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)

Endothelin-1 (ET-1), a member of a family of 21 amino acid peptides possessing vasoconstrictor properties, is known to stimulate mesangial cell proliferation. In this study, ET-1 (100 nm) induced a rapid activation of p21(ras) in human glomerular mesangial cells (HMC). Inhibition of Src family tyrosine kinase activation with [4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or chelation of intracellular free calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester significantly decreased ET-1dependent p21(ras) activation and suggested the involvement of the cytoplasmic proline-rich tyrosine kinase Pyk2. We have observed that Pyk2 was expressed in HMC and was tyrosine-phosphorylated within 5 min of ET-1 treatment. ET-1-induced activation of Pyk2 was further confirmed using phospho-specific anti-Pyk2 antibodies. Surprisingly, Src kinase activity was required upstream of ET-1-induced autophosphorylation of Pyk2. To determine whether Pyk2 autophosphorylation mediated ET-1-dependent p21(ras) activation, adenovirus-mediated transfer was employed to express a dominant-negative form of Pyk2 (CRNK). CRNK expression inhibited ET-1-induced endogenous Pyk2 autophosphorylation, but did not abolish ET-1-mediated increases in GTP-bound p21(ras) levels. ET-1-induced activation of the p38 MAPK (but not ERK) pathway was inhibited in HMC and in rat glomerular mesangial cells expressing the dominant-negative form of Pyk2. These findings suggest that the engagement of Pyk2 is important for ET-1-mediated p38 MAPK activation and hence the biological effect of this peptide in mesangial cells.
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PMID:Protein-tyrosine kinase Pyk2 mediates endothelin-induced p38 MAPK activation in glomerular mesangial cells. 1127 44

Here, we report the identification and characterization of a new member of the RalGDS-family, which is widely expressed and interacts strongly and selectively with the GTP-bound forms of M-Ras and p21 Ras. This Ras pathway modulator (RPM), also termed RGL3, exhibited Ras-binding and catalytic domains typical of the RalGDS-family of guanine nucleotide exchange factors, and was most similar to Rlf (RalGDS-like factor), but was distinguished by a unique proline-rich region with multiple candidate SH3-domain binding sites. RPM/RGL3 resembled AF-6 and Nore1 in interacting strongly with constitutively active M-Ras and p21 Ras. In contrast to Rlf, transiently expressed RPM/RGL3 did not activate an Elk-1-inducible reporter gene alone or in combination with activated p21 Ras, but strongly inhibited induction of this reporter gene by co-expression of activated H-Ras or MEKK-1. This inhibitory effect was independent of the Ras binding domain and required a second signal provided by p21 Ras or MEKK-1, but not Raf-1 or M-Ras. Expression of RPM/RGL3 also strongly inhibited cell growth of fibroblasts transformed by an activated Src Y527F. Thus, RPM/RGL3 is a novel potential effector of both p21 Ras and M-Ras with the novel function of negatively regulating Elk-1-dependent gene induction downstream of p21 Ras or MEKK-1.
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PMID:A novel potential effector of M-Ras and p21 Ras negatively regulates p21 Ras-mediated gene induction and cell growth. 1131 46

DOC-2/DAB2 (differentially expressed in ovarian carcinoma-2/disabled 2) appears to be a potential tumor suppressor gene with a growth inhibitory effect on several cancer types. Previously, we have shown that DOC-2/DAB2 suppresses protein kinase C-induced AP-1 activation, which is modulated by serine 24 phosphorylation in the N terminus of DOC-2/DAB2. However, the functional impact of the C terminus of DOC-2/DAB2, containing three proline-rich domains, has not been explored. In this study, we examined this functional role in modulating signaling mediated by peptide growth factor receptor tyrosine kinase, particularly because it involves the interaction with Grb2. Using sequence-specific peptides, we found that the second proline-rich domain of DOC-2/DAB2 is the key binding site to Grb2 in the presence of growth factors. Such elevated binding interrupts the binding between SOS and Grb2, which consequently suppresses downstream ERK phosphorylation. Reduced ERK phosphorylation was restored when the binding between DOC-2/DAB2 and Grb2 was interrupted by a specific peptide or by increasing the expression of Grb2. Furthermore, the C terminus of the DOC-2/DAB2 construct can inhibit the AP-1 activity elicited by growth factors. We conclude that DOC-2/DAB2, a potent negative regulator, can suppress ERK activation by interrupting the binding between Grb2 and SOS that is elicited by peptide growth factors. This study further illustrates that DOC-2/DAB2 has multiple effects on the RAS-mediated signal cascades active in cancer cells.
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PMID:The inhibitory role of DOC-2/DAB2 in growth factor receptor-mediated signal cascade. DOC-2/DAB2-mediated inhibition of ERK phosphorylation via binding to Grb2. 1137 63

