GADD45 binds to and activates MTK1.
Previously, we have shown that the GADD45 family proteins bind to a GADD45-binding domain (BD) (Fig. ) in the N-terminal regulatory region of MTK1 (34
). An MTK1 mutant protein that lacks the GADD45 BD cannot be activated by GADD45 proteins. In the following work, we used GADD45β as a representative member of the GADD45 family because GADD45β is most strongly induced by methyl methanesulfonate (MMS) stress and by TGF-β. Whenever we compared the three GADD45 proteins, however, there were no qualitative differences among them.
GADD45β is a small protein of 160 amino acid residues (Fig. ). To determine the region of GADD45β proteins that is directly involved in MTK1 activation, we constructed a series of deletion mutants, each of which has a deletion of ~10 amino acids. COS-7 cells were cotransfected with a plasmid encoding GFP-tagged GADD45β (GFP-GADD45β) or one of its deletion derivatives and another plasmid encoding full-length Flag-tagged MTK1 (Flag-MTK1). After transfection for 36 h, kinase activity of Flag-MTK1 was assayed in vitro. Coexpression of the full-length GADD45β increased Flag-MTK1 kinase activity more than 10-fold (Fig. ). Deletions at the N terminus (residues 1 to 12) or C terminus (residues 133 to 160) had relatively little effect on MTK1 activation. In contrast, the series of 10-aa deletion mutants between residue 13 and residue 132 was nonactivating, indicating that this region of GADD45β is necessary for MTK1 activation.
To determine whether the GADD45 mutants' failure to activate MTK1 reflected an inability to bind MTK1, binding was assessed by two-hybrid analysis (see Fig. S1 in the supplemental material) and coprecipitation assays (Fig. and data not shown). Whereas the full-length GADD45β and the activating GADD45β mutants could bind to MTK1, the nonactivating GADD45β mutants failed to do so. Neither the expression levels of the GADD45β deletion mutants (Fig. ) nor their subcellular localizations (data not shown) differed significantly from those of wild-type (WT) GADD45β. These results thus support our hypothesis that a direct binding of GADD45 to MTK1 is necessary to activate the MTK1 kinase domain.
GADD45 disrupts the MTK1 N-C association.
The deletion of the entire N-terminal regulatory segment of MTK1 constitutively activates its C-terminal kinase domain (33
), implying that the N-terminal segment contains an autoinhibitory domain (AID) that binds to and inhibits the kinase domain. If so, a possible mechanism by which GADD45 might activate MTK1 is by disruption of the N-C interaction in an MTK1 molecule. As a first step in testing this model, we examined whether the N-terminal and C-terminal segments of MTK1 can associate stably with each other in an in vitro binding assay (see Materials and Methods for details). The results in Fig. (lane 2) clearly demonstrate that Flag-MTK1-N (residues 22 to 994) bound to Myc-MTK1-C (residues 1247 to 1607). The N-C interaction, however, was completely disrupted when another cell lysate containing GADD45β was included in the incubation mixture (Fig. , lane 3). The addition of a lysate containing GADD45βΔ(53-62) (lacking residues 53 through 62), which cannot bind to MTK1, did not inhibit the interaction (Fig. , lane 4). Flag-MTK1-NΔBD, which lacks the GADD45 binding domain (residues 147 to 250), could bind Myc-MTK1-C (Fig. , lane 5), but the interaction was no longer susceptible to interference by GADD45β (lane 6). In summary, it is concluded that the binding of GADD45β to the N-terminal segment of MTK1 disrupts MTK1 N-C association.
Phosphorylation in the activation loop of the MTK1 kinase domain.
The activation of many protein kinases entails phosphorylation at a specific residue(s) in the activation loop (T-loop) between subdomains VII and VIII (21
). There are five potential phosphorylation sites in the MTK1 activation loop region (Ser-1484, Thr-1493, Ser-1500, Thr-1501, and Thr-1504) (Fig. ). To examine whether phosphorylation at any of these residues is required for MTK1 activation, we first individually mutated each of these residues by substitution with Ala. These Flag-MTK1 Ala substitution mutants were coexpressed with GADD45β, and their kinase activities were measured in vitro (Fig. ). Kinase activities of MTK1-S1484A, S1500A, and T1501A were only moderately lower than that of wild-type MTK1. In contrast, MTK1-T1504A was completely inactive, and MTK1-T1493A had only very weak activity.
