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

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 specific site within the αC-helix of EPKs, occupied by Arg in the MAPK Erk1/2 (Arg84/65), impacts spontaneous autophosphorylation capability. MAPKs lack spontaneous autoactivation, but we found that converting Erk1/2's Arg84/65 to various residues is sufficient to enable spontaneous autophosphorylation. Additionally, Erk1 molecules mutated in Arg84 are oncogenic. Arg84/65 thus obstructs the adoption of the ‘prone-to-autophosphorylate' conformation. All MAPKs, with no exception, harbor an Arg that is equivalent to Erks' Arg84/65, while Arg is rarely found at the equivalent position in other EPKs. Furthermore, we observed that Erk1/2's Arg84/65 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 another residue, 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 raising the possibility that not only TEY-phosphorylated molecules are active, but 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.