Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cell migration on extracellular matrix requires the turnover of integrin-dependent adhesions. The nonreceptor tyrosine kinases Src and FAK regulate focal-adhesion turnover by poorly understood mechanisms. ERK/MAP kinase-mediated activation of the protease Calpain 2 also promotes focal-adhesion turnover; however, it is not known if this is linked to the activities of Src and FAK. Calpain 2 has previously been demonstrated to colocalize with focal-adhesion structures and can cleave several focal-adhesion complex components, including FAK. Studies utilizing Calpain inhibitors or Calpain-deficient cells confirm that Calpain's role in regulating focal-adhesion turnover is necessary for cell migration. We have identified a novel and kinase-independent function for FAK as an adaptor molecule that mediates the assembly of a complex consisting of at least Calpain 2 and p42ERK. Mutation of proline residues (Pro2) in the amino-terminal region of FAK blocks direct binding with Calpain 2 and also prevents formation of the Calpain 2/p42ERK complex in cells. We show that both complex formation and MEK/ERK activity are associated with Calpain-mediated proteolysis of FAK and focal adhesion turnover during transformation and migration. Furthermore, FAK is necessary for recruiting both Calpain 2 and p42ERK/MAPK to peripheral adhesion sites facilitating maximal Calpain activity.
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PMID:A novel role for FAK as a protease-targeting adaptor protein: regulation by p42 ERK and Src. 1293 30

Identical proline-->arginine gain-of-function mutations in fibroblast growth factor receptor (FGFR) 1 (Pro252Arg), FGFR2 (Pro253Arg) and FGFR3 (Pro250Arg), result in type I Pfeiffer, Apert and Muenke craniosynostosis syndromes, respectively. Here, we characterize the effects of proline-->arginine mutations in FGFR1c and FGFR3c on ligand binding using surface plasmon resonance and X-ray crystallography. Both Pro252Arg FGFR1c and Pro250Arg FGFR3c exhibit an enhancement in ligand binding in comparison to their respective wild-type receptors. Interestingly, binding of both mutant receptors to FGF9 was notably enhanced and implicates FGF9 as a potential pathophysiological ligand for mutant FGFRs in mediating craniosynostosis. The crystal structure, of Pro252Arg FGFR1c in complex with FGF2, demonstrates that the enhanced ligand binding is due to an additional set of receptor-ligand hydrogen bonds, similar to those gain-of-function interactions that occur in the Apert syndrome Pro253Arg FGFR2c-FGF2 crystal structure. However, unlike the Apert syndrome Pro253Arg FGFR2c mutant, neither the Pfeiffer syndrome Pro250Arg FGFR1c mutant nor the Muenke syndrome Pro250Arg FGFR3c mutant bound appreciably to FGF7 or FGF10. This observation provides a potential explanation for why the limb phenotypes, observed in type I Pfeiffer and Muenke syndromes, are less severe than the limb abnormalities observed in Apert syndrome. Hence, although analogous proline-->arginine mutations in FGFR1-3 act through a common structural mechanism to result in gain-of-function, differences in the primary sequence among FGFRs result in varying effects on ligand binding specificity.
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PMID:Proline to arginine mutations in FGF receptors 1 and 3 result in Pfeiffer and Muenke craniosynostosis syndromes through enhancement of FGF binding affinity. 1461 73