FIG. 2. Phosphorylation of specific amino acid residues in the MTK1 kinase activation loop. (A) Amino acid sequence of the activation loop of the MTK1 kinase. The black dot indicates the activating phosphorylation site. (B) Effects of mutations at potential phosphorylation (more ...)
To examine whether Thr-1493 and/or Thr-1504 is phosphorylated in vivo, we developed specific antisera to the MTK1 T-loop peptides phosphorylated at Thr-1493 or at Thr-1504. To date, however, by using two different antisera, we have obtained no evidence that Thr-1504 is phosphorylated. It is possible that the mutation of Thr-1504 disturbs an essential secondary structure element unrelated to phosphorylation (45
). In contrast, we could detect stimulation-dependent phosphorylation at Thr-1493 using an anti-phospho-T1493 (αP-T1493) rabbit antibody. The αP-T1493 antibody reacted with MTK1 only when the latter was activated by GADD45β coexpression; unstimulated MTK1 was unreactive (Fig. , lanes 1 and 2). Furthermore, the antibody did not react with the MTK1-T1493A mutant protein whether it was stimulated by GADD45β coexpression or not (Fig. , lanes 4 and 3, respectively). These results indicate, on the one hand, that the αP-T1493 antibody is specific to phospho-T1493 and, on the other hand, that the activation of MTK1 by GADD45β induces Thr-1493 phosphorylation. Occasionally, but not always, a substitution of Thr or Ser by an acidic amino acid (Asp or Glu) mimics the effect of phosphorylation. Thus, we tested the substitution of Thr-1493 by Asp or Glu, but both T1493D and T1493E were inactive.
Next, we asked whether the phosphorylation of MTK1 at Thr-1493 is affected by the autophosphorylation of MTK1 or by another kinase using the catalytically inactive pFlag-MTK1-K/R construct. As shown in Fig. , no phosphorylation at Thr-1493 was observed for MTK1-K/R under the same conditions in which wild-type MTK1 was strongly phosphorylated. This result indicates that Thr-1493 phosphorylation is dependent on MTK1 kinase activity. Because Thr-1493 phosphorylation is also dependent on MTK1 homodimerization (see below), we conclude that the phosphorylation at Thr-1493 is mediated by the MTK1 kinase itself.
To test whether Thr-1493 phosphorylation plays a role in the activation of MTK1 in response to stress, we analyzed Thr-1493 phosphorylation following the addition of MMS to the human embryonic kidney HEK293 cells. MMS is a well-known cell stressor and a potent inducer of the GADD45 genes (34
). Strong Thr-1493 phosphorylation was observed after 3 h of exposure of the cells to MMS (Fig. and see Fig. ). Furthermore, this phosphorylation was dependent on MTK1 kinase activity because no Thr-1493 phosphorylation occurred when the kinase-defective Myc-MTK1-K/R mutant was used instead of wild-type Myc-MTK1. We also tested whether the endogenous MTK1 molecule is phosphorylated at Thr-1493 when cells are stimulated by stress. This is a difficult experiment because the amount of the endogenous MTK1 molecules is small, and the affinity of the available anti-MTK1 antibody is weak. Nonetheless, we could detect the MMS-induced phosphorylation of Thr-1493 (Fig. ). Thus, the phosphorylation at Thr-1493 is a physiologically relevant reaction to external stress stimuli.
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 μg/ml MMS for 180 min, and cell lysates were prepared. (more ...)
FIG. 8. GADD45 binding and subsequent MTK1 dimerization is required for stress-induced MTK1 autophosphorylation. Human embryonic kidney HEK293 cells were stably transfected with expression vectors for WT Myc-MTK1, GADD45 binding-defective MTK1-ΔBD, or (more ...) Phosphorylation at Thr-1493 occurs in trans.
We next determined whether MTK1 phosphorylates Thr-1493 by an intramolecular (cis) reaction or by an intermolecular (trans) reaction. We transfected COS-7 cells simultaneously with two different MTK1 constructs, namely, Flag-tagged, wild-type MTK1 and Myc-tagged, kinase-dead MTK1 (Myc-MTK1-K/R), together with pGADD45β or the empty vector (Fig. , lanes 1 and 2). Myc-MTK1-K/R contains five repeats of the Myc tag and is easily distinguishable from Flag-MTK1 by SDS gel electrophoresis. If the Thr-1493 phosphorylation occurs only in cis, Myc-MTK1-K/R will not be phosphorylated, even in the presence of the activated Flag-MTK1 protein. Thr-1493 phosphorylation was detected, however, both in Myc-MTK1-K/R (Fig. , lane 2) and in Flag-MTK1. When the tags were reversed and Myc-MTK1 and Flag-MTK1-K/R were used (Fig. , lanes 3 and 4), an essentially identical result was obtained. These data indicate that Thr-1493 phosphorylation is likely to be an intermolecular (trans) reaction.
