of MTK1 bind to each 18
other, and this association is disrupted by GADD45 binding to the N-terminal fragment (Figure 9D, 19
lanes 2 and 3). The constitutively active mutation L534Q in the MTK1-N terminal fragment 20
inhibited the N-C interaction, and this inhibition does not require GADD45 (Figure 9D, lane 4). 21
Thus, the constitutively active MTK1 mutations mimic the effects of GADD45 binding, including 22
the Thr-1493 autophosphorylation, MTK1 dimerization, and disruption of the N-C interaction, 23
suggesting that they are key residues in GADD45-mediated MTK1 activation. 24
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Discussion 1
2
The data from this study lead to the following model of the GADD45 mediated 3
activation of the MTK1 MAPKKK. Activation of MTK1 by GADD45 occurs through a series of 4
molecular steps (I through VI), as summarized in Figure 10. In brief, each step is as follows. (I) In 5
unstimulated cells, MTK1 is in a closed (inhibited) conformation in which the N-terminal 6
autoinhibitory domain (AID) blocks the C-terminal kinase catalytic domain (KD). (II) 7
Extracellular stimuli, such as MMS exposure, induce the expression of stress-inducible GADD45 8
proteins, which bind to the MTK1 N-terminal GADD45-binding domain (BD). (III) 9
GADD45-binding to MTK1 dissociates the latter’s AID from the C-terminal kinase catalytic 10
domain. (IV) At the same time, GADD45-binding unmasks the MTK1 dimerization domain, 11
inducing homo-dimer formation. At this stage, the MTK1 kinase domain is in open conformation 12
(i.e., not actively inhibited), but not yet fully active as a kinase. (V) Dimerized MTK1 becomes 13
fully activated when Thr-1493 is trans-autophosphorylated. (VI) GADD45 binding also unmasks a 14
site in the MTK1 kinase domain that interacts with the MAPKK DVD docking sites, allowing 15
MTK1 to interact with, and phosphorylate, the cognate MAPKKs, namely MKK3 and MKK6 (36). 16
Because the GADD45 proteins are highly conserved (34), it is likely that all three 17
members of the GADD45 family activate MTK1 by the same mechanism. Indeed, in our 18
investigation of GADD45-mediated MTK1 activation, we did not find any qualitative differences 19
among the three GADD45 proteins. We did find, however, that there are quantitative differences 20
among them; e.g., GADD45α does not stimulate MTK1 as efficiently as GADD45β or GADD45γ 21
(34). It is possible that they have slightly different affinities for MTK1. In terms of their function, 22
however, their varied expression pattern is perhaps more important. Expression of each member of 23
the GADD45 gene family is induced by a distinct set of environmental stresses and cytokines, in 24
different tissues. For example, expression of GADD45β, but not GADD45α nor γ, is induced by 25
TGF-β (35); and expression of GADD45α, but not GADD45β nor γ, is modulated by p53 (25). 26
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Consistently, the GADD45 proteins (GADD45α/β/γ) serve overlapping, but non-identical, 1
functions in different apoptotic and growth inhibitory pathways (25). 2
Full activation of MTK1 by GADD45 entails four different molecular mechanisms: 3
removal of the autoinhibitory domain; dimerization; phosphorylation of the activation loop; and 4
unmasking of the docking site for MAPKKs. Individually, these mechanisms are used by other 5
MAPKKKs. However, the details are different for each MAPKKK, reflecting their different 6
physiological roles. Therefore, in order to appreciate its physiological function, it is important to 7
analyze how the binding of one protein (GADD45) orchestrates these mechanisms, thereby 8
converting an inert enzyme (MTK1) to a fully active one. 9
Autoinhibition of the kinase catalytic domain by N-terminal (or sometimes C-terminal) 10
regulatory sequences is a mechanism employed by many MAPKKKs. Artificial deletion of such 11
autoinhibitory domains converts the kinase into a constitutively active one (5, 6, 11, 43, 46). In 12
physiologically relevant activation processes, however, release from autoinhibition is effected by a 13
different mechanism for each kinase. For example, MEKK1 is activated by a caspase-3 mediated 14
shedding of its N-terminal inhibitory domain (41), and Ste11 is activated by Ste20-mediated 15
phosphorylation of the autoinhibitory domain (39). In the case of MTK1, binding of GADD45 to 16
MTK1 seems to compete with the adjacent autoinhibitory domain for interaction with the MTK1 17
catalytic domain. 18
Dimerization is also frequently utilized to activate protein kinases including MAPKKKs. 19
For example, it has been reported that forced-dimerization of full-length MEKK1 induces its 20
autophosphorylation and subsequent phosphorylation of the MKK4 (SEK1) MAPKK (7). It was 21
also reported recently that dimerization of the MEKK4 (MTK1) kinase domain only (without the 22
N-terminal autoinhibitory domain) by AP20187 increases MEKK4 kinase activity (1). Unlike 23
these cases, however, artificial dimerization of full-length MTK1 using FKBP and AP20187 did 24
not enhance its in vitro catalytic activity, and did not lead to Thr-1493 phosphorylation in vivo. 25
Therefore, it is likely that dimerization is necessary but not sufficient for activation of full-length 26
