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)

Phorbol esters stimulate the phosphorylation of the insulin receptor on discrete serine and threonine residues in intact cells. Phosphorylation of the insulin receptor cytoplasmic domain on serine, threonine, and tyrosine residues regulates receptor tyrosine kinase activity and signaling. In these studies, we demonstrate that phorbol ester treatment of intact COS-1 cells transiently expressing the human insulin receptor stimulates phosphorylation of serine 1327 within the carboxyl-terminal tail of the insulin receptor beta subunit. Phosphopeptide maps of wild-type (Ser1327) and mutant (Ala1327) human insulin receptors revealed the absence of a single phosphopeptide in the Ala1327 receptors when compared with wild-type receptors from phorbol ester-treated cells. Phosphoamino acid analysis revealed phosphoserine within the phosphopeptide from wild-type receptors that is absent in the Ala1327 receptor. The synthetic peptide 1327S (KRSYEEHIPYTHMNGGKK) corresponding to amino acids 1325-1342 of the human insulin receptor is phosphorylated on serine by protein kinase C. After digestion with trypsin, the phosphorylated synthetic peptide comigrated with the serine-phosphorylated peptide isolated from wild-type insulin receptors that was absent from the Ala1327 mutant. Ser1327 is proximal to autophosphorylation sites Tyr1328 and Tyr1334. The potential effects of serine phosphorylation at position 1327 on subsequent phosphorylation of these tyrosines by the insulin receptor kinase were examined using synthetic peptides. The chemically modified peptide 1327S(P) was synthesized with the stoichiometric addition of phosphate to the side chain hydroxyl of a serine corresponding to position 1327 of the insulin receptor. Kinetic analysis revealed that the addition of phosphate to the serine improved substrate recognition by the insulin receptor tyrosine kinase almost 2-fold. The average Km was 1.44 mM for the peptide 1327S(P) versus 2.64 mM for peptide 1327S. However, when compared with the unphosphorylated control peptide, 1327S, the serine-phosphorylated peptide 1327S(P) also reduced the Vmax of the insulin receptor tyrosine kinase 53%. Radiosequence analysis revealed that the chemical addition of phosphate to the serine in peptide 1327S(P) inhibited insulin receptor-catalyzed phosphorylation of the tyrosine on 1327S(P) corresponding to Tyr1334 but not of the tyrosine corresponding to Tyr1328. These data suggest that the juxtaposition of a serine phosphorylation site adjacent to receptor tyrosine phosphorylation sites provides the potential for regulation of insulin receptor autophosphorylation and signaling through its carboxyl-terminal tail.
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PMID:Phorbol ester stimulates phosphorylation on serine 1327 of the human insulin receptor. 792 43

Rat connective tissue mast cells are known to store significant amounts of mast cell protease I (RMCP I), which suppresses normal cell growth and mediates cytotoxicity against tumor cell lines, including the fibrosarcoma cell line FL. To better define its effects on FL cells, RMCP I was added to FL cultures for 30 min. Analysis of de novo nuclear protein synthesis revealed that RMCP I suppressed the expression of three proteins (41, 46, and 69 kD) and enhanced the expression of two other proteins (25 and 32 kD). Treatment of FL cells with diisopropylfluorophosphate (DFP)-inactivated RMCP I proved that these effects were largely independent of the protease catalytic site. Western blot hybridization, using a monoclonal antibody to phosphotyrosine-containing proteins, revealed that RMCP I inhibited phosphorylation of a nuclear and a cytoplasmic 81-kD tyrosylprotein. Inhibition of nuclear tyrosine kinase activity by RMCP I appeared to be catalytic site dependent, whereas cytoplasmic tyrosine kinase inhibition was independent of RMCP I proteolytic activity. Biotinylated RMCP I was used to identify potential surface-binding proteins. Three specific binding complexes (130, 150, and 210 kD) were detected. The binding of biotinylated RMCP I to these surface proteins was inhibited by excess unlabeled RMCP I, but not by trypsin or chymotrypsin. We speculate that the binding proteins may be critical in initiating RMCP I-induced metabolic changes on FL cells. The ability of RMCP I to alter the metabolism of cells suggests that it may have an important role in regulating their functions.
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PMID:Rat mast cell protease I alters cell metabolism. 802 85