APS [for 'adapter protein with a pleckstrin homology (PH) and Src homology 2 (SH2) domain'] belongs to a family of adapter proteins involved in signalling by the receptors for insulin, insulin-like growth factor 1, platelet-derived growth factor and nerve growth factor. Other members include alternatively spliced SH2-B isoforms (SH2Balpha, SH2-Bbeta and SH2-Bgamma) and Lnk. These have a C-terminal SH2 domain, a central PH domain and an N-terminal proline-rich region. SH2Balpha, APS and Lnk have a conserved C-terminal tyrosine phosphorylation site, whereas the alternatively spliced SH2-Bbeta and SH2-Bgamma have distinct C-termini. There is considerable sequence similarity between APS, SH2-B and Lnk, particularly in the SH2 domain. Both APS and SH2-Balpha interact with the insulin-receptor activation loop phosphorylation sites and undergo insulin-stimulated tyrosine phosphorylation, although the phosphorylation of SH2-B is considerably weaker. APS couples c-Cbl to the insulin receptor, resulting in ubiquitination of the insulin receptor. We established cell lines [Chinese hamster ovary (CHO). T-APS and CHO. T-SH2-B cells] overexpressing APS and SH2-Balpha to study their roles in insulin receptor signalling. Either adapter protein enhances insulin receptor and ERK (extracellular-signal-regulated kinase) phosphorylation. In CHO. T-APS cells, Akt phosphorylation is observed earlier than in CHO.T-SH2-B cells. Both enhance insulin-stimulated Akt activation but APS seems to cause greater activation. Thus APS and SH2-B have similar effects on insulin receptor signalling, although the effects of SH2-B are independent of its phosphorylation.
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PMID:Functional effects of APS and SH2-B on insulin receptor signalling. 1149 22

The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The biology and pathogenesis of HEV remain poorly understood. We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins. This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85alpha regulatory subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma, and the adaptor protein Grb2. A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction. The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins. Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3.SH3 complex. In pORF3-expressing cells, pp60(src) was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells. However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells. These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling. The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection.
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PMID:The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK. 1151 2

The C-terminal region of mitogen-activated protein kinase kinase-1 and 2 (MKK1 and MKK2) may function in regulating interactions with upstream kinases or the magnitude and duration of ERK mitogen-activated protein kinase activity. The MKK C-terminal region contains a proline-rich region that reportedly functions in regulating interactions with the Raf-1 kinase and ERK activity. In addition, phosphorylation sites in the C terminus of MKK1 have been suggested to either sustain or attenuate MKK1 activity. To further understand how phosphorylation at the C terminus of MKK1 and protein interactions regulate MKK1 function, we have generated several MKK1 C-terminal deletion mutants and examined their function in regulating MKK1 localization, ERK protein activation, and cell growth. A deletion of C-terminal amino acids encompassing two putative alpha-helices between residues 330 and 379 caused a re-distribution of mutant MKK1 proteins to membrane compartments. Immunofluorescence analysis of MKK1 mutants revealed a loss of homogenous cytosolic distribution that is typically observed with MKK1 wild type, suggesting this region regulates MKK1 cellular localization. In contrast, MKK1 C-terminal deletion mutants localized to various sized punctate regions that overlapped with lysosome compartments. ERK activation in response to constitutively active Raf-1 or growth factor stimulus was attenuated in cells expressing MKK1 C-terminal deletion mutants. This could be partly explained by the inability of Raf-1 to phosphorylate MKK1 C-terminal deletion mutants even though the phosphorylation sites were intact in these mutants. Finally, we show that cells expressing MKK1 C-terminal deletion mutants displayed characteristic patterns of apoptotic cell death and reduced cell proliferation. These findings identify a novel C-terminal region between amino acid residues 330 and 379 on MKK1 that is necessary for regulating the cytoplasmic distribution and subsequent ERK protein activation necessary for cell survival and viability.
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PMID:Identification of a C-terminal region that regulates mitogen-activated protein kinase kinase-1 cytoplasmic localization and ERK activation. 1160 1

