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 addition to the conserved phosphorylation site, PIMT also contained putative MAPK docking sites; which frequently mediate high level interaction between MAPK kinase and its substrate. PIMT has one D domain ( 119 RNKVKIKKK) and one DEF motif ( 228 WNFP;Figure S1) [23,262728293031. These observations led us to examine whether PIMT is a substrate of MAPK/ERK kinase. "
[Show abstract][Hide abstract]ABSTRACT: PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser(298) and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMT(S298D)) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMT(S298D) but not PIMT(S298A) augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser(298) phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser(298) is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia.
"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). "
[Show abstract][Hide abstract]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.
"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
[Show abstract][Hide abstract]ABSTRACT: Gastric cancer (GC) is a threat to human health with increasing incidence and mortality worldwide. Down-regulation or absence of RAF kinase inhibitor protein (RKIP) was associated with the occurrence, differentiation, invasion, and metastasis of GC. This study aims to investigate the molecular mechanisms and biological functions of RKIP in the GC biology.
The fusion expression plasmid pcDNA3.1-RKIP-3xFLAG was transfected into SGC7901 cells, the RKIP fusion proteins were purified with anti-flag M2 magnetic beads, and the RKIP-interacting proteins were identified with tandem mass spectrometry (MS/MS), and were analyzed with bioinformatics tools. Western blot and co-immunoprecipitation were used to confirm the interaction complex.
A total of 72 RKIP-interacting proteins were identified by MS/MS. Those proteins play roles in enzyme metabolism, molecular chaperoning, biological oxidation, cytoskeleton organization, signal transduction, and enzymolysis. Three RKIP-interaction protein network diagrams were constructed with Michigan Molecular Interactions, functional linage network, and Predictome analysis to address the molecular pathways of the functional activity of RKIP. The MS/MS-characterized components of the existing interaction complex (RKIP, HSP90, 14-3-3epsilon, and keratin 8) were confirmed by Western blot analysis and co-immunoprecipitation.
This study is the first discovery of the interaction of RKIP with HSP90, 14-3-3, and keratin. The present data would provide insight into the molecular mechanisms of how RKIP inhibits the occurrence and development of GC.