Previous analyses have suggested that the CD45 tyrosine phosphatase activates src family tyrosine kinases p56lck and p59fyn by dephosphorylating regulatory COOH-terminal residues. We have examined the status of p56lck and p59fyn in murine and human CD45- T cell lines. Surprisingly, despite the fact that p56lck and p59fyn were spontaneously hyperphosphorylated, the tyrosine kinase activity of both enzymes was increased in CD45- versus CD45+ cells. In vitro exposure of hyperphosphorylated p56lck to CD45 decreased enzyme activity to near-basal levels. Lck from CD45- cells was hyperphosphorylated on the cyanogen bromide digestion fragment that contains the negative regulatory residue Tyr-505, and the identity of this site of phosphorylation was confirmed by trypsin digestion followed by high performance liquid chromatography. Loss of CD45 results, therefore, in a paradoxical hyperphosphorylation of the COOH-terminal tyrosine and increased src family kinase enzymatic activity.
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PMID:CD45 regulation of tyrosine phosphorylation and enzyme activity of src family kinases. 817 95

The ret proto-oncogene (proto-ret) encodes a receptor type tyrosine kinase with a cadherin-related sequence in the extracellular domain. To investigate whether the proto-Ret protein functions as a cell adhesion molecule like cadherins, we transfected the human proto-ret gene fused to the SV40 promoter or cytomegalovirus (CMV) promoter into mouse L cells in which cadherins are not expressed. Three transfectants with high levels of expression of the proto-Ret proteins were obtained. The proto-Ret proteins were expressed as 150 kDa and 170 kDa glycoproteins in transfectants as observed in human neuroblastoma cells. Cell fractionation experiments revealed that the 170 kDa protein but not the 150 kDa protein was detected predominantly in the plasma membrane fraction, indicating that the 170 kDa protein represents the mature glycosylated form of the proto-Ret protein present on the cell surface. Both 150 kDa and 170 kDa proto-Ret proteins showed tyrosine kinase activity in immunocomplex kinase assay. It is known that cadherins have Ca(2+)-dependent homophilic binding activity and are resistant to trypsinization in the presence of Ca2+. When L cells expressing the proto-Ret proteins were treated with trypsin in the presence of Ca2+, the 170 kDa protein was resistant to its digestion. On the other hand, it was completely digested in the presence of EGTA, suggesting the possibility that the proto-Ret protein interacts with Ca2+ like cadherins. However, the transfectants did not show clear adhesive properties in cell aggregation assays.
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PMID:Characterization of the ret proto-oncogene products expressed in mouse L cells. 841 95

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.
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PMID:Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg). 844 92

The retraction of axonal branches is a prominent feature of nervous system development and function. Although various biological and pathological signals can elicit retraction, little is known regarding their underlying mode of action. An in vitro assay using NG108-15 cells was used to demonstrate that rapid-onset neurites exposed acutely to trypsin, serum, lysophosphatidic acid, extracellular ATP, the phorbol ester phorbol 12-myristate 13-acetate, and nocodazole were all protected from retraction by the tyrosine kinase inhibitor genistein. This finding indicates that a common (genistein-sensitive) cellular event is involved in integrating the influence of multiple extrinsic and intrinsic signals and in regulating whether or not neurites will execute a retraction response.
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PMID:Acute neurite retraction elicited by diverse agents is prevented by genistein, a tyrosine kinase inhibitor. 851 81

We have studied the actions of the proteinase-activated-receptor-2 (PAR2)-activating polypeptide, SLIGRL-NH2 (SLI-NH2), in rat aorta and in gastric longitudinal muscle preparations. In the phenylephrine-precontracted aorta preparation, SLI-NH2 caused an endothelium-dependent relaxation that mimicked the action of low concentrations (0.5 U/mL) of trypsin and that was blocked by the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester. In endothelium-free aorta ring preparation, SLI-NH2 caused neither a relaxation nor a contraction. In the gastric longitudinal muscle preparation, SLI-NH2 caused a transient contraction that mimicked the action of trypsin (0.5 U/mL) and that was sensitive to inhibitors of cyclooxygenase (indomethacin) and tyrosine kinase (genistein). Further, using a reverse-transcriptase - polymerase chain reaction (RT-PCR) approach we detected, in both assay tissues, mRNA for the rat PAR2 receptor, and we ascertained, using a cloned receptor cDNA obtained from a rat intestinal cDNA library, that the putative N-terminal activating peptide sequence of the rat PAR2 receptor (SLIGRL) is identical with the one previously cloned from murine tissue. We concluded that, like the thrombin receptor, the PAR2 receptor may play a pathophysiologic role in the regulation of vascular and gastric smooth muscle contractility.
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PMID:Detection of functional receptors for the proteinase-activated-receptor-2-activating polypeptide, SLIGRL-NH2, in rat vascular and gastric smooth muscle. 856 91

