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

From the soluble protein fraction of lactating rat mammary epithelial cells, fatty acid-binding protein (FABP) was isolated by immunoaffinity chromatography. After digestion with trypsin, peptides were characterized with time-of-flight mass spectrometry and revealed identity with corresponding peptides derived from the heart-type FABP isolated from rat heart. In addition, by electrospray mass spectrometry the molecular mass has been determined to 14683.9 +/- 3 Da, further corroborating the identity. The content of FABP in mammary glands from virgin, pregnant and lactating rats was evaluated using two-dimensional gel electrophoresis and a FABP-specific immunosorbent assay. In the two-dimensional gels FABP was the apparently most abundant cytosolic protein in mammary epithelial cells from rats in late pregnancy as well as from lactating rats. The content of FABP was 59 +/- 19 microgram/mg (n = 11) of soluble proteins from the fully differentiated lactating mammary gland as determined by ELISA. This value represented an 80-fold increase compared with the FABP content of mammary gland from virgin rats, and is comparable with the level found in rat heart. Upon stimulation with insulin a small fraction of FABP was phosphorylated in lactating mammary epithelial cells. In conclusion, these findings indicate that the FABPs from rat mammary gland and heart are identical and further suggest that in mammary gland this FABP may play a role in signal transduction downstream from the insulin receptor.
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PMID:Differentiational regulation and phosphorylation of the fatty acid-binding protein from rat mammary epithelial cells. 811 46

We have previously expressed the human insulin receptor beta subunit domain containing transmembrane and cytoplasmic domains (IRTMTPK) in insect cells, and showed that the purified IRTMTPK was highly active (Li, S. L., Yan, P.-F., Pax, I. B., and Fujita-Yamaguchi, Y. (1992) Biochemistry 31, 12455-12462). To investigate the role of COOH-terminal and acidic domains of the insulin receptor kinase, we have expressed deletion mutants IRTMTPK delta CT (delta 76 amino acids) and IRTMTPK delta Acid (delta 19 amino acids). Both enzymes were purified by a one-step method using the same immunoaffinity column as used for IRTMTPK. While Km and Vmax for prephosphorylated IRTMTPK and delta Acid mutant enzyme determined using poly(Glu, Tyr)(4:1) were similar, catalytic efficiency of the delta CT mutant enzyme was significantly lower than those of IRTMTPK and delta Acid mutant enzyme as judged by Km and Vmax. Experiments for thermostability and susceptibility to proteases revealed that Tm of delta CT mutant enzyme was 3.5 degrees C lower than that of IRTMTPK enzyme (= 33.3 degrees C) and that delta CT mutant enzyme was digested by either trypsin or Lys-C into a 28,000 core domain much faster than IRTMTPK. Activation of delta CT mutant enzyme by polylysine was less significant than that of IRTMTPK and delta Acid mutant enzyme, approximately 4-versus approximately 17-fold. These studies suggested that the COOH-terminal domain plays important roles in both catalytic efficiency and stability of the insulin receptor kinase, and that the acidic domain by itself is not responsible for kinase activation by polylysine.
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PMID:The role of COOH-terminal and acidic domains in the activity and stability of human insulin receptor protein tyrosine kinase studied by purified deletion mutants of the beta subunit domain. 822 52

The functional significance of the insulin receptor on bovine aorta endothelial (BAE) cells is not well defined. The insulin receptor expressed on BAE cells does not mediate insulin hormonal effects and does not mediate the transcytosis of insulin from the apical to the basolateral domain of the cell monolayer. To assess the role of the insulin receptor on BAE cells, the physical characteristics of the BAE cell receptor were investigated, and the time-dependent interaction of insulin and insulin degradation products with BAE cell monolayers was quantitated. The BAE cell insulin receptor was found to be highly resistant to the proteolytic action of trypsin, pronase, and proteinase K at either 4 degrees C or 37 degrees C. This resistance may permit the receptor to maintain insulin binding capabilities in spite of the high concentrations of proteases which are normally present in blood. Scatchard analysis of cell-surface and total cellular insulin receptor demonstrated dissociation constants similar to values obtained with other cells and tissues. However, whereas other cells and tissues contain an intracellular pool of receptor that ranges from 20-40% of the total cellular receptor content, no intracellular population of insulin receptors was detected in BAE cells. Upon incubation of intact BAE cell monolayers with insulin, no endocytosis of cell-surface insulin receptor could be demonstrated. However, insulin degradation by the BAE cells was readily quantitated, at a rate of 16.3 fmol/10(6) cells/h at an insulin concentration of 2 nM. This rate of degradation was not inhibited by chloroquine, which inhibits insulin degradation in fibroblasts, hepatocytes, and adipocytes, nor by phenylarsine oxide, which inhibits endocytosis. Bacitracin inhibited insulin binding to the cell monolayers and inhibited insulin degradation with identical IC50 values (80 microM). These data suggest that in BAE cells, insulin degradation occurs in the absence of receptor-mediated endocytosis and is mediated by binding of insulin to its receptor. Therefore, it is concluded that the functional role of the insulin receptor expressed in BAE cells is to bind blood-borne insulin at the plasma membrane of the cell and thereby facilitate the degradation of insulin at the BAE cell plasma membrane.
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PMID:Insulin receptor characterization and function in bovine aorta endothelial cells: insulin degradation by a plasma membrane, protease-resistant insulin receptor. 822 65