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MTK1. Perhaps it is important for MTK1 activation that GADD45 dissociates the N-terminal 1
autoinhibitory domain from the C-terminal catalytic domain. The specific configuration of the 2
coiled-coil mediated dimerization might also be important. 3
Since GADD45 proteins can themselves form homo- and hetero-dimers (or oligomers) 4
(23), one possibility was that MTK1 indirectly dimerized through dimerization of the bound 5
GADD45 molecules. This is unlikely, however, for two reasons. First, MTK1 fragments that do 6
not contain the GADD45-BD can bind MTK1. Second, constitutively-active MTK1 mutants 7
dimerize in the absence of GADD45 proteins (Figure 9B). Nonetheless, it is possible that 8
GADD45 dimerization might serve to concentrate MTK1 molecules, hence enhancing the latter’s 9
dimerization. Finally, it should be noted that MTK1 is dimerized by other stimuli, such as TRAF4, 10
presumably via a GADD45-independent mechanism (1). 11
Phosphorylation of the activation loop is perhaps the most common activation 12
mechanism for diverse families of protein kinases (4, 15, 19, 42). However, different kinases 13
might use different mechanisms to achieve phosphorylation of the activation loop. Many kinases 14
are phosphorylated by their upstream activating kinase, in a similar manner to the phosphorylation 15
of MAPKKs by MAPKKKs, or the phosphorylation of MAPKs by MAPKKs. Other kinases 16
autophosphorylates the activation loop (12, 22). The Thr-1493 phosphorylation of MTK1 is 17
induced by expression of the GADD45 proteins, or by extracellular stimuli, such as MMS, that are 18
known to induce GADD45 expression. Dimerization-defective MTK1 mutants, however, cannot 19
autophosphorylate Thr-1493. Thus, stable dimerization induced by GADD45-binding is 20
responsible for efficient intermolecular Thr-1493 phosphorylation of MTK1. 21
Finally, the GADD45 proteins indirectly control the interaction between activated MTK1 22
and its substrates, i.e., MKK3/MKK6. Both MKK3 and MKK6 contain, at their C-terminus, an 23
~20 amino-acid peptide termed the DVD domain (36). Deletion and point mutations of the 24
MKK3/MKK6 DVD sequence inhibit stable interaction between MTK1 and MKK3/MKK6, and 25
consequently, inhibit activation of MKK3/MKK6 by MTK1. The DVD-mediated interaction 26
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between MKK6 and full-length MTK1 does not take place unless GADD45 is also present (28). It 1
is likely that the MTK1 N-terminal autoinhibitory domain prevents the MTK1 catalytic domain 2
from binding the MKK3/MKK6 DVD docking site. 3
With the detailed activation mechanism of MTK1 now available, it will be possible to 4
study this important but under-explored area of cellular signal transduction. 5
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Acknowledgments 1
We thank P. O’Grady for critical reading of the manuscript, M. Kitamura and N. Yoshida 2
for excellent technical assistance, and ARIAD Pharmaceuticals, Inc. for the gift of the ARGENT 3
Regulated Homodimerization Kit and the ARGENT Transcription Retrovirus Kit 4
(www/ariad.com/regulationkits). This work was supported in part by several Grants-in-Aid from 5
the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.S. and M.T.), 6
and a PRESTO program from the Japan Science and Technology Agency (to M.T.). 7
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References 1
2
1. Abell, A. N., and G. L. Johnson. 2005. MEKK4 is an effector of the embryonic TRAF4 for 3
JNK activation. J. Biol. Chem. 280:35793-35796. 4
2. Abell, A. N., J. A. Rivera-Perez, B. D. Cuevas, M. T. Uhlik, S. Sather, N. L. Johnson, S. K. 5
Minton, J. M. Lauder, A. M. Winter-Vann, K. Nakamura, T. Magnuson, R. R. 6
Vaillancourt, L. E. Heasley, and G. L. Johnson. 2005. Ablation of MEKK4 kinase activity 7
causes neurulation and skeletal patterning defects in the mouse embryo. Mol. Cell. Biol. 8
25:8948-8959. 9
3. Azam, N., M. Vairapandi, W. Zhang, B. Hoffman, and D. A. Liebermann. 2001. 10
Interaction of CR6 (GADD45γ) with proliferating cell nuclear antigen impedes negative 11
growth control. J. Biol. Chem. 276:2766-2774. 12
4. Bellon, S., M. J. Fitzgibbon, T. Fox, H. M. Hsiao, and K. P. Wilson. 1999. The structure of 13
phosphorylated p38γ is monomeric and reveals a conserved activation-loop conformation. 14
Structure 7:1057-1065. 15
5. Cairns, B. R., S. W. Ramer, and R. D. Kornberg. 1992. Order of action of components in 16
the yeast pheromone response pathway revealed with a dominant allele of the STE11 kinase 17
and the multiple phosphorylation of the STE7 kinase. Genes Dev. 6:1305-1318. 18
6. Cardone, M. H., G. S. Salvesen, C. Widmann, G. Johnson, and S. M. Frisch. 1997. The 19
regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell 90:315-323. 20
7. Chadee, D. N., T. Yuasa, and J. M. Kyriakis. 2002. Direct activation of mitogen-activated 21
protein kinase kinase kinase MEKK1 by the STE20p homologue GCK and the adapter protein 22
TRAF2. Mol. Cell. Biol. 22:737-749. 23
8. Chen, Z., T. B. Gibson, F. Robinson, L. Silvestro, G. Pearson, B. Xu, A. Wright, C. 24
Vanderbilt, and M. H. Cobb. 2001. MAP kinases. Chem. Rev. 101:2449-2476. 25
9. Chi, H., B. Lu, M. Takekawa, R. J. Davis, and R. A. Flavell. 2004. GADD45β/GADD45γ 26
ACCEPTED
on April 13, 2018 by guest
http://mcb.asm
.org/D
ownloaded from
Miyake et al.
