Mechanism of activation of ERK2 by dual phosphorylation.
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).
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Department of Molecular, Cellular and Developmental Biology, Interdepartmental Program in Biochemistry and Molecular Biology, University of California, Santa Barbara, California 93106, USA.
Lew
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012997:Solvents; D013816:Thermodynamics; D014783:Viscosity
D011994:Recombinant Proteins; D012997:Solvents; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D019950:Mitogen-Activated Protein Kinase 1; D000251:Adenosine Triphosphatases