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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We show that Escherichia coli produce a factor that inhibits the activity of tyrosine and serine/threonine protein kinases. The factor is a protein found in the periplasmic compartment and is also secreted into the culture medium. Using a particle concentration fluorescence immunoassay specific for tyrosine kinase activity and inhibition of the tyrosine kinase p56(lck), we purified this factor to apparent homogeneity. Analysis of trypsin-digested fragments by mass spectrometry identified the inhibitor as the bacterial periplasmic protein UDP-sugar hydrolase, an enzyme with potent and nonspecific 5'-nucleotidase activity. Overexpression of the enzyme in bacteria leads to coordinate increases in both 5'-nucleotidase and p56(lck) inhibitory activity, confirming the identity of the inhibitor. The kinase inhibitory activity appears to be due to the formation of adenosine, which we show is inhibitory for p56(lck), cAMP-dependent protein kinase, and casein kinase. Overexpression of UDP-sugar hydrolase leads to an increase in the recovery of enteropathogenic E. coli following infection of HeLa cell monolayers and corresponding alterations in tyrosine-phosphorylated host proteins. These results suggest that UDP-sugar hydrolase may be an important factor affecting host cell function following intracellular bacterial infection.
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PMID:Identification of a bacterial inhibitor of protein kinases. Mechanism and role in host cell invasion. 879 49

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95 kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, in in vitro fertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.
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PMID:Early steps of sperm-egg interactions during mammalian fertilization. 893 5

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140trk. These biological effects of NGF depend upon the signal-mediating function of p140trk substrates which are likely to differ from cell to cell. To define p140trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140trk cDNA sequence. Two stably transfected clones, one expressing p140trk with higher affinity (PC12EN-trk3; Kd 57.4 pM, Bmax 9.7 pmole/mg) and one expressing p140trk with a lower affinity (PC12EN-trk1; Kd 392.4 pM, Bmax 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140trk receptors show slow NGF-dissociation kinetics, are resistant to trypsin or Triton X-100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140trk receptors. NGF stimulates p140trk tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70S6k, SNT, and phospholipase C gamma, demonstrating that the major NGF-stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [3H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140trk receptor substrates.
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PMID:Expression of human p140trk receptors in p140trk-deficient, PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis. 921 24

We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.
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PMID:Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor. 1002 55

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and by tyrosine kinase. Phosphorylation by PKA occurred in the 110 kDa native form of GPI-PLD as well as in multiple proteolytic degradation products and caused a significant decrease in enzyme activity. Dephosphorylation by treatment with alkaline phosphatase completely restored GPI-PLD activity. In addition, incubation of GPI-PLD with trypsin, which results in the generation of distinct peptide fragments, resulted in complete dephosphorylation of radiolabeled GPI-PLD. The site of phosphorylation by PKA was assigned to Thr-286. Tyrosine phosphorylation was only observed in a proteolytically processed fragment of GPI-PLD but not in the 110 kDa native form and had no effect on GPI-PLD activity.
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PMID:In vitro phosphorylation of purified glycosylphosphatidylinositol-specific phospholipase D. 1038 65

We have developed a new cell culture substrate grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) using an electron beam irradiation method. These surfaces are hydrophobic in culture at 37 degrees C due to the hydration/dehydration changes intrinsic to PIPAAm at 32 degrees C, and they become highly hydrophilic below 32 degrees C. At 37 degrees C grafted and ungrafted surfaces showed no difference with regard to attachment, spreading, growth, confluent cell density, and morphology of bovine aortic endothelial cells. Stress fibers, peripheral bands, and focal contacts were established in similar ways. After the medium temperature was decreased to 20 degrees C, spread cells lost their flattened morphology, acquiring a rounded cell appearance similar to that of cells immediately after plating. After mild agitation cells floated free from the dish surface without trypsin treatment. Neither cell morphological changes nor cell detachment occurred on ungrafted surfaces. An ATP synthesis inhibitor, sodium azide, and a tyrosine kinase inhibitor, genistein, suppressed cell morphological changes and cell detachment while a protein synthesis inhibitor, cycloheximide, slightly enhanced cell detachment. An actin filament stabilizer, phalloidin, and its depolymerizer, cytochalasin D, also inhibited cell detachment. These findings suggest that cell detachment on grafted surfaces is mediated by intracellular signal transduction and reorganization of the cytoskeleton. While trypsinization causes damage to the cell membrane surface and extracellular matrix proteins, this alternative low temperature treatment is exceptionally noninvasive. The temperature-responsive cell culture surface also should prove useful for investigating the molecular machinery involved in cell-surface detachment.
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PMID:Signal transduction and cytoskeletal reorganization are required for cell detachment from cell culture surfaces grafted with a temperature-responsive polymer. 1039 3

Protease-activated receptor-2 (PAR-2) is distributed throughout the gastrointestinal systems. The present study investigated the role for PAR-2 in the rat salivary glands. PAR-2 mRNA was detected in the sublingual, submaxillary, and parotid glands by a reverse-transcriptase polymerase chain reaction. In the isolated sublingual gland that exhibited the strongest signal for PAR-2, Ser-Leu-Ile-Gly-Arg-Leu-NH(2), a PAR-2-activating peptide, and trypsin, a PAR-2-activating enzyme, but not thrombin that can activate PARs 1, 3, and 4, triggered secretion of N-acetylneuraminic acid, an indicator of mucin, that was a unique major sialic acid detectable after hydrolysis of the sublingual mucin with 0.1 N HCl. The PAR-2-mediated secretion of mucin was attenuated by genistein, a tyrosine kinase inhibitor, but not by inhibitors of protein kinase C and phosphatidyl inositol 3'-kinase. Thus, PAR-2 is expressed by the three distinct salivary glands in the rat, and sublingual PAR-2 appears to play a role in triggering mucin secretion, at least in part, via activation of tyrosine kinase.
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PMID:Activation of protease-activated receptor-2 (PAR-2) triggers mucin secretion in the rat sublingual gland. 1073 43