26
and MEKK4 comprise a genetic pathway mediating STAT4-independent IFNγ production in 1
T cells. EMBO J. 23:1576-1586. 2
10. Chi, H., M. R. Sarkisian, P. Rakic, and R. A. Flavell. 2005. Loss of mitogen-activated 3
protein kinase kinase kinase 4 (MEKK4) results in enhanced apoptosis and defective neural 4
tube development. Proc. Natl. Acad. Sci. USA 102:3846-3851. 5
11. Cutler, R. E. J., R. M. Stephens, M. R. Saracino, and D. K. Morrison. 1998. 6
Autoregulation of the Raf-1 serine/threonine kinase. Proc. Natl. Acad. Sci. USA 7
95:9214-9219. 8
12. Deak, J. C., and D. J. Templeton. 1997. Regulation of the activity of MEK kinase 1 9
(MEKK1) by autophosphorylation within the kinase activation domain. Biochem. J. 10
322:185-192. 11
13. Dohlman, H. G., and J. W. Thorner. 2001. Regulation of G protein-initiated signal 12
transduction in yeast: paradigms and principles. Annu. Rev. Biochem. 70:703-754. 13
14. Fornace, A. J. Jr., I. Jr. Alamo, and M. C. Hollander. 1988. DNA damage-inducible 14
transcripts in mammalian cells. Proc. Natl. Acad. Sci. USA 85:8800-8804. 15
15. Furdui, C. M., E. D. Lew, J. Schlessinger, and K. S. Anderson. 2006. Autophosphorylation 16
of FGFR1 kinase is mediated by a sequential and precisely ordered reaction. Mol. Cell 17
21:711-717. 18
16. Gerwins, P., J. L. Blank, and G. L. Johnson. 1997. Cloning of a novel mitogen-activated 19
protein kinase kinase kinase, MEKK4, that selectively regulates the c-Jun amino terminal 20
kinase pathway. J. Biol. Chem. 272:8288-8295. 21
17. Gupta, M., S. K. Gupta, A. G. Balliet, M. C. Hollander, A. J. Jr. Fornace, B. Hoffman, 22
and D. A. Liebermann. 2005. Hematopoietic cells from Gadd45a- and Gadd45b-deficient 23
mice are sensitized to genotoxic-stress-induced apoptosis. Oncogene 24:7170-7179. 24
18. Gustin, M. C., J. Albertyn, M. Alexander, and K. Davenport. 1998. MAP kinase pathways 25
in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62:1264-1300. 26
ACCEPTED
on April 13, 2018 by guest
http://mcb.asm
.org/D
ownloaded from
Miyake et al.
27
19. Jeffrey, P. D., A. A. Russo, K. Polyak, E. Gibbs, J. Hurwitz, J. Massagué, and N. P. 1
Pavletich. 1995. Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 2
complex. Nature 376:313-320. 3
20. Johnson, G. L., and R. Lapadat. 2002. Mitogen-activated protein kinase pathways mediated 4
by ERK, JNK, and p38 protein kinases. Science 298:1911-1912. 5
21. Johnson, L. N., M. E. M. Noble, and D. J. Owen. 1996. Active and inactive protein kinases: 6
structural basis for regulation. Cell 85:149-158. 7
22. Kishimoto, K., K. Matsumoto, and J. Ninomiya-Tsuji. 2000. TAK1 mitogen-activated 8
protein kinase kinase kinase is activated by autophosphorylation within its activation loop. J. 9
Biol. Chem. 275:7359-7364. 10
23. Kovalsky, O., F.-D. T. Lung, P. P. Roller, and A. J. Jr. Fornace. 2001. Oligomerization of 11
human GADD45a protein. J. Biol. Chem. 276:39330-39339. 12
24. Kyriakis, J. M., and J. Avruch. 2001. Mammalian mitogen-activated protein kinase signal 13
transduction pathways activated by stress and inflammation. Physiol. Rev. 81:807-869. 14
25. Liebermann, D. A., and B. Hoffman. 2002. Myeloid Differentiation (MyD)/Growth Arrest 15
DNA Damage (GADD) genes in tumor suppression, immunity and inflammation. Leukemia 16
16:527-541. 17
26. Lupus, A., M. van Dyke, and J. Stock. 1991. Predicting coiled coils from protein sequences. 18
Science 252:1162-1164. 19
27. Miller, J. H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, 20
Cold Spring Harbor, NY. 21
28. Mita, H., J. Tsutsui, M. Takekawa, E. A. Witten, and H. Saito. 2002. Regulation of 22
MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 23
binding. Mol. Cell. Biol. 22:4544-4555. 24
29. Nishina, H., T. Wada, and T. Katada. 2004. Physiological roles of SAPK/JNK signaling 25
pathway. J. Biochem (Tokyo) 136:123-126. 26
ACCEPTED
on April 13, 2018 by guest
http://mcb.asm
.org/D
ownloaded from
Miyake et al.