MET is a receptor protein tyrosine kinase for hepatocyte growth factor, a multifunctional cytokine controlling cell growth, morphogenesis, and motility. In our previous study, RanBPM/RanBP9, whose name originated from its ability to interact with Ran, was identified as a MET-interacting protein. RanBPM/RanBP9 activates the Ras/Erk signaling pathway by serving as an adaptor protein of MET to recruit Sos. In this study, we identify a protein sharing a high amino acid sequence identity with RanBPM/RanBP9, especially in its SPRY domain, the region responsible for MET binding. This protein lacks the N-terminal poly-proline and poly-glutamine (Poly-PQ) stretch present in RanBPM/RanBP9 and has less homology with RanBPM/RanBP9 in its mid-region. We subsequently named this protein RanBP10 after demonstrating its interaction with Ran. We show that, like RanBPM/RanBP9, RanBP10 interacts with the tyrosine kinase domain of MET via its SPRY domain and these two proteins can compete with each other to bind to MET. Interestingly, unlike RanBPM/RanBP9, overexpression of RanBP10 cannot induce Erk1/2 phosphorylation and serum response element-luciferase (SRE-LUC) reporter gene expression. More importantly, co-transfection of RanBPM/RanBP9 and RanBP10 significantly represses SRE-LUC reporter gene expression induced by overexpression of RanBPM/RanBP9. Additional binding assays demonstrate that RanBP10 fails to interact with Sos, which explains its inability to activate the Ras/Erk pathway. Furthermore, we show that the N-terminus of RanBPM/RanBP9 with the Poly-PQ stretch is required for recruiting Sos and a truncated RanBPM/RanBP9 lacking this region fails to recruit Sos, indicating that the functional difference between RanBP10 and RanBPM/RanBP9 lies in their sequence difference in their N-termini.
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PMID:A novel MET-interacting protein shares high sequence similarity with RanBPM, but fails to stimulate MET-induced Ras/Erk signaling. 1468 63

Hereditary medullary thyroid carcinoma, a tumor that arises from the parafollicular cells of the thyroid gland, occurs in isolation (as in familial medullary thyroid carcinoma), in association with hyperparathyroidism and pheochromocytoma (as in multiple endocrine neoplasia type 2A), or in association with pheochromocytoma, marfanoid habitus, and mucosal neuromas (as in multiple endocrine neoplasia type 2B). These genetic syndromes are associated with germline-activating mutations of the RET protooncogene, a cell surface tyrosine kinase receptor, which is believed to modulate specific intracellular signaling pathways involved in the regulation of C cell proliferation and apoptosis. RET-activating mutations involve two important functional areas of the receptor: the cysteine-rich extracellular domain and the intracellular tyrosine kinase domain. Multiple endocrine neoplasia type 2A and familial medullary thyroid carcinoma are more commonly associated with mutations in the cysteine-rich extracellular domain, whereas multiple endocrine neoplasia type 2B is exclusively associated with mutations involving the second intracellular tyrosine kinase domain. Here, we describe a novel missense mutation of the RET protooncogene that substitutes arginine for proline at codon 912 of the intracellular tyrosine kinase domain in a family with medullary thyroid carcinoma.
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PMID:A novel point mutation of the RET protooncogene involving the second intracellular tyrosine kinase domain in a family with medullary thyroid carcinoma. 1524 Jun 41

Prolidase [E.C. 3.4.13.9] is a cytosolic imidodipeptidase that plays an important role in collagen biosynthesis. The enzyme contributes to the recovery of proline from protein degradation products (mainly collagen) for collagen resynthesis. Prolidase activity and collagen biosynthesis are supposed to be regulated by beta(1)-integrins, which initiate a signaling pathway in which several kinases and intracellular proteins are involved, including focal adhesion kinase pp125(FAK) (FAK), Src, Shc, growth factor receptor bound protein 2 (Grb-2), son of sevenless protein (SOS), Ras, Raf and mitogen-activated protein kinases (MAPK), extracellular-signal regulated kinase 1 (ERK(1)) and kinase 2 (ERK(2)). We studied the effects of echistatin, a well-known disintegrin and thrombin, a serine protease capable of activation of platelet integrin alpha(2)beta(1) receptor on collagen production, prolidase activity, expression of prolidase, beta(1)-integrin receptor, FAK, SOS-protein and phosphorylated MAP-kinases (ERK(1) and ERK(2)) in confluent human dermal fibroblasts. It has been found that treatment of the cells with 100nM echistatin contributes to inhibition of collagen production, as well as prolidase activity and expression compared to control cells. These phenomena were accompanied by a decrease in the expression of FAK, SOS-protein and phosphorylated MAP-kinases, ERK(1) and ERK(2). An opposite phenomenon was observed in fibroblasts treated with 0.1IU thrombin. In this case, a significant increase in collagen production and prolidase activity, accompanied by a distinct raise in the expression of prolidase, FAK and phosphorylated MAP-kinases and a slight increase in expression of SOS compared to controls were found. The results suggest that regulation of prolidase activity and collagen biosynthesis in human dermal fibroblasts may involve beta(1)-integrin-dependent signaling.
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PMID:Differential effects of echistatin and thrombin on collagen production and prolidase activity in human dermal fibroblasts and their possible implication in beta1-integrin-mediated signaling. 1566 71