Human fetuin, [alpha2-Heremans Schmid Glycoprotein (alpha2-HSG)], is a natural inhibitor of insulin receptor tyrosine kinase activity (IR-TKA). Previously, we have demonstrated that alpha2-HSG inhibits the mitogenic pathway without affecting the metabolic arm of insulin signal transduction. In this study, we demonstrate the time-course and specificity of inhibition, its interaction with IR and probable physiological role. In intact rat1 fibroblasts overexpressing the human insulin receptor (HIRc B), incubation of recombinant human alpha2-HSGbac (1.8 microM) inhibited insulin-induced IR autophosphorylation by over 80%. This inhibitory effect of alpha2-HSGbac on insulin-induced IR autophosphorylation was blunted by half in 60 min. Interestingly, alpha2-HSGbac at similar concentrations (0.9 or 1.8 microM), had no effect on EGF- or IGF-I-induced cognate receptor autophosphorylation. Anti-alpha2-HSG immunoprecipitates of alpha2-HSGbac-treated HIRc B cell lysates demonstrated the presence of IR. Our data suggest that alpha2-HSGbac preferentially interacts with the activated IR. To further characterize the site(s) of interaction, the effect of alpha2-HSGbac on trypsin-treated IR autophosphorylation was studied. Trypsin-treatment of intact HIRc B cells results in proteolysis of the IR alpha-chain and constitutive activation of IR-TKA. We demonstrate that alpha2-HSGbac (0.1 microM) completely inhibited trypsin-activated IR autophosphorylation and TKA in vitro indicating that this effect was not mediated by its interaction with the proximal 576 amino acid residues of the IR alpha-subunit. The physiological relevance of these observations was explored by characterizing the effects of alpha2-HSG injection in rats. Alpha2-HSGbac (2 microM), acutely injected through the portal vein of normal rats, inhibited insulin-stimulated IR autophosphorylation and IRS-1 phosphorylation in liver and hindlimb muscle. Taken together our results suggest that alpha2-HSG, by interacting with IR, specifically inhibits insulin-stimulated IR autophosphorylation and may play a physiological role in the regulation of insulin signaling.
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PMID:Alpha2-HSG, a specific inhibitor of insulin receptor autophosphorylation, interacts with the insulin receptor. 1102 61

Airway remodeling with smooth muscle cell (SMC) hyperplasia is a feature of chronic asthma. We investigated the potential for tryptase, the major secretory product of human mast cells, to act as a growth factor for human airway SMCs. Because this serine protease can activate proteinase-activated receptor-2 (PAR-2), we also examined the actions of SLIGKV, a peptide agonist of PAR-2. Incubation with lung tryptase provoked a twofold increase in [(3)H]thymidine incorporation; a similar increase in cell numbers was found when we used the MTS assay. The effect was catalytic site dependent, being abolished by the protease inhibitors leupeptin and benzamidine and by heat inactivation of the enzyme. Tryptase-induced DNA synthesis was inhibited by preincubation of the cells with pertussis toxin, calphostin C, or genistein. Transduction mechanisms are thus likely to involve a pertussis toxin-sensitive G protein, protein kinase C, and tyrosine kinase. SLIGKV elicited a response on SMCs similar to that of tryptase. Tryptase could provide an important stimulus for SMC proliferation in asthmatic airways, by acting on PAR-2.
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PMID:Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells. 1150 38

Tyr phosphorylation of the multifunctional transmembrane protein band 3 has been implicated in several erythrocyte functions and disorders. We previously demonstrated that pervanadate treatment of human erythrocytes induces band-3 Tyr phosphorylation, which is catalyzed by the sequential action of tyrosine kinase Syk and tyrosine kinase(s) belonging to the Src family. In this study, we show that Tyr phosphorylation of band 3, elicited by pervanadate, N-ethylmaleimide, or diamide, greatly increases band-3 interaction with the tyrosine phosphatase SHP-2 in parallel with the translocation of SHP-2 to erythrocyte membranes. These events seem to be mediated by Src-like catalyzed phosphorylation of band 3 because both SHP-2 translocation to cellular membranes and its interaction with Tyr-phosphorylated protein are greatly counteracted by PP2, a specific inhibitor of Src kinases. Binding-competition experiments demonstrate that SHP-2 recruitment to band 3 occurs via its SH2 domain(s). In particular, our data support the view that SHP-2 docks specifically with P-Y359 of band 3. Experiments performed with intact erythrocytes in the presence of the SHP-2 inhibitor calpeptin suggest that, once recruited to Tyr-phosphorylated band 3, the tyrosine phosphatase dephosphorylates the protein. P-Y8, 21, and 904 are the residues affected by SHP-2, as judged by (32)P-peptide mapping of band 3 digested with trypsin. These results indicate that in treated erythrocytes, recruitment of cytosolic SHP-2 to band 3 is a prerequisite for the subsequent dephosphorylation of the transmembrane protein.
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PMID:Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes. 1207 37


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