28
30. Pearson, G., F. Robinson, T. B. Gibson, B.-E. Xu, M. Karandikar, K. Berman, and M. H. 1
Cobb. 2001. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological 2
functions. Endocrine Rev. 22:153-183. 3
31. Spencer, D. M., T. J. Wandless, S. L. Schreiber, and G. R. Crabtree. 1993. Controlling 4
signal transduction with synthetic ligands. Science 262:1019-1024. 5
32. Takekawa, M., T. Maeda, and H. Saito. 1998. Protein phosphatase 2Cα inhibits the human 6
stress-responsive p38 and JNK MAPK pathways. EMBO J. 17:4744-4752. 7
33. Takekawa, M., F. Posas, and H. Saito. 1997. A human homolog of the yeast Ssk2/Ssk22 8
MAP kinase kinase kinases, MTK1, mediates stress-induced activation of the p38 and JNK 9
pathways. EMBO J. 16:4973-4982. 10
34. Takekawa, M., and H. Saito. 1998. A family of stress-inducible GADD45-like proteins 11
mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 95:521-530. 12
35. Takekawa, M., K. Tatebayashi, F. Itoh, M. Adachi, K. Imai, and H. Saito. 2002. 13
Smad-dependent GADD45β expression mediates delayed activation of p38 MAP kinase by 14
TGF-β. EMBO J. 21:6473-6482. 15
36. Takekawa, M., K. Tatebayashi, and H. Saito. 2005. Conserved docking site is essential for 16
activation of mammalian MAP kinase kinases by specific MAP kinase kinase kinases. Mol. 17
Cell 18:295-306. 18
37. Tatebayashi, K., M. Takekawa, and H. Saito. 2003. A docking site determining specificity 19
of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway. EMBO J. 20
22:3624-3634. 21
38. Vairapandi, M., N. Azam, A. G. Balliet, B. Hoffman, and D. A. Liebermann. 2000. 22
Characterization of MyD118, Gadd45, and proliferating cell nuclear antigen (PCNA) 23
interacting domains: PCNA impedes MyD118 and Gadd45-mediated negative growth control. 24
J. Biol. Chem. 275:16810-16819. 25
39. van Drogen, F., S. M. O'Rourke, V. M. Stucke, M. Jaquenoud, A. M. Neiman, and M. 26
ACCEPTED
on April 13, 2018 by guest
http://mcb.asm
.org/D
ownloaded from
Miyake et al.
29
Peter. 2000. Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required 1
for MAP kinase signaling in vivo. Curr. Biol. 10:630-639. 2
40. Welm, B. E., K. W. Freeman, M. Chen, A. Contreras, D. M. Spencer, and J. M. Rosen. 3
2002. Inducible dimerization of FGFR1: development of a mouse model to analyze 4
progressive transformation of the mammary gland. J. Cell Biol. 157:703-714. 5
41. Widmann, C., P. Gerwins, N. L. Johnson, M. B. Jarpe, and G. L. Johnson. 1998. MEK 6
kinase 1, a substrate for DEVD-directed caspases, is involved in genotoxin-induced apoptosis. 7
Mol. Cell. Biol. 18:2416-2429. 8
42. Yamaguchi, H., and W. A. Hendrickson. 1996. Structural basis for activation of human 9
lymphocyte kinase Lck upon tyrosine phosphorylation. Nature 384:484-489. 10
43. Yamaguchi, K., K. Shirakabe, H. Shibuya, K. Irie, I. Oishi, N. Ueno, T. Taniguchi, E. 11
Nishida, and K. Matsumoto. 1995. Identification of a member of the MAPKKK family as a 12
potential mediator of TGF-β signal transduction. Science 270:2008-2011. 13
44. Zhan, Q., K. A. Lord, I. J. Alamo, M. C. Hollander, F. Carrier, D. Ron, K. W. Kohn, B. 14
Hoffman, D. A. Liebermann, and A. J. Jr. Fornace. 1994. The gadd and MyD genes define 15
a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell 16
growth. Mol. Cell. Biol. 14:2361-2371. 17
45. Zhang, D., V. Facchinetti, X. Wang, Q. Huang, J. Qin, and B. Su. 2006. Identification of 18
MEKK2/3 serine phosphorylation site targeted by the Toll-like receptor and stress pathways. 19
EMBO J. 25:97-107. 20
46. Zhang, H., and K. A. Gallo. 2001. Autoinhibition of mixed lineage kinase 3 through its Src 21
homology 3 domain. J. Biol. Chem. 276:45598-45603. 22
23
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Figure LegendsFigure LegendsFigure LegendsFigure Legends 1
2
FIG 1. GADD45 binds MTK1 and enhances MTK1 kinase activity. 3
(A) Domain structure of MTK1. The top cartoon schematically indicates the positions of the 4
GADD45 binding domain (BD), the autoinhibitory domain (AID), the dimerization domain (DD), 5
and the kinase domain (KD), in MTK1. The lower bars represent the MTK1-N and MTK1-C 6
constructs used in Figure 1E. (B) Structure of GADD45. The position of the MTK1 binding 7
domain in GADD45β is shown. (C) GADD45 region important for MTK1 activation. COS-7 cells 8
