Mechanism of activation of ERK2 by dual phosphorylation

The mitogen-activated protein (MAP) kinases ape characterized by their requ irement for dual phosphorylation at a conserved threonine and tyrosine resi due for catalytic activation. The structural consequences of dual-phosphory lation in the MAP kinase ERK2 (extracellular signal-regulated kinase 2) inc lude active site closure, alignment of key catalytic residues that interact with ATP, and remodeling of the activation loop. in this study, we report the specific effects of dual phosphorylation on the individual catalytic re action steps in ERK2, Dual phosphorylation leads to an increase in overall catalytic efficiency and turnover rate of approximately 600,000- and 50,000 -fold, respectively. Solvent viscosometric studies reveal moderate decrease s in the equilibrium dissociation constants (K-d) for both ATP and myelin b asic protein. However, the majority of the overall rate enhancement is due to an increase in the rate of the phosphoryl group transfer step by approxi mately 60,000-fold. By comparison, the rate of the same step in the ATPase reaction is enhanced only 2000-fold. This suggests that optimizing the posi tion of the invariant residues Lys(52) and Glu(69), which stabilize the pho sphates of ATP, accounts for only part of the enhanced rate of phosphoryl g roup transfer in the kinase reaction. Thus, significant stabilization of th e protein phosphoacceptor group must also occur. Our results demonstrate si milarities between the activation mechanisms of ERK2 and the cell cycle con trol enzyme, Cdk2 (cyclin-dependent kinase 2), Rather than dual phosphoryla tion, however, activation of the latter is controlled by cyclin binding fol lowed by phosphorylation at Thr(160).