The Raf-1 kinase is an important signaling molecule, functioning in the Ras pathway to transmit mitogenic, differentiative, and oncogenic signals to the downstream kinases MEK and ERK. Because of its integral role in cell signaling, Raf-1 activity must be precisely controlled. Previous studies have shown that phosphorylation is required for Raf-1 activation, and here, we identify six phosphorylation sites that contribute to the downregulation of Raf-1 after mitogen stimulation. Five of the identified sites are proline-directed targets of activated ERK, and phosphorylation of all six sites requires MEK signaling, indicating a negative feedback mechanism. Hyperphosphorylation of these six sites inhibits the Ras/Raf-1 interaction and desensitizes Raf-1 to additional stimuli. The hyperphosphorylated/desensitized Raf-1 is subsequently dephosphorylated and returned to a signaling-competent state through interactions with the protein phosphatase PP2A and the prolyl isomerase Pin1. These findings elucidate a critical Raf-1 regulatory mechanism that contributes to the sensitive, temporal modulation of Ras signaling.
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PMID:Regulation of Raf-1 by direct feedback phosphorylation. 1566 84

Protein kinases play important roles in many disease processes and are primary targets for drug development. Because cellular phosphorylation cascades are complex multidirectional pathways, the behavior of a drug in a biochemical enzyme assay may not accurately reflect its performance in the context of a whole cell. We have developed a near-infrared cytoblot assay that can be used to investigate both kinase signaling and effects of kinase inhibitors. Adherent cells were grown in either 96- or 384-well plates. Following stimulation, protein phosphorylation was detected immunohistochemically by simultaneous staining with two primary antibodies: a phospho-specific primary and normalization antibody that recognized either the target protein regardless of phosphorylation status (pan protein) or a housekeeping protein. Secondary antibodies labeled with two spectrally distinct near-infrared dyes were used for visualization. Nuclear staining with TO-PRO-3 was also used in place of the normalization antibody. Normalization for well-to-well variability was accomplished by ratiometric analysis of the two wavelengths. The near-infrared cytoblot was used to analyze phosphorylation of EGFR, Akt, Stat3, MEK 1, and ERK1/2. This assay format was also able to simultaneously assess the phosphorylation of multiple signaling proteins in response to known kinase inhibitors. We observed that the IC50 for the EGFR inhibitor PD168393 was similar for EGFR and Stat3 but was significantly higher for ERK1/2, a downstream modulator of EGFR function. The observation that the receptor and its effectors show different IC50 values for the same inhibitory drug could be important for target selection in drug development.
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PMID:A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy. 1570 44