were transiently co-transfected with an expression plasmid for Flag-MTK1 and another plasmid 9
encoding either full-length GFP-GADD45β (FL) or one of its deletion derivatives as indicated. 10
Flag-MTK1 was immunoprecipitated from cell extracts, and its kinase activity was assayed in vitro 11
using bacterially produced GST-MKK6 (K/A) as a specific substrate. An autoradiogram of 12
SDS-PAGE was digitally obtained using a PhosphoImager (top panel). Relative activity, 13
calculated versus the activity of mock-stimulated (by GFP) MTK1, are shown below the top panel 14
(fold activation). The average of three independent experiments is shown. Expression levels of 15
Flag-MTK1 and GFP-GADD45β derivatives were monitored by immunoblotting (middle and 16
bottom panels). (D) Binding of GADD45 to MTK1. Flag-MTK1 and either GADD45β or 17
GADD45β∆53-62 were transiently expressed in COS-7 cells. Flag-MTK1 was 18
immunoprecipitated from the cell extracts, and co-precipitation of GADD45β or 19
GADD45β∆53-62 was assayed by immunoblotting analysis (top panel). Expression levels of 20
GADD45β and Flag-MTK1were monitored by immunoblotting (middle and bottom panels). The 21
asterisk (*) indicates the expected position of GADD45β∆53-62. (E) Disruption of MTK1 N-C 22
interaction by GADD45. Flag-MTK1-N, Flag-MTK1-N∆BD, Myc-MTK1-C, GADD45β, and 23
GADD45β∆53-62 were individually expressed in COS-7 cells. Cell lysates were prepared, mixed 24
in vitro, and Flag-MTK1 or Flag-MTK1-N∆BD was immunoprecipitated. Co-precipitated 25
Myc-MTK1-C was detected by immunoblotting (top panel). Lower panels show the levels of 26
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protein expression. 1
2
FIG 2. Phosphorylation of specific amino acid residues in the MTK1 kinase activation loop. 3
(A) The amino acid sequence of the activation loop of the MTK1 kinase. The black dot indicates 4
the activating phosphorylation site. (B) The effect of mutations at potential phosphorylation sites 5
in the MTK1 activation loop. Wild-type Flag-MTK1, or MTK1 with an Ala-substitution at 6
potential phosphorylation site, was co-expressed with GADD45β in COS-7 cells. Flag-MTK1 was 7
immunoprecipitated from cell lysate, and subjected to an in vitro kinase assay using 8
GST-MKK6(K/A) as a substrate. Data were collected as in Figure 1C. Relative activity was 9
calculated using the activity of the wild-type MTK1 as 100%. The average of three independent 10
experiments is shown below the top panel (% activity). (C) COS-7 cells were transfected with the 11
wild-type (WT) Flag-MTK1 expression construct, or the T1493A phosphorylation site mutant, 12
together with either a GADD45β expression plasmid (+) or the empty vector pcDNA3 (-). 13
Immunoprecipitation was performed with an anti-Flag antibody, and the phosphorylation at 14
Thr-1493 in MTK1 was probed by immunoblotting analysis using the anti-phospho-Thr-1493 15
antiserum (αP-T1493). (D) COS-7 cells were transfected with the wild-type (WT) Flag-MTK1 16
expression construct or its kinase-dead derivative, K1371R (K/R), together with either a 17
GADD45β expression plasmid (+) or an empty vector (-). Phosphorylation at Thr-1493 was 18
probed as in (C). For (B)-(D), expression levels of Flag-MTK1 and GADD45β were monitored by 19
immunoblotting (middle and bottom panels). 20
21
FIG 3. 22
Induction of MTK1 Thr-1493 phosphorylation by MMS. (A) HEK293 cells stably expressing 23
either the wild-type Myc-MTK1 (WT) or the kinase-dead Myc-MTK1 (K/R) were stimulated with 24
100 µg/ml MMS (+) or without (-) for 180 min, and cell lysates were prepared. The expressed 25
Myc-MTK1 was affinity purified and its phosphorylation at Thr-1493 was probed using the 26
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anti-phospho Thr-1493 antiserum (αP-T1493). 1
(B) HEK293 cells were stimulated with 100 µg/ml MMS (+) or without (-) for 180 min, and cell 2
lysates were prepared. Endogenous MTK1 was immunoprecipitated by an affinity-purified 3
anti-MTK1 antibody (or by a control polyclonal anti-HA antibody), and its phosphorylation at 4
Thr-1493 was probed as in (A). 5
6
FIG 4. Trans-phosphorylation of Thr-1493 and dimerization of MTK1 are induced by 7
GADD45. 8
(A) Trans-phosphorylation of MTK1 Thr-1493 induced by GADD45β. COS-7 cells were 9
co-transfected with expression vectors for Myc-MTK1(K/R) and Flag-MTK1 (or for Myc-MTK1 10
and Flag-MTK1(K/R)), together with pGADD45β (+) or the empty vector pcDNA3 (-). 11
Flag-MTK1 and Myc-MTK1 were simultaneously immunoprecipitated using both anti-Flag and 12
anti-Myc antibodies. Phosphorylation of precipitated Flag-MTK1 and Myc-MTK1 at Thr-1493 13
was probed with an anti-phospho-Thr-1493 (αP-T1493) antibody. The asterisks (*) indicate the 14
positions of kinase-dead MTK1 proteins. (B) Dimerization of MTK1 induced by GADD45. 15
Flag-MTK1 and Myc-MTK1 were co-expressed in COS-7 cells, together with either GADD45α, 16
GADD45β, or GADD45γ. Flag-MTK1 was immunoprecipitated from cell lysates, and 17
co-precipitated Myc-MTK1 was probed by immunoblotting using an anti-Myc antibody (top 18
panel). (C) GADD45 binding is necessary for MTK1 dimerization. Flag-MTK1 and Myc-MTK1 19
were co-expressed in COS-7 cells, together with either GADD45β, or GADD45β∆53-62. 20
Flag-MTK1 was immunoprecipitated from cell lysates, and the co-precipitated Myc-MTK1 was 21
probed by immunoblotting using an anti-Myc antibody (top panel). For (B) and (C), the expression 22
levels of Myc-MTK1, Flag-MTK1, and GADD45 proteins are shown in the lower panels. 23
24
FIG 5. Mapping of the dimerization domain in MTK1. 25
(A) In vitro MTK1 dimerization assays. COS-7 cells were separately transfected with full-length 26
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(FL) Flag-MTK1 (Flag-MTK1-FL), one of Myc-MTK1 deletion mutants, or GADD45β. Cell 1
lysates were prepared, and extracts containing Flag-MTK1-FL, GADD45β, and one of the 2
Myc-MTK1 deletions were mixed in vitro, and subjected to immunoprecipitation with an anti-Flag 3
(αFlag) antibody. The presence or absence of co-precipitating Myc-MTK1 deletion mutant 4
proteins in the Flag-MTK1 immunoprecipitates was determined by immunoblotting analysis using 5
an anti-Myc antibody (top panel). The asterisks (*) indicate the expected positions of the proteins. 6
(B) In vivo dimerization of MTK1. COS-7 cells were co-transfected with expression plasmids for 7
Myc-MTK1(941-1321) and Flag-MTK1-FL, together with another plasmid encoding GADD45β 8
(+) or the empty vector pcDNA3 (-). Flag-MTK1 was immunoprecipitated from cell lysates, and 9
co-precipitated Myc-MTK1(941-1321) was probed with an anti-Myc antibody (top panel). The 10
expression level of each protein was monitored by immunoblotting of the whole extracts (lower 11
panels). 12
13
FIG 6. MTK1 dimerizes through the coiled-coil motif. 14
(A) The amino acid sequence of the putative coiled-coil region (black bar) in MTK1 and flanking 15
sequences. The three amino acid positions in which proline-substitution mutations were made are 16
indicated by dots. The ∆CC mutant lacks the entire coiled-coil region from residue 982 to 1012. 17
PP is L997P/I1001P double proline-substitution mutation, and PPP is L997P/I1001P/V1008P triple 18
proline-substitution mutation. (B) The coiled-coil region is necessary for MTK1 dimerization. 19
COS-7 cells were co-transfected with expression plasmids encoding the wild-type or coiled-coil 20
mutant versions of Myc-MTK1 and Flag-MTK1 as indicated, together with a GADD45β 21
expression plasmid (+) or the empty vector pcDNA3 (-). Flag-MTK1 was immunoprecipitated 22
from cell lysates, and co-precipitated Myc-MTK1 was probed with an anti-Myc antibody (top 23
panel). 24
25
FIG 7. MTK1 dimerization is essential for Thris essential for Thris essential for Thris essential for Thr----1493 autophosphorylation and for 1493 autophosphorylation and for 1493 autophosphorylation and for 1493 autophosphorylation and for 26
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MTK1 activation.MTK1 activation.MTK1 activation.MTK1 activation. 1
(A and B) Dimerization-defective MTK1 mutants cannot autophosphorylate. COS-7 cells were 2
co-transfected with either wild-type or mutant Flag-MTK1, as indicated, together with a 3
GADD45β expression vector (+) or the empty control vector (-), and cell extracts were prepared 36 4
h after transfection. Immunoprecipitation was performed with an anti-Flag antibody, and Thr-1493 5
phosphorylation of the precipitated Flag-MTK1 was analyzed by immunoblotting using an 6
