Substrate Recognition Domains within Extracellular Signal-regulated Kinase Mediate Binding and Catalytic Activation of Mitogen-activated Protein Kinase Phosphatase-3

Harvard University, Cambridge, Massachusetts, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2000; 275(32):24613-24621. DOI: 10.1074/jbc.M001515200


Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that inactivates extracellular
signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERK and the MKP-3 amino terminus
with consequent phosphatase activation and dephosphorylation of the bound MAP kinase. We have used a series of p38/ERK chimeric
molecules to identify domains within ERK necessary for binding and catalytic activation of MKP-3. These studies demonstrate
that ERK kinase subdomains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains
constitute the major COOH-terminal structural lobe of ERK. p38/ERK chimeras possessing these regions display increased sensitivity
to inactivation by MKP-3. These data also reveal an overlap between ERK domains interacting with MKP-3 and those known to
confer substrate specificity on the ERK MAP kinase. Consistent with this, we show that peptides representing docking sites
within the target substrates Elk-1 and p90rskinhibit ERK-dependent activation of MKP-3. In addition, abolition of ERK-dependent phosphatase activation following mutation
of a putative kinase interactionmotif (KIM) within the MKP-3 NH2 terminus suggests that key sites of contact for the ERK COOH-terminal structural lobe include residues localized between
the Cdc25 homology domains (CH2) found conserved between members of the DSP gene family.

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    • "The MAPK pathway controls a vast array of physiological processes, including cell proliferation, cell cycle arrest, cell survival and cell motility (3–6). Specifically, DUSP6 dephosphorylates the threonine and tyrosine residues of extracellular signal-regulated kinase (ERK) 1/2, and inactivates ERK1/2 in a feedback loop (7–9). Disruption of this feedback loop may give rise to increased exposure to growth factors, resulting in neoplastic or even malignant transformation (2,10,11). "
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    ABSTRACT: Dual-specificity phosphatase 6 (DUSP6), a specific negative feedback regulator of phosphorylated extracellular signal-regulated kinase, was found to play an important role in numerous types of solid tumors as a tumor suppressor. In this study, 64.2% (61/95) of esophageal squamous cell carcinoma (ESCC) specimens studied exhibited reduced DUSP6 protein expression, compared with 91% (81/89) of normal esophageal specimens that displayed moderate or strong DUSP6 protein expression in tissue microarray analysis. In total, 36.8% (7/19) of the tumor biopsies displayed at least two-fold downregulation of DUSP6 compared with their paired normal counterparts, by qPCR. Significant loss of DUSP6 was observed in EC9706 and KYSE150 ESCC cell lines by immunoblotting assay. Low DUSP6 protein expression was significantly associated with pathological grade in ESCC by immunohistochemistry (P<0.05). Treatment with 5-aza-2'-deoxycytidine restored DUSP6 expression in the two ESCC cell lines, and the expression varied according to the drug concentration. Methylation-specific PCR analysis showed methylation-specific products in the two ESCC cell lines. We observed significant differences in the early and total apoptotic proportion between the control and experimental groups of the two ESCC cell lines and their transfectants (P<0.001) by annexin/propidium iodide assay. The presence of cleaved PARP product, a marker of caspase-mediated apoptosis, expressed in the two pCMV-DUSP6 transfectants in marked contrast to the parental and pCMV-transfected EC9706 and KYSE150 cells, was observed by immunoblotting. Overall, our results support the role of DUSP6 as a novel candidate tumor suppressor gene in ESCC, which may be a potential prognostic marker for ESCC.
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    • "Extracellular signal-regulated kinase (ERK) is a member of the MAPK family. Its RAS/RAF/MEK/ERK signal transmission pathway is the core of the signal network, which is involved in the regulation of cell growth, development, and division [9,17,28]. "
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    • "These enzymes all display a high degree of substrate selectivity for the ERKs (extracellular-signal-regulated kinases) 1 and 2 in vitro and in vivo [3,4]. Specific recognition and binding to ERK2 is mediated by a conserved KIM (kinase interaction motif) within the N-terminal non-catalytic domain of DUSP6/MKP-3 and this region of the protein also contains a conserved NES (nuclear export signal), which is responsible for the cytoplasmic localization of this phosphatase [5,6]. The specificity of DUSP6/MKP-3 for dephosphorylation and inactivation of the ERK1 and ERK2 MAPKs is enhanced further by ERK-induced conformational change within the catalytic domain of MKP-3, which leads to greatly enhanced phosphatase activity in vitro [7,8]. "
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    ABSTRACT: DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown. In the present study, we demonstrate, using pharmacological inhibitors and analysis of the murine DUSP6/MKP-3 gene promoter, that the ERK pathway is critical for FGF-induced DUSP6/MKP-3 transcription. Furthermore, we show that this response is mediated by a conserved binding site for the Ets (E twenty-six) family of transcriptional regulators and that the Ets2 protein, a known target of ERK signalling, binds to the endogenous DUSP6/MKP-3 promoter. Finally, the murine DUSP6/MKP-3 promoter coupled to EGFP (enhanced green fluorescent protein) recapitulates the specific pattern of endogenous DUSP6/MKP-3 mRNA expression in the chicken neural plate, where its activity depends on FGFR (FGF receptor) and MAPK signalling and an intact Ets-binding site. These findings identify a conserved Ets-factor-dependent mechanism by which ERK signalling activates DUSP6/MKP-3 transcription to deliver ERK1/2-specific negative-feedback control of FGF signalling.
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