FIG. 4. trans phosphorylation of Thr-1493 and dimerization of MTK1 are induced by GADD45. (A) trans phosphorylation of MTK1 Thr-1493 induced by GADD45β. COS-7 cells were cotransfected with expression vectors for Myc-MTK1-K/R and Flag-MTK1 (or for Myc-MTK1 (more ...) GADD45 enhances MTK1 oligomerization.
We tested whether MTK1 can form a stable homo-oligomer by coexpression of Myc-MTK1 and Flag-MTK1. Flag-MTK1 was immunoprecipitated from cell lysates, and the presence of coprecipitated Myc-MTK1 was probed by immunoblotting. As shown in Fig. , lane 2, Flag-MTK1 and Myc-MTK1 coprecipitated only very weakly from unstimulated cells. However, when GADD45α/β/γ was also expressed in the same cells, interaction between Myc-MTK1 and Flag-MTK1 was much enhanced (Fig. , lanes 3 to 5). GADD45βΔ53-62, which cannot bind to MTK1 (Fig. ), could not enhance the Myc-MTK1/Flag-MTK1 interaction (Fig. , lane 4). These results prove that MTK1 oligomerizes when stimulated by GADD45. Because full-length MTK1 is a very large molecule, it is difficult to measure the extent of oligomerization with any accuracy by gel filtration or other methods. Gel filtration analyses of a shorter fragment that contains the oligomerization domain (see below) were consistent with a dimer formation, but a trimer formation could not be excluded (data not shown). With this caveat, we conclude that MTK1 dimerizes when stimulated by GADD45.
MTK1 contains a dimerization domain.
GADD45 proteins are known to form homo- and hetero-oligomeric complexes (23
). In principle, therefore, MTK1 dimerization could be a consequence of dimerization of the associated GADD45. Alternatively, a GADD45-induced conformational change might unmask a latent dimerization domain in MTK1. To distinguish these possibilities, we undertook the experiments described below.
Initially, using a full-length Flag-MTK1 and a series of Myc-MTK1 truncation constructs, we mapped the region in MTK1 that is responsible for dimerization. Full-length Myc-MTK1 coprecipitated with full-length Flag-MTK1 in the presence of GADD45β (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 bind to 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 hypothesis that MTK1 dimerization is an indirect consequence of the GADD45 dimerization.
Mapping of the dimerization domain in MTK1.
The MTK1 dimerization domain was further mapped using additional deletion 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, indicating that the sequence between amino acid residues 941 and 953 is essential for dimerization (Fig. , left panel). The deletion of MTK1(941-1341) from its C-terminal side showed that the shortest segment that can bind full-length MTK1 is MTK1(941-1272) (Fig. , right panel). Thus, we conclude that the minimal region required for MTK1 dimerization is the region between amino acid residues 941 and 1272, although a slightly longer segment (residues 941 to 1321) binds full-length MTK1 more efficiently.
FIG. 5. Mapping of the dimerization domain in MTK1. (A) In vitro MTK1 dimerization assays. COS-7 cells were separately transfected with full-length Flag-MTK1, one of Myc-MTK1 deletion mutants, or GADD45β. Cell lysates were prepared, and extracts containing (more ...)
Because the dimerization assay in the above mapping was performed 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 in either the presence or the absence of GADD45β (Fig. ). In this in vivo experiment, as in the in vitro experiments, Myc-MTK1(941-1321) and full-length Flag-MTK1 dimerized, but only in the presence of GADD45β.
Coiled-coil motif in the dimerization domain.
A database search revealed no significant candidates that are structurally similar to the MTK1 dimerization domain (residues 941 to 1272), except the corresponding regions in MTK1 orthologs of vertebrates, insects, and nematodes. The COILS program (26
), however, predicted that the short region of residues 982 to 1012 may form a coiled-coil structure (Fig. ). A coiled-coil is made of two α-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 conserved in the MTK1 orthologs of evolutionarily diverse organisms (see Fig. S3 in the supplemental material).
