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.
Chen
P
P
Laboratory of Cellular and Molecular Biology, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
Hutter
D
D
Yang
X
X
Gorospe
M
M
Davis
R J
RJ
Liu
Y
Y
D019943:Amino Acid Substitution; D001665:Binding Sites; D002460:Cell Line; D054637:Dual-Specificity Phosphatases; D004789:Enzyme Activation; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D048055:Mitogen-Activated Protein Kinase 8; D054639:Mitogen-Activated Protein Kinase Phosphatases; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D054639:Mitogen-Activated Protein Kinase Phosphatases; D054648:Protein Phosphatase 2; C490583:DUSP4 protein, human; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases