Discordance between the binding affinity of mitogen-activated protein kinase subfamily members for MAP kinase phosphatase-2 and their ability to activate the phosphatase catalytically.

MKP-2 is a member of the mitogen-activated protein (MAP) kinase phosphatase family which has been suggested to play an important role in the feedback control of MAP kinase-mediated gene expression. Although MKP-2 preferentially inactivates extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) MAP kinase subfamilies, the mechanisms underlying its ...
own regulation remain unclear. In this report, we have examined the MKP-2 interaction with and catalytic activation by distinct MAP kinase subfamilies. We found that the catalytic activity of MKP-2 was enhanced dramatically by ERK and JNK but was affected only minimally by p38. By contrast, p38 and ERK bound MKP-2 with comparably strong affinities, whereas JNK and MKP-2 interacted very weakly. Through site-directed mutagenesis, we defined the ERK/p38-binding site as a cluster of arginine residues in the NH(2)-terminal domain of MKP-2. Mutation of the basic motif abrogated its interaction with both ERK and p38 and severely compromised the catalytic activation of MKP-2 by these kinases. Unexpectedly, such mutations had little effect on JNK-triggered catalytic activation. Both in vitro and in vivo, wild type MKP-2 effectively inactivated ERK2 whereas MKP-2 mutants incapable of binding to ERK/p38 did not. Finally, in addition to its role as a docking site for ERK and p38, the MKP-2 basic motif plays a role in regulating its nuclear localization. Our studies provided a mechanistic explanation for the substrate preference of MKP-2 and suggest that catalytic activation of MKP-2 upon binding to its substrates is crucial for its function.
Mesh Terms:
Amino Acid Substitution, Binding Sites, Cell Line, Dual-Specificity Phosphatases, Enzyme Activation, Genetic Vectors, Glutathione Transferase, Hela Cells, Humans, Kinetics, MAP Kinase Signaling System, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinase 8, Mitogen-Activated Protein Kinase Phosphatases, Mitogen-Activated Protein Kinases, Mutagenesis, Site-Directed, Protein Phosphatase 2, Protein Tyrosine Phosphatases, Recombinant Fusion Proteins, Recombinant Proteins, Transfection, p38 Mitogen-Activated Protein Kinases
J. Biol. Chem.
Date: Aug. 03, 2001
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