FIG. 6. MTK1 dimerizes through the coiled-coil motif. (A) Amino acid sequence of the putative coiled-coil region (black bar) in MTK1 and flanking sequences. The three amino acid positions in which proline substitution mutations were made are indicated by large (more ...)
To examine whether the coiled-coil motif plays a role in MTK1 dimerization and/or activation, we constructed in vivo coimmunoprecipitation assays using four MTK1 mutants in the putative coiled-coil motif. The first mutant, MTK1-ΔCC, lacks the entire coiled-coil motif (amino acids 982 to 1012). In the other three mutants, one or more positions (Leu-997, Ile-1001, and Val-1008) are substituted by a helix-disrupting proline; the L997P/I1001P double mutant is abbreviated as PP, and the L997P/I1001P/V1008P triple mutant is abbreviated as PPP. When the entire coiled-coil region was deleted (MTK1-ΔCC) 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 GADD45β (Fig. ).
From these results, we conclude that the coiled-coil formation is critical for GADD45-induced MTK1 dimerization. We also conclude that the dimerization domain in full-length MTK1 is usually masked and is uncovered only in the presence of the GADD45 proteins.
MTK1 dimerization is required for Thr-1493 autophosphorylation.
Both dimerization and Thr-1493 autophosphorylation of MTK1 are induced by the coexpression of GADD45, suggesting that these events might be causally related. More specifically, we hypothesized that MTK1 dimerization is a prerequisite for autophosphorylation at Thr-1493. To test this hypothesis, we examined whether dimerization-defective MTK1 mutants can autophosphorylate at Thr-1493. GADD45-stimulated phosphorylation of Thr-1493 was undetectable in MTK1-ΔCC, compared to the robust Thr-1493 phosphorylation of the wild-type MTK1 molecule (Fig. ). PP and PPP substitution mutants are also completely defective in autophosphorylation, although the L997P single mutant could weakly autophosphorylate at Thr-1493 (Fig. ). Perhaps consistent with these findings, the L997P single mutant did not inhibit MTK1 dimerization to an appreciable degree (data not shown). Thus, Thr-1493 autophosphorylation requires MTK1 dimerization.
FIG. 7. MTK1 dimerization is essential for Thr-1493 autophosphorylation and for MTK1 activation. (A and B) Dimerization-defective MTK1 mutants cannot autophosphorylate. COS-7 cells were cotransfected with either wild-type or mutant Flag-MTK1, as indicated, together (more ...)
Earlier, we suggested that Thr-1493 autophosphorylation is an intermolecular, or trans, reaction between a pair of MTK1 molecules in a dimer. To further examine whether Thr-1493 phosphorylation is an intermolecular reaction, we transfected COS-7 cells with two MTK1 constructs: a Myc-tagged, catalytically active, full-length molecule (Myc-MTK1) and a Flag-tagged, catalytically defective molecule, without or with a mutation in the coiled-coil motif (Flag-MTK1-K/R or Flag-MTK1-PP-K/R, respectively). Because Flag-MTK1-K/R has no catalytic activity, the only way its Thr-1493 can be phosphorylated by the coexpressed Myc-MTK1 is in trans. When Myc-MTK1 and Flag-MTK1-K/R were coexpressed, immunoprecipitated Flag-MTK1-K/R was phosphorylated at Thr-1493 when GADD45β was also expressed (Fig. , lanes 1 and 2). However, when Myc-MTK1 and the dimerization-defective Flag-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 GADD45β (Fig. , lane 4). From these results, we conclude that the MTK1 dimerization is required for Thr-1493 phosphorylation in trans between two MTK1 molecules.
MTK1 dimerization is required for the activation of the MTK1 kinase domain.
So far, we have shown separately that MTK1 dimerization is required for Thr-1493 phosphorylation and that Thr-1493 phosphorylation precedes the full activation of the MTK1 kinase. Thus, we can predict that dimerization is required for the full activation of the MTK1 kinase catalytic domain. This prediction was tested using an in vitro kinase assay (Fig. ). MTK1-L997P, which weakly autophosphorylates in the presence of GADD45β (Fig. ), also had a weaker kinase activity than wild-type MTK1 did (Fig. , lanes 4 and 2, respectively). MTK1-PP and MTK1-PPP, which are both completely defective in dimerization (Fig. and data not shown) and in autophosphorylation at Thr-1493 (Fig. ), are completely defective in kinase activity (Fig. , lanes 6 and 8, respectively). Thus, there is a very strong correlation between dimerization, autophosphorylation, and catalytic activation of MTK1.
