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A conserved arginine within the αC-helix of Erk1/2 is a latch of autoactivation and of oncogenic capabilities.

ABSTRACT: Eukaryotic protein kinases (EPKs) adopt an active conformation following phosphorylation of a particular activation loop residue. Most EPKs spontaneously autophosphorylate this residue. While structure-function relationships of the active conformation are essentially understood, those of the "prone-to-autophosphorylate" conformation are unclear. Here, we propose that a site within the αC-helix of EPKs, occupied by Arg in the mitogen-activated protein kinase (MAPK) Erk1/2 (Arg84/65), impacts spontaneous autophosphorylation. MAPKs lack spontaneous autoactivation, but we found that converting Arg84/65 of Erk1/2 to various residues enables spontaneous autophosphorylation. Furthermore, Erk1 molecules mutated in Arg84 are oncogenic. Arg84/65 thus obstructs the adoption of the "prone-to-autophosphorylate" conformation. All MAPKs harbor an Arg that is equivalent to Arg84/65 of Erks, whereas Arg is rarely found at the equivalent position in other EPKs. We observed that Arg84/65 of Erk1/2 interacts with the DFG motif, suggesting that autophosphorylation may be inhibited by the Arg84/65-DFG interactions. Erk1/2s mutated in Arg84/65 autophosphorylate not only the TEY motif, known as critical for catalysis, but also on Thr207/188. Our MS/MS analysis revealed that a large proportion of the Erk2^R65H population is phosphorylated on Thr188 or on Tyr185 + Thr188, and a small fraction is phosphorylated on the TEY motif. No molecules phosphorylated on Thr183 + Thr188 were detected. Thus, phosphorylation of Thr183 and Thr188 is mutually exclusive suggesting that not only TEY-phosphorylated molecules are active but perhaps also those phosphorylated on Tyr185 + Thr188. The effect of mutating Arg84/65 may mimic a physiological scenario in which allosteric effectors cause Erk1/2 activation by autophosphorylation.

SUBMITTER: Soudah N

PROVIDER: S-EPMC10458722 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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A conserved arginine within the αC-helix of Erk1/2 is a latch of autoactivation and of oncogenic capabilities.

Soudah Nadine N Baskin Alexey A Smorodinsky-Atias Karin K Beenstock Jonah J Ganon Yifat Y Hayouka Ruchama R Aboraya Mohammed M Livnah Oded O Ilouz Ronit R Engelberg David D

The Journal of biological chemistry 20230718 9

Eukaryotic protein kinases (EPKs) adopt an active conformation following phosphorylation of a particular activation loop residue. Most EPKs spontaneously autophosphorylate this residue. While structure-function relationships of the active conformation are essentially understood, those of the "prone-to-autophosphorylate" conformation are unclear. Here, we propose that a site within the αC-helix of EPKs, occupied by Arg in the mitogen-activated protein kinase (MAPK) Erk1/2 (Arg84/65), impacts spon ...[more]

PMID: 37474104

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Project description:Transmembrane domains of membrane proteins sometimes contain conserved charged or ionizable residues which may be essential for protein function and regulation. This work examines the molecular interactions of single Arg residues within a highly dynamic transmembrane peptide helix. To this end, we have modified the GW4,20ALP23 (acetyl-GGAW4(AL)7AW20AGA-amide) model peptide framework to incorporate Arg residues near the center of the peptide. Peptide helix formation, orientation and dynamics were analyzed by means of solid-state NMR spectroscopy to monitor specific 2H- or 15N-labeled residues. GW4,20ALP23 itself adopts a tilted orientation within lipid bilayer membranes. Nevertheless, the GW4,20ALP23 helix exhibits moderate to high dynamic averaging of NMR observables, such as 2H quadrupolar splittings or 15N-1H dipolar couplings, due to competition between the interfacial Trp residues on opposing helix faces. Here we examine how the helix dynamics are impacted by the introduction of a single Arg residue at position 12 or 14. Residue R14 restricts the helix to low dynamic averaging and a well-defined tilt that varies inversely with the lipid bilayer thickness. To compensate for the dominance of R14, the competing Trp residues cause partial unwinding of the helix at the C-terminal. By contrast, R12GW4,20ALP23 exits the DOPC bilayer to an interfacial surface-bound location. Interestingly, multiple orientations are exhibited by a single residue, Ala-9. Quadrupolar splittings generated by 2H-labeled residues A3, A5, A7, and A9 do not fit to the α-helical quadrupolar wave plot defined by residues A11, A13, A15, A17, A19, and A21. The discontinuity at residue A9 implicates a helical swivel distortion and an apparent 310-helix involving the N-terminal residues preceding A11. These molecular features suggest that, while arginine residues are prominent factors controlling transmembrane helix dynamics, the influence of interfacial tryptophan residues cannot be ignored.

Dataset Information

A conserved arginine within the αC-helix of Erk1/2 is a latch of autoactivation and of oncogenic capabilities.

Publications

A conserved arginine within the αC-helix of Erk1/2 is a latch of autoactivation and of oncogenic capabilities.

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