Mechanism of Activation of ERK2 by Dual Phosphorylation

Abstract

The mitogen-activated protein (MAP) kinases are characterized by their requirement for dual phosphorylation at a conserved threonine and tyrosine residue for catalytic activation. The structural consequences of dual-phosphorylation in the MAP kinase ERK2 (extracellular signal-regulated kinase 2) include 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 reaction 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 decreases in the equilibrium dissociation constants (K d ) for both ATP and myelin basic protein. However, the majority of the overall rate enhancement is due to an increase in the rate of the phosphoryl group transfer step by approximately 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 position of the invariant residues Lys 52 and Glu 69 , which stabilize the phosphates of ATP, accounts for only part of the enhanced rate of phosphoryl group transfer in the kinase reaction. Thus, significant stabilization of the protein phosphoacceptor group must also occur. Our results demonstrate similarities between the activation mechanisms of ERK2 and the cell cycle control enzyme, Cdk2 (cyclin-dependent kinase 2). Rather than dual phosphorylation, however, activation of the latter is controlled by cyclin binding followed by phosphorylation at Thr 160 .Members of the family of protein kinases referred to as the MAP 1 kinases are critical components of the biochemical processes that define the essence of life, the ability of cells to sense their external environment and respond. The prototype member of the MAP kinase family that mediates signaling by all polypeptide mitogens is the extracellular signal-regulated kinase, ERK2. Like all MAP kinases, ERK2 participates in a three-tier protein phosphorylation cascade that, in response to growth factor receptor signaling, is activated by dual phosphorylation catalyzed by an upstream activating kinase, MEK1/ MEK2. Among the targets of ERK2 are downstream kinases involved in cellular growth control as well as nuclear transcription factors. Thus, ERK2 provides an essential link in transducing the diverse signals from transmembrane growth factor receptors into gene regulatory events (for review see Refs. 1 and 2).The x-ray crystallographic structure of ERK2 (357 amino acids) reveals a conserved catalytic core (residues 22-311) flanked by N-and C-terminal extensions that lie on the surface of the molecule. Like all protein kinases, the catalytic core is globular, consisting of an N-and C-terminal lobe, whose interface defines the active site cleft. Within the active site, the adenine ring of ATP is deeply buried, with the ␥-phosphate pointing toward the mouth of the ac...

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