Next, we tested whether the dimerization of MTK1 by itself is sufficient to achieve kinase domain activation by using FK506-binding protein 12 (FKBP12)-mediated dimerization of MTK1. An FKBP12 domain can be induced to dimerize by adding the dimeric FK506 reagent AP20187 to the medium (31
). MTK1 phosphorylation at Thr-1493 could not be induced by AP20187 at any concentration between 0 and 100 nM (see Fig. S4 in the supplemental material) The coexpression of GADD45β induced Thr-1493 phosphorylation of FKBP-MTK1, showing that the fusion protein is capable of autophosphorylation, but only after binding of GADD45. Thus, dimerization is necessary but not sufficient for MTK1 activation. Perhaps the AP20187-induced dimer has a less optimal orientation of the coiled-coil segments for activation.
To test whether GADD45 binding and MTK1 dimerization play roles in the phosphorylation of MTK1 Thr-1493 in response to stress, we analyzed Thr-1493 phosphorylation following the addition of MMS to the cells (Fig. ). HEK293 cells were stably transfected with a wild type, GADD45-binding site deletion (ΔBD) mutant, or coiled-coil defective (PP) mutant version of a Myc-MTK1 construct. MTK1 phosphorylation at Thr-1493 was monitored before and after the exposure of these cells to MMS (Fig. ). Strong Thr-1493 phosphorylation of the wild-type MTK1 was observed after 3 h of exposure to MMS. In contrast, Thr-1493 phosphorylation was completely absent in ΔBD and PP mutants, indicating that both GADD45 binding and MTK1 dimerization are important for phosphorylation at Thr-1493 by extracellular stress stimuli.
Constitutively active MTK1 mutants revisited.
Previously, we described constitutively active MTK1 mutants that can bind to and phosphorylate MKK6 in the absence of GADD45 (28
). The mechanism of activation of these MTK1 mutants in the absence of GADD45 binding should shed further light on how GADD45 activates the MTK1 kinase. Thus, we investigated whether these constitutively active mutations can mimic MTK1 activation by GADD45 binding. Specifically, we tested three constitutively active mutants (L534Q, Q637P, and I1360M) that can phosphorylate the MKK6 substrate, both in vivo and in vitro, in the absence of GADD45 expression. The fourth mutant, V1300F, can bind but does not phosphorylate MKK6 in the absence of GADD45.
First, we tested MTK1 Thr-1493 phosphorylation in these mutants. As shown in Fig. , 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-V1300F was not (lanes 1 and 5, respectively). Next, we tested whether these mutants were dimerized by coexpressing Myc-tagged and Flag-tagged versions of each mutant. The three constitutively active mutants (L534Q, Q637P, and I1360M) were dimerized in the absence of GADD45 (Fig. , lanes 3, 4, and 6), whereas MTK1-V1300F was dimerized only at the level of the wild-type MTK1 protein without GADD45 (lane 5). Thr-1493 phosphorylation of the constitutively active mutants was dependent on the MTK1 dimerization domain because the dimerization-defective PP mutation completely abolished Thr-1493 phosphorylation of the L534Q mutant (Fig. ). Lastly, we tested the effects of constitutively active mutations on MTK1 N-C interaction. As we showed earlier in this paper, N- and C-terminal segments of MTK1 bind to each other and this association is disrupted by GADD45 binding to the N-terminal fragment (Fig. , lanes 2 and 3). The constitutively active mutation L534Q in the MTK1 N-terminal fragment inhibited the N-C interaction, and this inhibition does not require GADD45 (Fig. , lane 4). Thus, the constitutively active MTK1 mutations mimic the effects of GADD45 binding, including the Thr-1493 autophosphorylation, MTK1 dimerization, and disruption of the N-C interaction, suggesting that they are key residues in GADD45-mediated MTK1 activation.
FIG. 9. Constitutively active MTK1 mutants. (A) Constitutively active MTK1 mutants are phosphorylated at Thr-1493 in the absence of GADD45 binding. COS-7 cells were transfected with an expression plasmid for wild-type Myc-MTK1 or its constitutively active mutant (more ...)