anti-phospho Thr-1493 (αP-T1493) antibody (top panel). (C) MTK1 autophosphorylation is an 7
intermolecular reaction. COS-7 cells were co-transfected with expression vectors for Myc-MTK1 8
and either Flag-MTK1-K/R or Flag-MTK1-PP-K/R, together with a GADD45β expression vector 9
(+) or the empty vector pcDNA3 (-). Immunoprecipitation was performed with an anti-Flag 10
antibody, and phosphorylation of the precipitated Flag-MTK1-K/R (or Flag-MTK1-PP-K/R) was 11
detected by immunoblotting analysis using an anti-phospho-Thr-1493 (αP-T1493) antibody (top 12
panel). (D) Dimerization-defective MTK1 mutants cannot be activated by GADD45. COS-7 cells 13
were transfected with either an expression plasmid for Flag-MTK1 or one of its coiled-coil motif 14
mutants, together with another plasmid encoding GADD45β (+) or the empty vector pcDNA3 (-). 15
Cell lysates were subjected to an in vitro kinase assay using GST-MKK6(K/A) as a specific 16
substrate. An autoradiogram of SDS-PAGE was digitally obtained using a PhosphoImager (top 17
panel). Relative activity (fold activation) was calculated versus the activity of unstimulated MTK1 18
(the leftmost sample). The average of two independent experiments is shown below the top panel. 19
For (A-D), the expression level of each protein was monitored by immunoblotting, and shown in 20
the lower panels. 21
22
FIG 8. GADD45 binding and subsequent MTK1 dimerization is required for stress-induced 23
MTK1 autophosphorylation. 24
Human embryonic kidney HEK293 cells were stably transfected with expression vectors for 25
wild-type Myc-MTK1 (WT), GADD45-binding defective MTK1-∆BD, or dimerization defective 26
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MTK1-PP, respectively. Transfected cells were treated without (-) or with (+) 100 µg/ml MMS for 1
180 min before preparation of cell extracts. Myc-MTK1 was affinity purified, and its 2
phosphorylation status at Thr-1493 was probed using the αP-T1493 antibody (upper panel). The 3
same filter was reprobed with anti-Myc antibody (bottom panel). 4
5
FIG 9. Constitutively active MTK1 mutants. 6
(A) Constitutively active MTK1 mutants are phosphorylated at Thr-1493 in the absence of 7
GADD45 binding. COS-7 cells were transfected with an expression plasmid for wild-type 8
Myc-MTK1 or its constitutively active mutant versions, together with a second plasmid encoding 9
GADD45β (+) or the control empty vector pcDNA3 (-). Immunoprecipitation was carried out with 10
an anti-Myc antibody, and the phosphorylation status of Thr-1493 was probed with the αP-T1493 11
antibody (top panel). (B) Constitutively active MTK1 mutants are dimerized in the absence of 12
GADD45 binding. Wild-type or mutant version of Flag-MTK1 and Myc-MTK1, and GADD45β, 13
were co-expressed in COS-7 cells as indicated in the figure. Cell lysates were prepared and 14
subjected to immunoprecipitation with an anti-Flag antibody. The presence of Myc-MTK1 in the 15
Flag-MTK1 precipitates was probed by an anti-Myc antibody (top panel). (C) The dimerization 16
domain is essential for GADD45-independent Thr-1493 autophosphorylation in a constitutively 17
active MTK1 mutant. COS-7 cells were transfected with an expression plasmid for either 18
wild-type Myc-MTK1, constitutively active Myc-MTK1 L534Q, or the dimerization-defective 19
Myc-MTK1 L534Q-PP, together with a second plasmid encoding GADD45β (+) or the control 20
pcDNA3 empty vector (-). Immunoprecipitation was performed with an anti-Myc antibody, and 21
Thr-1493 phosphorylation of the precipitates was detected by immunoblotting analysis with the 22
αP-T1493 antibody. (D) A constitutively active MTK1 mutation disrupts the MTK1 N-C 23
interaction independent of GADD45 binding. Flag-MTK1-N, Flag-MTK1-N-L534Q, 24
Myc-MTK1-C, or GADD45β were separately expressed in COS-7 cells. Cell lysates were 25
prepared, and mixed in vitro as indicated. Flag-MTK1 or Flag-MTK1-N-L534Q was 26
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immunoprecipitated and co-precipitated Myc-MTK1-C was detected by immunoblotting. For 1
(A-D), protein expression levels are shown in the lower panels. 2
3
FIG 10. Schematic model of MTK1 activation. 4