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for endothelial cells. VEGF is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia. VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The higher molecular weight forms of VEGF can be cleaved by plasmin to release a diffusible form(s) of VEGF. We characterized the proteolysis of VEGF by plasmin and other proteases. Thrombin, elastase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with soluble VEGF receptors, and ability to promote endothelial cell mitogenesis. Plasmin yields two fragments of VEGF as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111. A heterodimeric form of VEGF165/110, was isolated from partial plasmin digests of VEGF165. The carboxyl-terminal polypeptide (111-165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors. The various isoforms of VEGF (165, 165/110, and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM). In contrast, soluble FLT-1 receptor differentiated VEGF isoforms (165, 165/110, 110, and 121) with apparent affinities of 10, 30, 120, and 200 pM, respectively. Endothelial cell mitogenic potencies of VEGF110 and VEGF121 were decreased more than 100-fold compared to that of VEGF165. The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity.
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PMID:The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. 863 22

Histone H4 stimulates the uptake of glucose in rat adipocytes and muscle cells. However, the mechanism of this unusual activity is not known. Therefore, we have begun to investigate the mechanism by which histone H4 stimulates the glucose uptake in rat adipocytes. We report that histone H4 requires 15-20 min to achieve its maximum effect and its time course is virtually indistinguishable from the time course of insulin itself. Reduction of the concentration of insulin receptors on the surface of adipocytes, either by trypsin digestion of the receptor, or by insulin-induced down regulation of the receptor, reduced the histone H4 effect as well as the insulin effects. Also, quercetin, a bioflavenoid that inhibits the insulin receptor tyrosine kinase activity, inhibits the actions of both histone H4 and insulin. However, histone H4 activity is somewhat more resistant to these interventions than insulin activity. In contrast to the activity of insulin, histone H4 does not appear to be able to down regulate the insulin receptor, since the pretreatment of adipocytes with histone H4 did not affect the subsequent actions of either insulin or histone H4. Finally, Scatchard analysis of the binding of 125I-insulin in the presence and absence of histone H4 increases the specific binding of insulin in a concentration dependent fashion. Histone H2b, a histone that does not have insulin-like activity, does not affect insulin binding. Taken together, these data suggest that the greatest portion of the insulin-like activity of histone H4 is initiated at the insulin receptor. However, the interaction of histone H4 and the insulin receptor is more complex than a simple binding of H4 to the insulin binding site. These studies may provide additional insight into alternate mechanisms for activation of the insulin receptor.
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PMID:Histone H4 stimulates glucose uptake through the insulin receptor. 872 9

1. The biological activities of the proteinase-activated receptor number 2 (PAR-2)-derived peptides, SLIGRL (PP6) SLIGRL-NH2 (PP6-NH2) and SLIGR-NH2 (PP5-NH2) were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR-2-derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR-NH2 (TP5-NH2). 2. From a neonatal rat intestinal cDNA library, and from intestinal and kidney-derived cDNA, the coding region of the rat PAR-2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR-2 receptor; and this information, along with a reverse-transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR-2 and thrombin receptor mRNA in both tissues. 3. In the mouse and rat gastric preparations, the PAR-2-derived polypeptides, PP6, PP6-HN2 and PP5-NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml-1; 10 nM) and that were equivalent to contractions caused by TP5-NH2. 4. The cumulative exposure of the rat LM tissue to PP6-NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5-NH2 and vice versa, so as to indicate a lack of cross-desensitization between the receptors responsive to the PAR-2 and thrombin receptor-derived peptides. 5. In the rat gastric preparation, the potencies of the PAR-2-activating peptides were lower than the potency of TP5-NH2 (potency order: TP5-NH2 > > PP6-NH2 > or = PP6 > PP5-NH2); PP6 was a partial agonist in this preparation. 6. The contractile actions of PP6 and PP6-NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo-oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7. In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR-2-derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME) but not by D-NAME; in an endothelium-free preparation, which possessed mRNA for both the PAR-2 and thrombin receptors, the PAR-2-activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5-NH2. The relaxation response to PP6-NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8. In the rat aortic preparation, the potencies of PP6, PP6-NH2 and PP5-NH2 were greater than those of the thrombin receptor activating peptide, TP5-NH2 (potency order: PP6-NH2 > or = PP6 > PP5-NH2 > TP5-NH2). 9. In the rat aortic preparation, the relaxant actions of the PAR-2-derived peptides were mimicked by trypsin, at concentrations (0.5-1 u ml-1; 1-2 nM) lower than those that can activate the thrombin receptor. 10. The bioassay data obtained with the PAR-2 peptides and with trypsin, along with the molecular cloning/RT-PCR analysis, point to the presence of functional PAR-2 receptors that can activate distinct responses in the gastric and vascular smooth muscle preparations. These responses were comparable to those resulting from thrombin receptor activation in the same tissues, so as to suggest that the receptor for the PAR-2-activating peptides may play a physiological role as far reaching as the one proposed for the thrombin receptor.
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PMID:Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue. 876 73


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