Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain

Abstract

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45alpha/beta/gamma). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45.

FIG. 1.

GADD45 binds MTK1 and enhances MTK1 kinase activity. (A) Domain structure of MTK1. The top cartoon schematically indicates the positions of the GADD45 binding domain, the autoinhibitory domain, the dimerization domain (DD), and the kinase domain (KD) in MTK1. The lower bars represent the MTK1-N and MTK1-C constructs used for Fig. 1E. (B) Structure of GADD45. The position of the MTK1 binding domain in GADD45β is shown. (C) GADD45 region important for MTK1 activation. COS-7 cells were transiently cotransfected with an expression plasmid for Flag-MTK1 and another plasmid encoding either full-length (FL) GFP-GADD45β or one of its deletion derivatives as indicated. Flag-MTK1 was immunoprecipitated from cell extracts, and its kinase activity was assayed in vitro using bacterially produced GST-MKK6(K/A) as a specific substrate. An autoradiogram of SDS-PAGE was digitally obtained using a PhosphorImager (top panel). Relative activity, calculated versus the activity of mock-stimulated (by GFP) MTK1, is shown below the top panel (Fold activation). The average of three independent experiments is shown. Expression levels of Flag-MTK1 and GFP-GADD45β derivatives were monitored by immunoblotting (middle and bottom panels). (D) Binding of GADD45 to MTK1. Flag-MTK1 and either GADD45β or GADD45βΔ53-62 were transiently expressed in COS-7 cells. Flag-MTK1 was immunoprecipitated from the cell extracts, and the coprecipitation of GADD45β or GADD45βΔ53-62 was assayed by immunoblot analysis (top panel). Expression levels of GADD45β and Flag-MTK1were monitored by immunoblotting (middle and bottom panels). The star indicates the expected position of GADD45βΔ53-62. (E) Disruption of MTK1 N-C interaction by GADD45. Flag-MTK1-N, Flag-MTK1-NΔBD, Myc-MTK1-C, GADD45β, and GADD45βΔ53-62 were individually expressed in COS-7 cells. Cell lysates were prepared and mixed in vitro, and Flag-MTK1 or Flag-MTK1-NΔBD was immunoprecipitated. Coprecipitated Myc-MTK1-C was detected by immunoblotting (top panel). Lower panels show the levels of protein expression. α, anti; IB, immunoblot; IP, immunoprecipitate.

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 sites in the MTK1 activation loop. Wild-type Flag-MTK1 (or MTK1 with an Ala substitution at the potential phosphorylation site) was coexpressed with GADD45β in COS-7 cells. Flag-MTK1 was immunoprecipitated from cell lysate and subjected to an in vitro kinase assay using GST-MKK6(K/A) as a substrate. Data were collected as described for Fig. 1C. Relative activity was calculated using the activity of the wild-type MTK1 as 100%. The average of three independent experiments is shown below the top panel (% activity). (C) COS-7 cells were transfected with the WT Flag-MTK1 expression construct or the T1493A phosphorylation site mutant, together with either a GADD45β expression plasmid (+) or the empty vector pcDNA3 (−). Immunoprecipitation was performed with an anti-Flag antibody, and the phosphorylation at Thr-1493 in MTK1 was probed by immunoblot analysis using the antiserum αP-T1493. (D) COS-7 cells were transfected with the WT Flag-MTK1 expression construct or its kinase-dead derivative, K1371R (K/R), together with either a GADD45β expression plasmid (+) or an empty vector (−). The phosphorylation at Thr-1493 was probed as for panel C. (B through D) Expression levels of Flag-MTK1 and GADD45β were monitored by immunoblot analysis (middle and bottom panels). α, anti; IB, immunoblot; IP, immunoprecipitate.

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. The expressed Myc-MTK1 was affinity purified, and its phosphorylation at Thr-1493 was probed using the antiserum αP-T1493. (B) HEK293 cells were stimulated with (+) or without (−) 100 μg/ml MMS for 180 min, and cell lysates were prepared. Endogenous MTK1 was immunoprecipitated by an affinity-purified anti-MTK1 antibody (or by a control polyclonal anti-HA antibody), and its phosphorylation at Thr-1493 was probed as described for panel A. α, anti; Ab, antibody; IB, immunoblot; IP, immunoprecipitate.

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 and Flag-MTK1-K/R), together with pGADD45β (+) or the empty vector pcDNA3 (−). Flag-MTK1 and Myc-MTK1 were simultaneously immunoprecipitated using both anti-Flag and anti-Myc antibodies. Phosphorylation of precipitated Flag-MTK1 and Myc-MTK1 at Thr-1493 was probed with αP-T1493 antibody. The stars indicate the positions of kinase-dead MTK1 proteins. (B) Dimerization of MTK1 induced by GADD45. Flag-MTK1 and Myc-MTK1 were coexpressed in COS-7 cells, together with GADD45α, GADD45β, or GADD45γ. Flag-MTK1 was immunoprecipitated from cell lysates, and coprecipitated Myc-MTK1 was probed by immunoblotting using an anti-Myc antibody (top panel). (C) GADD45 binding is necessary for MTK1 dimerization. Flag-MTK1 and Myc-MTK1 were coexpressed in COS-7 cells, together with either GADD45β, or GADD45βΔ53-62. Flag-MTK1 was immunoprecipitated from cell lysates, and the coprecipitated Myc-MTK1 was probed by immunoblotting using an anti-Myc antibody (top panel). For panels B and C, the expression levels of Myc-MTK1, Flag-MTK1, and GADD45 proteins are shown in the lower portions. α, anti; IB, immunoblot; IP, immunoprecipitate.

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 full-length Flag-MTK1, GADD45β, and one of the Myc-MTK1 deletions were mixed in vitro and subjected to immunoprecipitation with an anti-Flag antibody (αFlag). The presence or absence of coprecipitating Myc-MTK1 deletion mutant proteins in the Flag-MTK1 immunoprecipitates was determined by immunoblot analysis using an anti-Myc antibody (top panel). Stars indicate the expected positions of the proteins. (B) In vivo dimerization of MTK1. COS-7 cells were cotransfected with expression plasmids for Myc-MTK1(941-1321) and full-length Flag-MTK1, together with another plasmid encoding GADD45β (+) or the empty vector pcDNA3 (−). Flag-MTK1 was immunoprecipitated from cell lysates, and coprecipitated Myc-MTK1(941-1321) was probed with an anti-Myc antibody (top panel). The expression level of each protein was monitored by immunoblot analysis of the whole extracts (lower panels). α, anti; IB, immunoblot; IP, immunoprecipitate.

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 black dots. The ΔCC mutant lacks the entire coiled-coil region from residues 982 to 1012. (B) The coiled-coil region is necessary for MTK1 dimerization. COS-7 cells were cotransfected with expression plasmids encoding the wild-type or coiled-coil mutant versions of Myc-MTK1 and Flag-MTK1 as indicated, together with a GADD45β expression plasmid (+) or the empty vector pcDNA3 (−). Flag-MTK1 was immunoprecipitated from cell lysates, and coprecipitated Myc-MTK1 was probed with an anti-Myc antibody (top panel). α, anti; IB, immunoblot; IP, immunoprecipitate.

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 with a GADD45β expression vector (+) or the empty control vector (−), and cell extracts were prepared 36 h after transfection. Immunoprecipitation was performed with an anti-Flag antibody, and Thr-1493 phosphorylation of the precipitated Flag-MTK1 was analyzed by immunoblotting using αP-T1493 antibody (top panel). (C) MTK1 autophosphorylation is an intermolecular reaction. COS-7 cells were cotransfected with expression vectors for Myc-MTK1 and either Flag-MTK1-K/R or Flag-MTK1-PP-K/R, together with a GADD45β expression vector (+) or the empty vector pcDNA3 (−). Immunoprecipitation was performed with an anti-Flag antibody, and phosphorylation of the precipitated Flag-MTK1-K/R (or Flag-MTK1-PP-K/R) was detected by immunoblot analysis using αP-T1493 antibody (top panel). (D) Dimerization-defective MTK1 mutants cannot be activated by GADD45. COS-7 cells were transfected with either an expression plasmid for Flag-MTK1 or one of its coiled-coil motif mutants, together with another plasmid encoding GADD45β (+) or the empty vector pcDNA3 (−). Cell lysates were subjected to an in vitro kinase assay using GST-MKK6(K/A) as a specific substrate. An autoradiogram of SDS-PAGE was digitally obtained using a PhosphorImager (top panel). Relative activity (Fold activation) was calculated versus the activity of unstimulated MTK1 (the leftmost sample). The average of two independent experiments is shown below the top panel. For panels A through D, the expression level of each protein was monitored by immunoblotting, and shown in the lower portions. α, anti; IB, immunoblot; IP, immunoprecipitate.

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 dimerization-defective MTK1-PP. Transfected cells were treated without (−) or with (+) 100 μg/ml MMS for 180 min before preparation of cell extracts. Myc-MTK1 was affinity purified, and its phosphorylation status at Thr-1493 was probed using the αP-T1493 antibody (upper panel). The same filter was reprobed with anti-Myc antibody (bottom panel). α, anti; IB, immunoblot; IP, immunoprecipitate.

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 versions, together with a second plasmid encoding GADD45β (+) or the control empty vector pcDNA3 (−). Immunoprecipitation was carried out with an anti-Myc antibody, and the phosphorylation status of Thr-1493 was probed with the αP-T1493 antibody (top panel). (B) Constitutively active MTK1 mutants are dimerized in the absence of GADD45 binding. Wild-type or mutant versions of Flag-MTK1 and Myc-MTK1 and GADD45β were coexpressed in COS-7 cells as indicated. Cell lysates were prepared and subjected to immunoprecipitation with an anti-Flag antibody. The presence of Myc-MTK1 in the Flag-MTK1 precipitates was probed by an anti-Myc antibody (top panel). (C) The dimerization domain is essential for GADD45-independent Thr-1493 autophosphorylation in a constitutively active MTK1 mutant. COS-7 cells were transfected with an expression plasmid for either wild-type Myc-MTK1, constitutively active Myc-MTK1 L534Q, or the dimerization-defective Myc-MTK1 L534Q-PP, together with a second plasmid encoding GADD45β (+) or the control pcDNA3 empty vector (−). Immunoprecipitation was performed with an anti-Myc antibody, and Thr-1493 phosphorylation of the precipitates was detected by immunoblot analysis with the αP-T1493 antibody. (D) A constitutively active MTK1 mutation disrupts the MTK1 N-C interaction independent of GADD45 binding. Flag-MTK1-N, Flag-MTK1-N-L534Q, Myc-MTK1-C, or GADD45β was separately expressed in COS-7 cells. Cell lysates were prepared and mixed in vitro as indicated. Flag-MTK1 or Flag-MTK1-N-L534Q was immunoprecipitated, and coprecipitated Myc-MTK1-C was detected by immunoblotting. For panels A through D, protein expression levels are shown in the lower portions. α, anti; IB, immunoblot; IP, immunoprecipitate.

FIG. 10.

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