Activation of MTK1 by GADD45 can be dissected into several stages, as indicated by the roman 5
numerals in the scheme (I through VI). See text for details. 6
7
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Fig. 1
C∆
1-1
2
∆1
3-2
2
∆3
3-4
2
∆5
3-6
2
∆2
3-3
2
GF
P
FL
∆4
3-5
2
∆6
3-7
2
∆8
3-9
2
∆9
3-1
02
∆1
13
-122
∆7
3-8
2
∆1
03
-112
GF
P
FL
∆1
33
-142
∆1
23
-132
∆1
53
-160
∆1
43
-152
GF
P
FL
GFP-GADD45β GFP-GADD45β GFP-GADD45β
MTK1 activity
Flag-MTK1
GFP-GADD45β
GST-MKK6(K/A)
Fold activation 111 2221131 241422181 7562161
21 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
D E
IP:αFlagIB:αMyc
Flag-MTK1-N
Myc-MTK1-C
Flag-MTK1-N
GADD45β
Myc-MTK1-C
GADD45β
++++++++ ∆53-62
∆BD
−−−−−−−− −−−−++++++++ ++++ ++++ ++++ ++++
∆BD++++ ++++ ++++−−−−
21 3 4 5 6
A B
GADD45β13
216
013
MTK1 BD
GADD45β
Flag-MTK1
IP:αFlag
IB:αGADD45β
GADD45β ++++ ++++ ∆53-62
Flag-MTK1 ++++ ++++−−−−
21 3
MTK1-N MTK1-C
22 994 1247 1607
MTK1 DDAIDBD14
725
013
4216
0712
7294
1
KD
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Fig. 2
C D
IP: αFlagIB: αP-T1493
Flag-MTK1
GADD45β
GADD45β
Flag-MTK1
- + +WT WT K/R
IP: αFlag
IB: αP-T1493
Flag-MTK1
GADD45β
GADD45β
Flag-MTK1
+ +- -
WT
WT
T1493
A
T1493
A
21 3 4
B
GST-
MKK6(K/A)
(%) 100 3 74 8 62 38 2
Flag-MTK1
WT
K/R
S1484
A
T1493
A
S1500
A
T1501
A
T1504
A
Flag-MTK1
MTK1 Activity
GADD45β
A
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A
MMS
Myc-MTK1
- +
K/R
- +
WT
Myc-MTK1
IP: αMycIB: αP-T1493
αP-T1493
Anti-MTK1
IP: Anti-MTK1Control Ab
MMS −−−− ++++ −−−− ++++
B
Fig. 3
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Fig. 4
B
IP:αFlagIB:αMyc
Myc-MTK1
Flag-MTK1
GADD45β
GADD45α
GADD45γ
Flag-MTK1 ++++ ++++ ++++−−−− ++++
Myc-MTK1 ++++ ++++ ++++++++ ++++
GADD45 α β γ−−−− −−−−
21 3 4 5
A
++++ ++++−−−− −−−−GADD45β
K/R K/R++++ ++++Flag-MTK1
K/R K/R ++++ ++++Myc-MTK1
Flag-MTK1
Myc-MTK1
Flag-MTK1
Myc-MTK1
IP: αFlag & αMyc
IB: αP-T1493
GADD45β
MTK1
21 3 4
CMyc-MTK1 ++++++++++++ ++++Flag-MTK1 ++++++++ ++++−−−−GADD45β ∆53-62++++−−−−−−−−
IP:αFlag
IB:αMyc
Myc-MTK1
Flag-MTK1
GADD45β
21 3 4
Relative intensity 1 3.5 0.6
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Fig. 5
B
Myc-MTK1(941-1321) ++++++++++++Flag-MTK1-FL ++++++++−−−−
GADD45β ++++−−−− −−−−IP:αFlag IB:αMyc
Myc-MTK1(941-1321)
GADD45β
Flag-MTK1-FL
A
IP:αFlag
IB:αMyc
Flag-MTK1-FL
Myc-MTK1
GADD45β
85
3-1
341
90
1-1
341
94
1-1
341
92
1-1
341
FL
Ve
cto
r
95
3-1
341
Myc-MTK1 Myc-MTK1
94
1-1
341
94
1-1
301
94
1-1
252
94
1-1
272
FL
Ve
cto
r
94
1-1
321
178
61
17819
kDa
61
178
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Fig. 6
A
B
IP:αFlagIB:αMyc
Flag-MTK1
Myc-MTK1
Myc-MTK1
Flag-MTK1
GADD45β
GADD45β
∆CC
∆CC
∆CC
∆CC
PP
PP
PP
PP
+ + +
+ +
+++
-
- - - -
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Fig. 7
A
C D
IP: αFlag
IB: αP-T1493
Flag-MTK1
GADD45β
GADD45β
Flag-MTK1 ∆CC ∆CC
++++ ++++−−−− −−−−
++++ ++++
IP: αFlagIB: αP-T1493
Flag-MTK1
GADD45β
GADD45β
Flag-MTK1L997
P
L997
PPP PP++++++++
++++ ++++−−−− −−−− ++++−−−− ++++−−−−
PPP PPP
Myc-MTK1
GADD45β
Myc-MTK1
GADD45β
IP:αFlagIB:αP-T1493
Flag-MTK1(K/R)
Flag-MTK1-K/R
++++++++++++
++++++++++++
++++++++
−−−− −−−−
PP PP
1 2 3 4
MTK1 activity
Flag-MTK1
GADD45β
GADD45β
Flag-MTK1
Fold activation
GST-
MKK6(K/A)
1 1 1 1 1 114 3
++++ ++++−−−− −−−− ++++−−−− ++++−−−−
L997
P
L997
PPP PP PPP PPP++++++++
1 2 3 4 5 6 7 8
B
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Fig. 8
WT ∆BD PP WT ∆BD PP
MMS
Myc-MTK1
Myc-MTK1
1 2 3 4 5 6
IP:αMycIB:αP-T1493
−−−− ++++
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Fig. 9
A
B
C
D1 2 3 4 5 6
IP:αMyc IB:αP-T1493
Myc-MTK1
GADD45β
Myc-MTK1
GADD45β −−−− −−−− −−−− −−−− −−−−++++
++++L534
Q
Q637
PV1300
F++++I1360
M
IP:αFlag IB:αMyc
Myc-MTK1
Flag-MTK1
GADD45β
GADD45β ++++−−−− −−−− −−−−−−−−−−−−−−−−
Myc-MTK1 ++++++++ ++++L534
Q
Q637
P
V1300
FI1360
M
Flag-MTK1 ++++ ++++−−−−L534
Q
Q637
P
V1300
FI1360
M
1 2 3 4 5 6C
Myc-MTK1
GADD45β
Myc-MTK1
GADD45β
IP:αMyc IB:αP-T1493
++++
++++++++
−−−− −−−− −−−−
L534
Q
L534Q
-PP
1 2 3 4
IP:αFlag IB:αMyc
Flag-MTK1-N
Myc-MTK1-C
Flag-MTK1-N
GADD45β
Myc-MTK1-C
GADD45β
−−−−
−−−− −−−− −−−−
++++ ++++
++++
++++
++++
++++
++++
++++++++
L534
Q
L534
Q
1 2 3 4 5
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Fig. 10
P
P
(I) (II) (III)
(IV)(V)
(VI)
P
P
MKK6P
MTK1
KD
AID
GADD45α/β/γ
BDcoiled-coil
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