The Mitogen-activated Protein Kinase Phosphatase-3 N-terminal Noncatalytic Region Is Responsible for Tight Substrate Binding and Enzymatic Specificity

Abstract

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3⌬C) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3⌬N) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPK␤. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3⌬N than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3⌬N and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPK␤. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.The extracellular signal-regulated kinase (ERK), 1 c-Jun Nterminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) represent three major classes of mitogen-activated protein (MAP) kinase (1-4). Different cell stimuli appear to activate distinct MAP kinases preferentially. Hence, while growth factors and some oncogenes are linked to activation of ERK, inflammatory cytokines and cell stresses lead to activation of JNK/SAPK and p38 (1-4). MAP kinases are known to phosphorylate several key regulatory proteins including additional kinases, cytoskeletal proteins, nuclear receptors, as well as several transcription factors, indicating a central role in controlling cell function (1, 5-10). Indeed, ERK has been shown to be important in processes leading to neuronal differentiation, mitogenesis, and oncogenic transformation (1, 11-15), while JNK/SAPK and p38 MAP kinases play critical roles in pathways leading to the generation of inflammatory cytokines and apoptotic death (1, 16 -20). MAP kinase activation requires phosphorylation on Thr and Tyr residues located within the motif TXY of kinase domain VIII (1, 2, 4, 7). While several upstream kinases catalyze this modification on specific MAP kinases, an emerging family of dual-specificity phosphatase dephosphorylate both threonine and tyrosine residues and appear likely to inactivate MAP kinases effect...

Interaction of Mitogen-activated Protein Kinases with the Kinase Interaction Motif of the Tyrosine Phosphatase PTP-SL Provides Substrate Specificity and Retains ERK2 in the Cytoplasm

“…The association of ERK1/2 with the dual-specificity phosphatase MKP-3/Pyst1/rVH6 has been recently reported (22,55), and such an association was found to cause catalytic activation of this phosphatase, indicating a model of phosphatase catalytic regulation by substrate binding (56); however, we have not detected changes in the in vitro tyrosine phosphatase activity of PTP-SL for pNPP upon binding to purified recombinant FIG. 7.…”

Reciprocal Regulation between Slt2 MAPK and Isoforms of Msg5 Dual-specificity Protein Phosphatase Modulates the Yeast Cell Integrity Pathway

“…The N-terminal non-catalytic region of DSPs has been shown to be responsible for binding to MAPKs (40). Most mammalian DSPs possess two motifs designated CH2 (Cdc25 homology regions) at the N-terminal half of the protein.…”

Activation of p42 Mitogen-activated Protein Kinase (MAPK), but not c-Jun NH2-Terminal Kinase, Induces Phosphorylation and Stabilization of MAPK PhosphataseXCL100 inXenopusOocytes

“…Jacobs et al (1999) demonstrated that FXFP is an evolutionarily conserved motif that mediates binding of p42 MAPK to various substrate proteins. The discovery that both substrate specificity and tight substrate binding are conferred by the amino terminus of the related phosphatase MKP-3 (Camps et al, 1998;Muda et al, 1998) supports the hypothesis that the FNFP motif within XCL100 mediates its role as a p42 MAPK substrate. In addition, the absence of an analogous experimentally defined JNK docking site (Yang et al, 1998a,b;Jacobs et al, 1999) in XCL100 is consistent with our conclusion that this phosphatase is a poor substrate for JNK.…”