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Mechanism and consequence of the autoactivation of p38α mitogen-activated protein kinase promoted by TAB1.

De Nicola GF, Martin ED, Chaikuad A, Bassi R, Clark J, Martino L, Verma S, Sicard P, Tata R, Atkinson RA, Knapp S, Conte MR, Marber MS - Nat. Struct. Mol. Biol. (2013)

Bottom Line: TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif, allowing autophosphorylation in cis.Interference with p38α recognition by TAB1 abolishes its cardiac toxicity.Such intervention could potentially circumvent the drawbacks of clinical pharmacological inhibitors of p38 catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: 1] King's College London British Heart Foundation Centre of Excellence, Rayne Institute, London, UK. [2] Randall Division of Cell and Molecular Biophysics, King's College London, London, UK.

ABSTRACT
p38α mitogen-activated protein kinase (p38α) is activated by a variety of mechanisms, including autophosphorylation initiated by TGFβ-activated kinase 1 binding protein 1 (TAB1) during myocardial ischemia and other stresses. Chemical-genetic approaches and coexpression in mammalian, bacterial and cell-free systems revealed that mouse p38α autophosphorylation occurs in cis by direct interaction with TAB1(371-416). In isolated rat cardiac myocytes and perfused mouse hearts, TAT-TAB1(371-416) rapidly activates p38 and profoundly perturbs function. Crystal structures and characterization in solution revealed a bipartite docking site for TAB1 in the p38α C-terminal kinase lobe. TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif, allowing autophosphorylation in cis. Interference with p38α recognition by TAB1 abolishes its cardiac toxicity. Such intervention could potentially circumvent the drawbacks of clinical pharmacological inhibitors of p38 catalytic activity.

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Verification of the residues within TAB1 responsible for p38α autoactivation and their discriminatory effect on p38β. (a) ITC analysis of the interaction between p38α and TAB1(371-416) mutants; TAB1(371-416A), TAB1(371-416B) or TAB1(371-416A-B) (see text). Raw data of the heat produced by p38α titrated into a solution of either TAB1(371-416A) (offset ~ −0.045μcal/sec), TAB1(371-416B) (offset ~ −0.2μcal/sec) or TAB1(371-416A-B) (see Table 1 and text). (b) ITC analysis of the interaction between p38β and wild-type TAB1(371-416) and mutants. Raw data of the heat produced by p38β titrated into a solution of either wild type TAB1(371-416) (offset ~−0. 09μcal/sec), TAB1(371-416A) (offset ~−0. 45μcal/sec) or TAB1(371-416B). (c) The residues crucial for the TAB1 mediated p38 autoactivation in vitro are investigated. Western blot analysis of in vitro kinase reaction with WTp38α in the absence (−) or presence (+) of peptides corresponding to TAB1(371-416), TAB1(371-416A), TAB1(371-416B) or TAB1(371-416A-B). The pattern differs markedly from that seen with p38β (see supplementary fig. 5a). (d) The residues crucial for the TAB1 mediated p38 autoactivation are confirmed in E.coli. Co-expression of p38α and TAB1 in E.coli transformed using the following pET duet vector constructs; p38α alone, p38α/TAB1(1-418), p38α/TAB1(1-418A), p38α/TAB1(1-418B) and p38α/TAB1(1-418A-B). (e) The residues crucial for the TAB1 mediated p38 autoactivation are confirmed in a human cell line. Co transfection into HEK293 cells of p38α with TAB1 (1-418), TAB1 (1-418A), TAB1 (1-418B) or TAB1 (1-418A-B). The arrowheads indicates ectopic WTp38α with an HA tag. Blot is aligned with quantitative data derived from three separate transfection which appear in Supplementary Fig.6. Bars represent mean ± s.d. * = P< 0.05 vs p38α. †= P< 0.05 vs p38α+TAB1 (1-418). The uncropped images from which the immunoblots are derived appear in Supplementary Fig.6.
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Figure 5: Verification of the residues within TAB1 responsible for p38α autoactivation and their discriminatory effect on p38β. (a) ITC analysis of the interaction between p38α and TAB1(371-416) mutants; TAB1(371-416A), TAB1(371-416B) or TAB1(371-416A-B) (see text). Raw data of the heat produced by p38α titrated into a solution of either TAB1(371-416A) (offset ~ −0.045μcal/sec), TAB1(371-416B) (offset ~ −0.2μcal/sec) or TAB1(371-416A-B) (see Table 1 and text). (b) ITC analysis of the interaction between p38β and wild-type TAB1(371-416) and mutants. Raw data of the heat produced by p38β titrated into a solution of either wild type TAB1(371-416) (offset ~−0. 09μcal/sec), TAB1(371-416A) (offset ~−0. 45μcal/sec) or TAB1(371-416B). (c) The residues crucial for the TAB1 mediated p38 autoactivation in vitro are investigated. Western blot analysis of in vitro kinase reaction with WTp38α in the absence (−) or presence (+) of peptides corresponding to TAB1(371-416), TAB1(371-416A), TAB1(371-416B) or TAB1(371-416A-B). The pattern differs markedly from that seen with p38β (see supplementary fig. 5a). (d) The residues crucial for the TAB1 mediated p38 autoactivation are confirmed in E.coli. Co-expression of p38α and TAB1 in E.coli transformed using the following pET duet vector constructs; p38α alone, p38α/TAB1(1-418), p38α/TAB1(1-418A), p38α/TAB1(1-418B) and p38α/TAB1(1-418A-B). (e) The residues crucial for the TAB1 mediated p38 autoactivation are confirmed in a human cell line. Co transfection into HEK293 cells of p38α with TAB1 (1-418), TAB1 (1-418A), TAB1 (1-418B) or TAB1 (1-418A-B). The arrowheads indicates ectopic WTp38α with an HA tag. Blot is aligned with quantitative data derived from three separate transfection which appear in Supplementary Fig.6. Bars represent mean ± s.d. * = P< 0.05 vs p38α. †= P< 0.05 vs p38α+TAB1 (1-418). The uncropped images from which the immunoblots are derived appear in Supplementary Fig.6.