Growth factor receptor-binding protein-2 (Grb2) plays a key role in signal transduction initiated by Bcr/Abl oncoproteins and growth factors, functioning as an adaptor protein through its Src homology 2 and 3 (SH2 and SH3) domains. We found that Grb2 was tyrosine-phosphorylated in cells expressing BCR/ABL and in A431 cells stimulated with epidermal growth factor (EGF). Phosphorylation of Grb2 by Bcr/Abl or EGF receptor reduced its SH3-dependent binding to Sos in vivo, but not its SH2-dependent binding to Bcr/Abl. Tyr209 within the C-terminal SH3 domain of Grb2 was identified as one of the tyrosine phosphorylation sites, and phosphorylation of Tyr209 abolished the binding of the SH3 domain to a proline-rich Sos peptide in vitro. In vivo expression of a Grb2 mutant where Tyr209 was changed to phenylalanine enhanced BCR/ABL-induced ERK activation and fibroblast transformation, and potentiated and prolonged Grb2-mediated activation of Ras, mitogen-activated protein kinase and c-Jun N-terminal kinase in response to EGF stimulation. These results suggest that tyrosine phosphorylation of Grb2 is a novel mechanism of down-regulation of tyrosine kinase signaling.
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PMID:Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling. 1172 15

Exercise increases glucose transport in muscle by activating 5'-AMP-activated protein kinase (AMPK), but subsequent events are unclear. Presently, we examined the possibility that AMPK increases glucose transport through atypical protein kinase Cs (aPKCs) by activating proline-rich tyrosine kinase-2 (PYK2), ERK pathway components, and phospholipase D (PLD). In mice, treadmill exercise rapidly activated ERK and aPKCs in mouse vastus lateralis muscles. In rat extensor digitorum longus (EDL) muscles, (a) AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-d-riboside (AICAR), activated PYK2, ERK and aPKCs; (b) effects of AICAR on ERK and aPKCs were blocked by tyrosine kinase inhibitor, genistein, and MEK1 inhibitor, PD98059; and (c) effects of AICAR on aPKCs and 2-deoxyglucose (2-DOG) uptake were inhibited by genistein, PD98059, and PLD-inhibitor, 1-butanol. Similarly, in L6 myotubes, (a) AICAR activated PYK2, ERK, PLD, and aPKCs; (b) effects of AICAR on ERK were inhibited by genistein, PD98059, and expression of dominant-negative PYK2; (c) effects of AICAR on PLD were inhibited by MEK1 inhibitor UO126; (d) effects of AICAR on aPKCs were inhibited by genistein, PD98059, 1-butanol, and expression of dominant-negative forms of PYK2, GRB2, SOS, RAS, RAF, and ERK; and (e) effects of AICAR on 2DOG uptake/GLUT4 translocation were inhibited by genistein, PD98059, UO126, 1-butanol, cell-permeable myristoylated PKC-zeta pseudosubstrate, and expression of kinase-inactive RAF, ERK, and PKC-zeta. AMPK activator dinitrophenol had effects on ERK, aPKCs, and 2-DOG uptake similar to those of AICAR. Our findings suggest that effects of exercise on glucose transport that are dependent on AMPK are mediated via PYK2, the ERK pathway, PLD, and aPKCs.
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PMID:Activation of the ERK pathway and atypical protein kinase C isoforms in exercise- and aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR)-stimulated glucose transport. 1197 88

Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the alpha5beta1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the alpha5beta1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCdelta inhibitor rottlerin. Furthermore, PKCdelta translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after cotransfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves FN-f stimulation of the alpha5beta1 integrin and activation of the nonreceptor tyrosine kinase PYK2 by PKC, most likely PKCdelta
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PMID:Fibronectin fragment activation of proline-rich tyrosine kinase PYK2 mediates integrin signals regulating collagenase-3 expression by human chondrocytes through a protein kinase C-dependent pathway. 1273 Feb 23

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.
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PMID:Antibody-mediated targeting of replication-competent retroviral vectors. 1280 41


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