Surfactant deficiency contributes to acute lung injury and may result from the elaboration of bioactive lipids such as oxysterols. We observed that the oxysterol 22-hydroxycholesterol (22-HC) in combination with its obligate partner, 9-cis-retinoic acid (9-cis-RA), decreased surfactant phosphatidylcholine (PtdCho) synthesis by increasing phosphorylation of the regulatory enzyme CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Phosphorylation of CCTalpha decreased its activity. 22-HC/9-cis-RA inhibition of PtdCho synthesis was blocked by PD98059 or dominant-negative ERK (p42 kinase). Overexpression of constitutively active MEK1, the kinase upstream of p42 kinase, increased CCTalpha phosphorylation. Expression of truncated CCTalpha mutants lacking proline-directed sites within the C-terminal phosphorylation domain partially blocked oxysterol-mediated inhibition of PtdCho synthesis. Mutagenesis of Ser315 within CCTalpha was both required and sufficient to confer significant resistance to 22-HC/9-cis-RA inhibition of PtdCho synthesis. A novel putative ERK-docking domain N-terminal to this phosphoacceptor site was mapped within the CCTalpha membrane-binding domain (residues 287-300). The results are the first demonstration of a physiologically relevant phosphorylation site and docking domain within CCTalpha that serve as targets for ERKs, resulting in inhibition of surfactant synthesis.
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PMID:Oxysterols inhibit phosphatidylcholine synthesis via ERK docking and phosphorylation of CTP:phosphocholine cytidylyltransferase. 1578 6

Rho GTPases are important regulators for cell dynamics. They are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We recently identified a novel RhoGAP, BPGAP1, that uses the BNIP-2 and Cdc42GAP homology (BCH) domain, RhoGAP domain and proline-rich region to regulate cell morphology and migration. To further explore its roles in intracellular signaling, we employed protein precipitations and matrix-assisted laser desorption/ionization mass-spectrometry and identified EEN/endophilin II as a novel partner of BPGAP1. EEN is a member of the endocytic endophilin family but its function in regulating endocytosis remains unclear. Pull-down and co-immunoprecipitation studies with deletion mutants confirmed that EEN interacted directly with BPGAP1 via its Src homology 3 (SH3) domain binding to the proline-rich region 182-PPPRPPLP-189 of BPGAP1, with prolines 184 and 186 being indispensable for this interaction. Overexpression of EEN or BPGAP1 alone induced EGF-stimulated receptor endocytosis and ERK1/2 phosphorylation. These processes were further enhanced when EEN was present together with the wildtype but not with the non-interactive proline mutant of BPGAP1. However, EEN lacking the SH3 domain served as a dominant negative mutant that completely inhibited these effects. Furthermore, BPGAP1 with a catalytically inactive GAP domain also blocked the effect of EEN and/or BPGAP1 in EGF receptor endocytosis and concomitantly reduced their level of augmentation for ERK1/2 phosphorylation. Our findings reveal a concomitant activation of endocytosis and ERK signaling by BPGAP1 via the coupling of its proline-rich region, which targets EEN and its functional GAP domain. BPGAP1 could therefore provide an important link between cytoskeletal network, endocytic trafficking and Ras/MAPK signaling.
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PMID:Activation of EGF receptor endocytosis and ERK1/2 signaling by BPGAP1 requires direct interaction with EEN/endophilin II and a functional RhoGAP domain. 1594 98

TRIP6 (thyroid receptor-interacting protein 6), also known as ZRP-1 (zyxin-related protein 1), is a member of the zyxin family that has been implicated in cell motility. Previously we have shown that TRIP6 binds to the LPA2 receptor and associates with several components of focal complexes in an agonist-dependent manner and, thus, enhances lysophosphatidic acid (LPA)-induced cell migration. Here we further report that the function of TRIP6 in LPA signaling is regulated by c-Src-mediated phosphorylation of TRIP6 at the Tyr-55 residue. LPA stimulation induces tyrosine phosphorylation of endogenous TRIP6 in NIH 3T3 cells and c-Src-expressing fibroblasts, which is virtually eliminated in Src-null fibroblasts. Strikingly, both phosphotyrosine-55 and proline-58 residues of TRIP6 are required for Crk binding in vitro and in cells. Mutation of Tyr-55 to Phe does not alter the ability of TRIP6 to localize at focal adhesions or associate with actin. However, it abolishes the association of TRIP6 with Crk and p130cas in cells and significantly reduces the function of TRIP6 to promote LPA-induced ERK activation. Ultimately, these signaling events control TRIP6 function in promoting LPA-induced morphological changes and cell migration.
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PMID:c-Src-mediated phosphorylation of TRIP6 regulates its function in lysophosphatidic acid-induced cell migration. 1598 3


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