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

ABSTRACT 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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    • "In particular, it identifies Arg65 of DUSP6/MKP-3 and Asp319 of ERK2 as providing the vast majority of side chain contacts in the ERK2-DUSP6/MKP-3 complex. This latter result is entirely consistent with previous biochemical and genetic studies where mutation of Arg65 causes a dramatic reduction in the affinity of DUSP6/MKP-3 for ERK2 and mutation of Asp319 to Asn in the sevenmaker gain of function mutant in Drosophila ERK/Rolled results in resistance of ERK to inactivation by protein phosphatases (Bott et al., 1994; Chu et al., 1996; Nichols et al., 2000). With respect to potential determinants of docking specificity, this study reinforces the concept that although MAP kinases may employ a "
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    ABSTRACT: The regulated dephosphorylation of mitogen-activated protein kinases (MAPKs) plays a key role in determining the magnitude and duration of kinase activation and hence the physiological outcome of signalling. In mammalian cells, an important component of this control is mediated by the differential expression and activities of a family of 10 dual-specificity (Thr/Tyr) MAPK phosphatases (MKPs). These enzymes share a common structure in which MAPK substrate recognition is determined by sequences within an amino-terminal non-catalytic domain whereas MAPK binding often leads to a conformational change within the C-terminal catalytic domain resulting in increased enzyme activity. MKPs can either recognize and inactivate a single class of MAP kinase, as in the specific inactivation of extracellular signal regulated kinase (ERK) by the cytoplasmic phosphatase DUSP6/MKP-3 or can regulate more than one MAPK pathway as illustrated by the ability of DUSP1/MKP-1 to dephosphorylate ERK, c-Jun amino-terminal kinase and p38 in the cell nucleus. These properties, coupled with transcriptional regulation of MKP expression in response to stimuli that activate MAPK signalling, suggest a complex negative regulatory network in which individual MAPK activities can be subject to negative feedback control, but also raise the possibility that signalling through multiple MAPK pathways may be integrated at the level of regulation by MKPs.
    Oncogene 06/2007; 26(22):3203-13. DOI:10.1038/sj.onc.1210412 · 8.56 Impact Factor
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    • "Selective low molecular weight inhibitors of MEK1/2 (the kinases that phosphorylate Erk1/2) have proved very useful in dissecting the roles of the Erks in cellular regulation [16]. Activation of Erk can be reversed by dual-specificity protein phosphatases [37], e.g., MAP kinase phosphatase (MKP)-3 [11] [55]. MEK1/2 are also activated by phosphorylation, catalysed by members of the Raf group of kinases, and Raf is activated by binding RasÁGTP. "
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    Cardiovascular Research 09/2004; 63(3):403-13. DOI:10.1016/j.cardiores.2004.02.003 · 5.81 Impact Factor
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    • "In particular, the members of the dual-speci®city phosphatase family such as CL100 (MKP-1), VH2 (MKP-2) or Pyst1 (MKP-3) can remove phosphate groups from both the critical threonine and tyrosine residues of activated MAPK (Alessi et al., 1993; Guan and Butch, 1995; Groom et al., 1996; Muda et al., 1996). The MAPK phosphatases (MKPs) contain a speci®c MAPK-binding domain located in the N-terminal half and a catalytic phosphatase domain in the C-terminal portion (Muda et al., 1998; Nichols et al., 2000). The binding of a dual-speci®city MKP to an activated MAPK induces a conformational change that stimulates the catalytic MKP activity (Camps et al., 1998). "
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    ABSTRACT: In the Caenorhabditis elegans hermaphrodite germline, spatially restricted mitogen-activated protein kinase (MAPK) signalling controls the meiotic cell cycle. First, the MAPK signal is necessary for the germ cells to progress through pachytene of meiotic prophase I. As the germ cells exit pachytene and enter diplotene/diakinesis, MAPK is inactivated and the developing oocytes arrest in diakinesis (G(2)/M arrest). During oocyte maturation, a signal from the sperm reactivates MAPK to promote M phase entry. Here, we show that the MAPK phosphatase LIP-1 dephosphorylates MAPK as germ cells exit pachytene in order to maintain MAPK in an inactive state during oocyte development. Germ cells lacking LIP-1 fail to arrest the cell cycle at the G(2)/M boundary, and they enter a mitotic cell cycle without fertilization. LIP-1 thus coordinates oocyte cell cycle progression and maturation with ovulation and fertilization.
    The EMBO Journal 09/2002; 21(16):4317-26. DOI:10.1093/emboj/cdf430 · 10.75 Impact Factor
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