Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation

Juyoux, Pauline; Galdadas, Ioannis; Gobbo, Dorothea; Velsen, Jill von; Pelosse, Martin; Tully, Mark D.; Vadas, Oscar; Gervasio, Francesco Luigi; Pellegrini, Erika; Bowler, Matthew W.

doi:10.1101/2022.07.04.498667

Cited by 4 publications

(5 citation statements)

References 108 publications

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“…Suggestively, a recent cryo-EM structure of the upstream kinase (MKK6 DD ) trapped in complex with the substrate analogue p38α T180V (PDB: 8A8M), similarly showed the p38α T180V activation-loop redirected towards the orientation that we report for p38α-2p bound to phosphatase stimulating compounds 26 (Fig. 5C).…”

Section: Discussionsupporting

confidence: 72%

Dual-Action Kinase Inhibitors Influence p38α MAP Kinase Dephosphorylation

Stadnicki,

Ludewig,

Kumar

et al. 2024

Preprint

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Reversible protein phosphorylation directs essential cellular processes including cell-division, cell-growth, cell-death, inflammation, and differentiation. Because of this, small molecule ATP-competitive kinase inhibitors have achieved remarkable clinical success, often achieving target specificity by binding to inactive kinase conformations (type II inhibitors). A key to determining kinase activity, and thus signaling outcome, is the conformational state of kinase activation-loops. Activation-loop phosphorylation is a common regulatory mechanism that promotes kinase activity, while type II inhibitors commonly select inactive activation-loop conformations. We hypothesized that protein phosphatases, which can act as the cell's natural kinase inhibitors, may recognize distinct activation-loop conformations. Using a set of type II inhibitors targeting the MAP kinase p38α, we identified inhibitors that modulate dephosphorylation of the activation-loop phospho-threonine by WIP1. Our X-ray crystal structures of dual phosphorylated p38α bound to the kinase inhibitors pexmetinib, nilotinib and BIRB796 reveal a shared flipped conformation of the activation-loop with a fully accessible phospho-threonine for WIP1. In contrast, our X-ray structure of apo p38α reveals a very different activation-loop conformation with an inaccessible phospho-threonine, thereby explaining increased rate of dephosphorylation upon inhibitor binding. These findings reveal an unexpected dual-action mechanism for kinase inhibitors combining direct active site kinase inhibition with a global decrease in active phosphorylated kinase species, suggesting a new approach to achieve improved potency and specificity.

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Section: Discussionsupporting

confidence: 72%

Dual-Action Kinase Inhibitors Influence p38α MAP Kinase Dephosphorylation

Stadnicki,

Ludewig,

Kumar

et al. 2024

Preprint

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“…Protein complex samples were used in analyses immediately after formation. Constitutively active MKK6DD (S207D/T211D mutant) and WT p38α were produced as described in 31 .…”

Section: Methodsmentioning

confidence: 99%

The structural mechanism of MCIA complex assembly links mitochondrial redox pathways

McGregor

Acajjaoui

Desfosses

et al. 2023

Preprint

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The mitochondrial Complex I assembly (MCIA) complex is an essential player in the biogenesis of respiratory Complex I (CI), the multiprotein complex responsible for the initiation of oxidative phosphorylation (OXPHOS). It is not well understood how MCIA facilitates the assembly of CI. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, resulting in efflux of the FAD cofactor and redeployment of ACAD9 from fatty acid β-oxidation (FAO) to CI assembly. We identify an adjacent α-helix as a key structural element that specifically enables the CI assembly functionality of ACAD9, distinguishing it from its closely related VLCAD counterpart. Furthermore, we show that ECSIT is phosphorylated in vitro and ex cellulo and provide evidence that phosphorylation downregulates its association with ACAD9. Interestingly, ECSIT has previously been linked to the pathogenesis of Alzheimer's disease and here we show that ECSIT phosphorylation in neuronal cells is reduced upon exposure to amyloid-β (Aβ) oligomers. These findings shed light on the assembly of the MCIA complex and implicate ECSIT as a potential reprogrammer of bioenergetic metabolic pathways that can be altered when mitochondria are affected by Aβ toxicity, a hallmark of Alzheimer's disease.

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“…Constitutively active MKK6DD (S207D/T211D mutant) and WT p38α were produced as described in ref. 34.…”

Section: Recombinant Protein Expression and Purificationmentioning

confidence: 99%

“…P38α MAP kinase was activated with the active (DD) MKK6 kinase form following a similar procedure as in ref. 34. Protein samples were prepared on ice (MKK6DD:p38α:ECSIT) in 10 µl phosphorylation reaction buffer.…”

Section: Phosphorylation Assays In Vitromentioning

confidence: 99%

The assembly of the Mitochondrial Complex I Assembly complex uncovers a redox pathway coordination

McGregor,

Acajjaoui,

Desfosses

et al. 2023

Nat Commun

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The Mitochondrial Complex I Assembly (MCIA) complex is essential for the biogenesis of respiratory Complex I (CI), the first enzyme in the respiratory chain, which has been linked to Alzheimer’s disease (AD) pathogenesis. However, how MCIA facilitates CI assembly, and how it is linked with AD pathogenesis, is poorly understood. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, releasing the FAD cofactor and converting ACAD9 from a fatty acid β-oxidation (FAO) enzyme to a CI assembly factor. We provide evidence that ECSIT phosphorylation downregulates its association with ACAD9 and is reduced in neuronal cells upon exposure to amyloid-β (Aβ) oligomers. These findings advance our understanding of the MCIA complex assembly and suggest a possible role for ECSIT in the reprogramming of bioenergetic pathways linked to Aβ toxicity, a hallmark of AD.

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Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation

Cited by 4 publications

References 108 publications

Dual-Action Kinase Inhibitors Influence p38α MAP Kinase Dephosphorylation

Dual-Action Kinase Inhibitors Influence p38α MAP Kinase Dephosphorylation

The structural mechanism of MCIA complex assembly links mitochondrial redox pathways

The assembly of the Mitochondrial Complex I Assembly complex uncovers a redox pathway coordination

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