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)

Separation and purification of rSj32 were carried out with SDS-PAGE and electroelution technique. The purified rSj32, AWA and SEA were used to examine sera of patients with schistosomiasis, healthy individuals and patients with other parasitic diseases by ELISA. The results showed that the molecule weight of rSj32 was 37 kD. The sensitivity and specificity of rSj32 as a coating antigen in ELISA were as good as those of AWA and SEA. It is suggested that rSj32 antigen is a potential antigen for immunodiagnosis of schistosomiasis japonica.
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PMID:[Schistosoma japonicum: purification and diagnostic application of recombinant 32 kD antigen]. 1118 85

Macrophage colony stimulating factor (M-CSF or CSF-1) acts to regulate the development and function of cells of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with the CSF-1 receptor (FD/WT) adopt a macrophage-like morphology when cultured in CSF-1. This process is abrogated in FDC-P1 cells transfected with the CSF-1 receptor with a tyrosine to phenyalanine substitution at position 807 (FD/807), suggesting that a molecular interaction critical to differentiation signaling is lost (Bourette, R. P., Myles, G. M., Carlberg, K., Chen, A. R., and Rohrschneider, L. R. (1995) Cell Growth Differ. 6, 631--645). A detailed examination of lysates of CSF-1-treated FD/807 cells by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins whose degree of tyrosine phosphorylation was modulated by the Y807F mutation. Included in this category were three phosphorylated proteins that co-migrated with p46/52(Shc). Immunoprecipitation, Western blotting, and in vitro binding studies suggest that they are indeed p46/52(Shc). A key regulator of differentiation in a number of cell systems, ERK was observed to exhibit an activity that correlated with the relative degree of differentiation induced by CSF-1 in the two cell types. Transfection of cells with a non-tyrosine-phosphorylatable form of p46/52(Shc) prevented the normally observed CSF-1-mediated macrophage differentiation as determined by adoption of macrophage-like morphology and expression of the monocyte/macrophage lineage cell surface marker, Mac-1. These results are the first to suggest that p46/52(Shc) may play a role in CSF-1-induced macrophage differentiation. Additionally, a number of proteins were identified by two-dimensional SDS-PAGE whose degree of tyrosine phosphorylation is also modulated by the Y807F substitution. This group of molecules may contain novel signaling molecules important in macrophage differentiation.
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PMID:Proteomic analysis of macrophage differentiation. p46/52(Shc) Tyrosine phosphorylation is required for CSF-1-mediated macrophage differentiation. 1129 Jul 43

The 35-residue peptide corresponding to the very hydrophobic transmembrane region of the tyrosine kinase receptor neu, Neu(TM35), has been synthesized. The peptide can be solubilized in millimolar concentrations in TFE or incorporated into an SDS-water micellar solution or into well-hydrated DMPC/DCPC bicelles. In all these media, circular dichroism demonstrated that the peptide adopts a helical structure for about 80% of its amino acids. The peptide is monomeric below 2 mM in TFE, as also determined by variable concentration experiments. The three-dimensional solution structure in TFE has been obtained by homonuclear proton NMR and shows a well-defined alpha-helix from residues 4 to 21, then a pi-bulge from Ile(22) to Gly(28), and a final short alpha-helix from positions 29 to 32. This experimental finding is in agreement with structures predicted recently by molecular dynamics calculations in a vacuum [Sajot, N., and Genest, M. (2000) Eur. Biophys. J. 28, 648-662]. The biological implications of a possible retention of this structure in a membrane environment are finally discussed.
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PMID:Evidence for an alpha-helix --> pi-bulge helicity modulation for the neu/erbB-2 membrane-spanning segment. A 1H NMR and circular dichroism study. 1137 Dec 17

The cDNA fragment of the first 3 loops of VEGF receptor, KDR, was cloned by PCR and inserted into a baculovirus expression plasmid pFASTBACI. The competent E. coli DH10BAC cell, which contain another plasmid with baculovirus genome in it, was transformed with pFASTBACI-KDRn3. Homologous recombination in the prokaryotic cells resulted in a recombinant plasmid containing KDRn3 in baculovirus genome. Transfection of the insect cell SF-9 with above plasmid generated a recombinant baculorvirus contain target gene fragment. SDS-PAGE and Western blot analysis of the supernatant of the infected SF-9 cell showed that KDRn3 was secreted in the medium. The recombinant protein was verified with Western blot and tested for their binding activity with VEGF. Its anti-angiogenic activity was assayed on chorionic allantoic membrane(CAM) of fertilized egg. The results showed that the recombinant protein could inhibit new vessel formation on CAM of fertilized eggs.
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PMID:[Cloning of VEGF receptor KDR and its expression in insect cells]. 1141 Dec 19