Mentions: To assess the individual role of the two recognition sites in the p38:TAB1 complex, we prepared the following TAB1 variants, in which residues were mutated in pairs as follows; V390A Y392A (designated TAB1(371-416A) to disrupt lower, non-canonical site); V408G M409A (designated TAB1(371-416B) to disrupt upper, canonical site); V390A Y392A V408G M409A (designated TAB1(371-416A-B) to disrupt both sites). We used ITC to examine the binding behaviour of these TAB1 mutants to p38α. Fig. 5a shows that no binding was detected with TAB1(371-416A-B), whereas the individual mutation pairs exhibited changes in the thermodynamic signature of binding but with a similar dissociation constant to that of wild-type TAB1 (Table 1). However, whilst the wild-type TAB1 interaction was enthalpically driven (with a negative entropic contribution), with TAB1(371-416A) and TAB1(371-416B) the interaction was entropically driven (with a smaller enthalpic contribution), reflecting differences in the interaction and/or its consequences.


Mechanism and consequence of the autoactivation of p38α mitogen-activated protein kinase promoted by TAB1.

De Nicola GF, Martin ED, Chaikuad A, Bassi R, Clark J, Martino L, Verma S, Sicard P, Tata R, Atkinson RA, Knapp S, Conte MR, Marber MS - Nat. Struct. Mol. Biol. (2013)

Verification of the residues within TAB1 responsible for p38α autoactivation and their discriminatory effect on p38β. (a) ITC analysis of the interaction between p38α and TAB1(371-416) mutants; TAB1(371-416A), TAB1(371-416B) or TAB1(371-416A-B) (see text). Raw data of the heat produced by p38α titrated into a solution of either TAB1(371-416A) (offset ~ −0.045μcal/sec), TAB1(371-416B) (offset ~ −0.2μcal/sec) or TAB1(371-416A-B) (see Table 1 and text). (b) ITC analysis of the interaction between p38β and wild-type TAB1(371-416) and mutants. Raw data of the heat produced by p38β titrated into a solution of either wild type TAB1(371-416) (offset ~−0. 09μcal/sec), TAB1(371-416A) (offset ~−0. 45μcal/sec) or TAB1(371-416B). (c) The residues crucial for the TAB1 mediated p38 autoactivation in vitro are investigated. Western blot analysis of in vitro kinase reaction with WTp38α in the absence (−) or presence (+) of peptides corresponding to TAB1(371-416), TAB1(371-416A), TAB1(371-416B) or TAB1(371-416A-B). The pattern differs markedly from that seen with p38β (see supplementary fig. 5a). (d) The residues crucial for the TAB1 mediated p38 autoactivation are confirmed in E.coli. Co-expression of p38α and TAB1 in E.coli transformed using the following pET duet vector constructs; p38α alone, p38α/TAB1(1-418), p38α/TAB1(1-418A), p38α/TAB1(1-418B) and p38α/TAB1(1-418A-B). (e) The residues crucial for the TAB1 mediated p38 autoactivation are confirmed in a human cell line. Co transfection into HEK293 cells of p38α with TAB1 (1-418), TAB1 (1-418A), TAB1 (1-418B) or TAB1 (1-418A-B). The arrowheads indicates ectopic WTp38α with an HA tag. Blot is aligned with quantitative data derived from three separate transfection which appear in Supplementary Fig.6. Bars represent mean ± s.d. * = P< 0.05 vs p38α. †= P< 0.05 vs p38α+TAB1 (1-418). The uncropped images from which the immunoblots are derived appear in Supplementary Fig.6.
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Figure 5: Verification of the residues within TAB1 responsible for p38α autoactivation and their discriminatory effect on p38β. (a) ITC analysis of the interaction between p38α and TAB1(371-416) mutants; TAB1(371-416A), TAB1(371-416B) or TAB1(371-416A-B) (see text). Raw data of the heat produced by p38α titrated into a solution of either TAB1(371-416A) (offset ~ −0.045μcal/sec), TAB1(371-416B) (offset ~ −0.2μcal/sec) or TAB1(371-416A-B) (see Table 1 and text). (b) ITC analysis of the interaction between p38β and wild-type TAB1(371-416) and mutants. Raw data of the heat produced by p38β titrated into a solution of either wild type TAB1(371-416) (offset ~−0. 09μcal/sec), TAB1(371-416A) (offset ~−0. 45μcal/sec) or TAB1(371-416B). (c) The residues crucial for the TAB1 mediated p38 autoactivation in vitro are investigated. Western blot analysis of in vitro kinase reaction with WTp38α in the absence (−) or presence (+) of peptides corresponding to TAB1(371-416), TAB1(371-416A), TAB1(371-416B) or TAB1(371-416A-B). The pattern differs markedly from that seen with p38β (see supplementary fig. 5a). (d) The residues crucial for the TAB1 mediated p38 autoactivation are confirmed in E.coli. Co-expression of p38α and TAB1 in E.coli transformed using the following pET duet vector constructs; p38α alone, p38α/TAB1(1-418), p38α/TAB1(1-418A), p38α/TAB1(1-418B) and p38α/TAB1(1-418A-B). (e) The residues crucial for the TAB1 mediated p38 autoactivation are confirmed in a human cell line. Co transfection into HEK293 cells of p38α with TAB1 (1-418), TAB1 (1-418A), TAB1 (1-418B) or TAB1 (1-418A-B). The arrowheads indicates ectopic WTp38α with an HA tag. Blot is aligned with quantitative data derived from three separate transfection which appear in Supplementary Fig.6. Bars represent mean ± s.d. * = P< 0.05 vs p38α. †= P< 0.05 vs p38α+TAB1 (1-418). The uncropped images from which the immunoblots are derived appear in Supplementary Fig.6.
Mentions: To assess the individual role of the two recognition sites in the p38:TAB1 complex, we prepared the following TAB1 variants, in which residues were mutated in pairs as follows; V390A Y392A (designated TAB1(371-416A) to disrupt lower, non-canonical site); V408G M409A (designated TAB1(371-416B) to disrupt upper, canonical site); V390A Y392A V408G M409A (designated TAB1(371-416A-B) to disrupt both sites). We used ITC to examine the binding behaviour of these TAB1 mutants to p38α. Fig. 5a shows that no binding was detected with TAB1(371-416A-B), whereas the individual mutation pairs exhibited changes in the thermodynamic signature of binding but with a similar dissociation constant to that of wild-type TAB1 (Table 1). However, whilst the wild-type TAB1 interaction was enthalpically driven (with a negative entropic contribution), with TAB1(371-416A) and TAB1(371-416B) the interaction was entropically driven (with a smaller enthalpic contribution), reflecting differences in the interaction and/or its consequences.

Bottom Line: TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif, allowing autophosphorylation in cis.Interference with p38α recognition by TAB1 abolishes its cardiac toxicity.Such intervention could potentially circumvent the drawbacks of clinical pharmacological inhibitors of p38 catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: 1] King's College London British Heart Foundation Centre of Excellence, Rayne Institute, London, UK. [2] Randall Division of Cell and Molecular Biophysics, King's College London, London, UK.

ABSTRACT
p38α mitogen-activated protein kinase (p38α) is activated by a variety of mechanisms, including autophosphorylation initiated by TGFβ-activated kinase 1 binding protein 1 (TAB1) during myocardial ischemia and other stresses. Chemical-genetic approaches and coexpression in mammalian, bacterial and cell-free systems revealed that mouse p38α autophosphorylation occurs in cis by direct interaction with TAB1(371-416). In isolated rat cardiac myocytes and perfused mouse hearts, TAT-TAB1(371-416) rapidly activates p38 and profoundly perturbs function. Crystal structures and characterization in solution revealed a bipartite docking site for TAB1 in the p38α C-terminal kinase lobe. TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif, allowing autophosphorylation in cis. Interference with p38α recognition by TAB1 abolishes its cardiac toxicity. Such intervention could potentially circumvent the drawbacks of clinical pharmacological inhibitors of p38 catalytic activity.

Show MeSH
Related in: MedlinePlus