Mitogen activation of resting lymphocytes induces expression of high affinity insulin receptors on the plasma membrane. The mechanism underlying this effect on insulin receptor expression was examined by comparing levels of insulin receptor mRNA and protein in resting and mitogen-activated rodent lymphocytes. Analysis of RNA levels indicated that resting and concanavalin A-activated lymphocytes contained equivalent amounts of insulin receptor mRNA with predominant transcripts of 7.9 and 9.5 kilobases. Although little or no insulin binding was detectable on intact resting lymphocytes, detergent solubilization of these cells resulted in the appearance of readily detectable insulin binding activity that could be immunoprecipitated with anti-insulin receptor antibodies. Detergent-solubilized resting and mitogen-activated lymphocytes expressed equivalent amounts of insulin receptors that bound insulin with similar affinity (KD = 90 pM) and migrated on reduced SDS-polyacrylamide gels with apparent masses of approximately 130 and approximately 95 kDa. Insulin receptors from resting lymphocytes appeared to be associated with the plasma membrane since 125I labeling of intact lymphocytes radiolabeled the insulin receptor, insulin binding activity was detected in membrane fractions of hypotonically lysed cells, and trypsin treatment of intact cells destroyed > 90% of the insulin binding activity in detergent extracts. These results suggest that resting lymphocytes express insulin receptor mRNA and protein and that mitogen activation exposes cryptic insulin receptors present in the plasma membrane of resting lymphocytes.
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PMID:Mitogen activation of resting lymphocytes exposes cryptic insulin receptors. 844 Jul 5

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

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

To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor.
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PMID:Inhibition of clathrin-mediated endocytosis selectively attenuates specific insulin receptor signal transduction pathways. 963 70

The cell-free examination of the human insulin receptor during biogenesis may provide a greater understanding of the elements that contribute to the acquisition of receptor function. The insulin receptor precursor components were produced in a cell-free system and the insulin binding ability of the [35S]methionine-labeled translation products was determined. The processed proreceptor represented by a 190 kDa band was retained on insulin-linked biotin-streptavidin agarose or an insulin column. The insulin binding 190 kDa band migrated slower than the non-binding 190 kDa band on SDS-PAGE which suggests that covalent modifications account for these differences. The trypsin-digested product of the 190 kDa proreceptor was also retained on insulin-linked biotin-streptavidin agarose, however the alpha-subunit precursor was retained on insulin agarose to a much lesser degree. We conclude that a significant fraction of the processed, in vitro translated insulin proreceptor acquires insulin binding ability.
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PMID:The in vitro synthesized and processed human insulin receptor precursor binds insulin. 1048 54

Low catalytic efficiency of basal-state protein kinases often depends on activation loop residues blocking substrate access to the catalytic cleft. Using the recombinant soluble form of the insulin receptor's kinase domain (IRKD) in its unphosphorylated state, activation loop conformation was analyzed by limited proteolysis. The rate of activation loop cleavage by trypsin is slow in the apo-IRKD. Bound Mg-adenine nucleoside di- and triphosphates increased the cleavage rate with half-maximal effects observed at 0.4-0.9 mM nucleotide. Adenosine monophosphate at concentrations up to 10 mM was not bound appreciably by the IRKD and had virtually no impact on activation loop cleavage. Amino-terminal and carboxy-terminal core-flanking regions of the IRKD had no statistically significant impact on the ligand-dependent or -independent activation loop cleavages. Furthermore, the core-flanking regions did not change the inherent conformational stability of the active site or the global stability of the IRKD, as determined by guanidinium chloride-induced denaturation. These measurements indicate that the intrasterically inhibitory conformation encompasses > or =90% of the ligand-free basal state kinase. However, normal intracellular concentrations of Mg-adenine nucleotides, which are in the millimolar range, would favor a basal-state conformation of the activation loop that is more accessible.
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PMID:Conformational changes in the activation loop of the insulin receptor's kinase domain. 1054 62


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