The protein release profiles and the morphology of poly(D,L-lactide-co-glycolide) (PLG) and poly(epsilon-caprolactone) (PCL) microcapsules were investigated. The microcapsules were prepared by the (oil(1)-in-oil2)-in-water emulsion solvent evaporation method using bovine serum albumin (BSA) as a model protein. The internal and external morphologies of the microcapsules were examined using a light microscope, scanning electron microscope and a laser scanning confocal microscope. A Coulter counter was used to determine particle size and particle size distribution. Protein quantitation and molecular integrity were performed by the bicinchoninic acid protein assay micro-method and SDS-PAGE, respectively. Microcapsules with a polymeric wall surrounding an oily core containing the protein were formed. The encapsulation efficiency (39-96%) for PLG and (13-90%) for PCL increased with polymer molecular weight and particle volume mean diameter (Vmd). Vmd ranged from 87-128 to 42-157 microm for PLG and PCL, respectively. The protein release profile for PLG microcapsules was either continuous or irregularly pulsatile depending on particle morphology and was completed after cavity breakdown. However, that of PCL microcapsules was essentially irregularly pulsatile and was completed after a longer period of time without cavity breakdown but with significant swelling. There was no detectable cleavage of the protein during 6 months storage of PLG and PCL microcapsules at 4 degrees C. Furthermore, insignificant degradation of protein occurred during in vitro release from PCL microcapsules. In contrast, significant degradation occurred in PLG microcapsules. This approach to microencapsulation of a protein may be promising for the controlled delivery of protein vaccines, and the oil core may enhance the immunogenicity of some weak subunit vaccine candidates.
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PMID:Protein release profiles and morphology of biodegradable microcapsules containing an oily core. 1157 45

An expression vector (pJW4) for a human epidermal growth factor (hEGF)-CH1 fusion protein was constructed by fusing the gene for hEGF with the gene for CH1 of murine IgG1 with/without a peptide linker sequence [(GGGGS)3] and inserting the recombinant gene into vector pGEX2T. Expression vector pGEX2T was transfected into E. coli (BL-21) and hEGF-CH1 expressed by induction of the lac Iq promotor with 50 microM isopropyl beta-D-thiogalactopyranoside (IPTG). hEGF- CH1 fused to glutathione S-transferase (GST) was isolated and purified by affinity chromatography. GST was cleaved using thrombin. SDS-PAGE demonstrated a protein with the expected M(r) (18 kDa) positive for hEGF by Western blot. hEGF-linker-CH1 exhibited preserved binding to A431 (2-3 x 10(6) EGFR/cell) and MDA-MB-468 breast cancer cells (1-2 x 10(6) EGFR/cell). hEGF-CH1 without the linker exhibited poor receptor binding. hEGF-linker-CH1 also exhibited strong binding to soluble EGFR equivalent to that of hEGF. The tumor and normal tissue distribution of hEGF-linker-CH1 labeled with 123I was compared with 123 I-hEGF at 24 h after i.v. injection to mice implanted with s.c. MDA-MB-468 xenografts. Fusion of hEGF with CH1 increased its retention in the blood 14-fold but did not significantly increase tumor uptake. Tumor/blood ratios were higher for hEGF than for hEGF-linker-CH1. We conclude that hEGF is more attractive than hEGF-linker-CH1 for imaging EGFR-positive tumors.
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PMID:Fusion of the CH1 domain of IgG1 to epidermal growth factor (EGF) prolongs its retention in the blood but does not increase tumor uptake. 1253 70

We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK >> p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK.
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PMID:Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase, at Ser-446. 1279 87

