Constitutive activation of extracellular signal-regulated kinase 2 by synergistic point mutations

Constitutively active mutant forms of signaling enzymes provide insight int o mechanisms of activation as well as useful molecular tools for probing do wnstream targets. In this study, point mutations in ERK2 at conserved resid ues L73P and S151D were identified that individually led to 8-12-fold incre ased specific activity and in combination reached 50-fold, indicating syner gistic interactions between these residues. Examination by mass spectrometr y, phosphatase sensitivity, and Western blotting revealed that the mutation s enhanced ERK2 activity by facilitating intramolecular autophosphorylation predominantly at Tyr-185 and to a lesser extent at Thr-183 and that phosph orylation at both sites is required for activation. A set of short molecula r dynamics simulations were carried out using different random seeds to sam ple locally accessible configurations. Simulations of the active mutant sho wed potential hydrogen bonding interactions between the phosphoryl acceptor and catalytic nucleophile, which could account for enhanced intramolecular autophosphorylation. In intact cells, the ERK2 mutants were functionally a ctive in phosphorylating Elk-1 and RSK1 and activating the e-fos promoter. This activity was only partially reduced upon treatment of cells with the M KK1/2 inhibitor, U0126, indicating that in vivo the mechanism of ERK2 activ ation occurs substantially through autophosphorylation and partially throug h phosphorylation by MKK1/2.