The nucleic acid sequence of the human gene, MUC17, indicates that this mucin contains an SEA domain, a transmembrane domain, and putative N-glycosylation sites in the carboxyl terminus. Mucins that possess an SEA domain are usually proteolytically cleaved within that domain to yield two subunits, the smaller of which is associated with the surface membrane. Homogenates of ASPC-1 pancreatic cancer cells showed three main bands of immunoreactivity with alpha-SEA (a polyclonal antibody directed against a site downstream of the postulated cleavage site) after SDS-PAGE and Western blotting (38, 45, and 49 kDa). Experiments utilizing N-glycan specific hydrolases showed that the 38 kDa band contained high mannose glycans whereas the 45 and 49 kDa bands contained complex-type glycans. Only two smaller alpha-SEA reactive bands (30 and 32 kDa) were present after cells had been treated with the N-glycosylation inhibitor tunicamycin. Surface biotinylation studies showed that only the forms possessing complex-type N-glycans were localized to the cell surface. Both tunicamycin and brefeldin A, an inhibitor of protein transport, reduced surface localization. In summary, our results indicate that the surface localization of the smaller subunit of MUC17 is dependent on its N-glycosylation status.
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PMID:N-glycosylation is required for the surface localization of MUC17 mucin. 1288 91

Growth hormone (GH), prostaglandins F (PGF) and prostaglandins E (PGE) are important regulators of ovarian function. Therefore, interrelationships between GH and these substances and their intracellular mechanisms might be of physiological significance in the ovary. The aims of this study on cultured porcine ovarian granulosa cells were to determine the effect of GH on the secretion of oxytocin (OT), PGF and PGE and whether MAP kinase could be involved in the mediation of GH action. Experiments were carried out with cultured porcine granulosa cells to investigate the effects of exogenous pGH (1-100 ng/ml) on the expression of MAP kinase (ERK-1, -2) and of PGH (1-100 ng/ml) and the MAP kinase blocker PD 98059 (1 microg/ml) on the secretion of PGF, PGE and OT. The cellular content of ERK-1 and -2 was analyzed by Western immunoblotting and immunocytochemistry, whilst PGF, PGE and OT accumulation in the medium was measured by RIA. Addition of GH to culture medium significantly altered the pattern of ovarian ERK MAP kinase on SDS-PA gels: the 44 and 42 kDa bands were reduced and additional 50 and 48 kDa bands appeared. Moreover, there was an increase in the percentage of cells containing ERK MAP kinase. GH stimulated the secretion of PGF (at a concentration of 1 ng GH per ml medium) and OT (100 ng GH per ml), but not PGE. The MAP kinase blocker alone did not affect PGF, PGE and OT secretion but did prevent the stimulatory effects of GH on PGF and induced stimulatory action of GH (10 ng/ml) on PGE. GH-stimulated OT secretion was unaffected. These observations confirm the role of GH in regulating porcine ovarian PGF, PGE and OT secretion and the presence of ERK MAP kinase in porcine granulosa cells. Furthermore, our studies demonstrate that MAP kinase-dependent intracellular mechanisms are dependent on GH, and that these mechanisms are involved in the mediation of GH action on ovarian PGF and PGE but not OT secretion.
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PMID:Involvement of MAP kinase in the mediation of GH action on ovarian granulosa cells. 1289 May 81

Fibroblast growth factor (FGF) receptor substrate 2 (FRS2) is a membrane-anchored docking protein that has been shown to play an important role in linking FGF, nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) receptors with the Ras/mitogen-activated protein (MAP) kinase signaling cascade. Here we provide evidence that FRS2 can also play a role in epidermal growth factor (EGF) signaling. Upon EGF stimulation, FRS2 mediates enhanced MAPK activity and undergoes phosphorylation on tyrosine as well as serine/threonine residues. This involves the direct interaction of the FRS2 PTB domain with the EGFR and results in a significantly altered mobility of FRS2 in SDS-PAGE which is also observed in FGF stimulated cells. This migration shift of FRS2 is completely abrogated by U0126, a specific MAPK kinase 1 (MEK1) inhibitor, suggesting that ERK1/2 acts as serine/threonine kinase upstream of FRS2. Indeed, we show that the central portion of FRS2 constitutively associates with ERK1/2, whereas the FRS2 carboxy-terminal region serves as substrate for ERK2 phosphorylation in response to EGF and FGF stimulation. Notably, tyrosine phosphorylation of FRS2 is enhanced when ERK1/2 activation is inhibited after both EGF and FGF stimulation. These results indicate a ligand-stimulated negative regulatory feedback loop in which activated ERK1/2 phosphorylates FRS2 on serine/threonine residues thereby down-regulating its tyrosine phosphorylation. Our findings support a broader role of FRS2 in EGFR-controlled signaling pathways in A-431 cells and provide insight into a molecular mechanism for ligand-stimulated feedback regulation with FRS2 as a central regulatory switch point.
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PMID:EGFR and FGFR signaling through FRS2 is subject to negative feedback control by ERK1/2. 1297 90


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