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)

Trypsin treatment of a partially purified insulin receptor preparation from rat adipocytes stimulated the phosphorylation of 90,000- and 72,000-Da polypeptides immunoprecipitated by anti-insulin receptor antibody. The phosphorylation of tyrosine residues alone was observed in both polypeptides. Trypsin concentrations which stimulated insulin receptor phosphorylation were the same as those previously shown to activate rat adipocyte glycogen synthase. Trypsin treatment of the insulin receptor fraction also stimulated the phosphorylation of an exogenous substrate of tyrosine kinase similarly to insulin treatment. Trypsin treatment of a highly purified insulin receptor from human placenta also activated the phosphorylation of the receptor-derived peptides. These results suggest that the insulin-stimulated protein kinase, a component of the insulin receptor, was activated by tryptic digestion to phosphorylate polypeptides derived from the insulin receptor itself. Thus, it is suggested that stimulation by trypsin of phosphorylation of the insulin receptor may be related to the insulin-like metabolic actions of trypsin observed in rat adipocytes.
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PMID:Insulin-like effect of trypsin on the phosphorylation of rat adipocyte insulin receptor. 641 37

Glycogen synthase I in a homogenate of human polymorphonuclear leukocytes was phosphorylated under imitated physiological conditions utilizing the endogenous protein kinases. At subsequent steps of phosphorylation the 32P-labelled synthase was purified and characterized. Limited tryptic hydrolysis of the 32P-labelled synthase released four phosphopeptides (t-A, t-B, t-C, t-D) and subsequent chymotrypsinization of the trypsin resistant core released three phosphopeptides (c-A, c-B, c-C). One Pi/subunit was incorporated within 8-10 min and 2.2 Pi/subunit within 60 min increasing the Kc for Glc-6-P to 4-6 mM. The initial phosphorylation up to 0.8 Pi/subunit occurred mainly in peptide c-A and a linear relation between ratio of independence (RI) of glycogen synthase in the interval RI 0.85 to RI 0.05 and phosphorylation of this peptide of 0.5 Pi was observed. Phosphorylation of this peptide is responsible for the decrease in ratio of independence. From experiments with inhibitors and activators, the initial phosphorylation was found predominantly catalysed by the endogenous cAMP independent synthase kinase, however, the endogenous cAMP dependent protein kinase and phosphorylase kinase also phosphorylate endogenous glycogen synthase I to a minor degree. Circumstantial evidence for a Ca-dependent synthase kinase different from phosphorylase kinase is presented. The endogenous Glc-6-P dependent glycogen synthase occurring in a homogenate of leukocytes disrupted in the presence of NaF incorporated 1.07 Pi/subunit and Kc for Glc-6 was increased from 6-8 mM to 20 mM. From the present and previous experiments [7] a total of 8 major phosphorylatable sites have been defined, one on each of the peptides t-A, t-B, c-B, c-C and two on peptide c-A, which in addition may contain a third site for phosphorylase kinase. Assuming identical subunits, only 13 out of 32 sites are thus covalently modified at maximum phosphorylation. The operational defined synthase R (Kc for Glc-6-P 0.5 mM) and D (Kc for Glc-6-P 2-8 mM) activities correspond to synthase with about 0.8 Pi and 1.8-2.3 Pi/subunit, respectively.
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PMID:Phosphorylation of glycogen synthase in a homogenate of human polymorphonuclear leukocytes. 678 73

Brief treatment of rat adipocytes with low concentration of trypsin activated both cell membrane and intracellular insulin-sensitive functions in marked contrast H2O2 (1), increase in pH, and oxidized glutathione (papers I and II). Glucose oxidation was activated maximally by trypsin in 30 s and preceded maximal activation of glycogen synthase, which occurred in 60s. Trypsin action to activate glycogen synthase was further enhanced by insulin. Mitochondrial pyruvate dehydrogenase was also rapidly activated by trypsin. With both insulin and trypsin action, mediator generation was directly demonstrated by glycogen synthase phosphoprotein phosphatase activation. Trypsin is thus the most insulin-like of these four agents studied since it acts by the formation of chemical mediator peptide(s) which are similar but not identical to those produced by insulin.
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PMID:Independent control of selected insulin-sensitive cell membrane and intracellular functions-the linkage of cell membrane and intracellular events controlled by insulin. III. The influence of trypsin on cell membrane hexose transport and on glycogen synthase and mitochondrial pyruvate dehydrogenase activation. 679 3

Glycogen synthase is a substrate for five distinct protein kinases in skeletal muscle which phosphorylate seven different serine residues on the enzyme. Cyclic-AMP-dependent protein kinase phosphorylates sites 1a, 1b and 2, phosphorylase kinase, site 2, glycogen synthase kinase 3, sites 3a, 3b and 3c, glycogen synthase kinase 4, site 2 and glycogen synthase kinase 5 site 5. Site 2 is seven residues from the N-terminus of glycogen synthase and is located in a cyanogen bromide peptide termed CB1 (apparent Mr = 9000). The other six phosphorylation sites are located in a cyanogen bromide peptide termed CB2 (apparent Mr = 24 000) at the C-terminal end of the molecule. The sequence of the N-terminal 123 residues of peptide CB2, has been completed. Sites 3a, 3b, 3c, 5, 1a and 1b are located at residues 30, 34, 38, 46, 87 and 100 from the N-terminus of CB2 respectively. Site 1a is the next serine residue after site 5. The region surrounding sites 3a, 3b and 3c is very rich in proline residues while that surrounding sites 1a and 1b contains many serine and threonine residues. The 23 residues following site 5 contain 15 aspartic acid and glutamic acid residues, while the region immediately N-terminal to site 1a is very basic. The whole region is remarkably hydrophilic and is the region at which the native enzyme is attacked by proteinases. The sites at which glycogen synthase is cleaved by trypsin, chymotrypsin and thermolysin have been identified. The finding that trypsin cleaves the enzyme C-terminal to site 3c while chymotrypsin cleaves N-terminal to site 3a has formed the basis of a simple procedure for determining the state of phosphorylation of the seven serine residues in vivo [Parker, P.J., Embi, N., Caudwell, F.B., and Cohen, P. (1982) Eur. J. Biochem. 124, 47-55].
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PMID:Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Organisation of the seven sites in the polypeptide chain. 680 97

A cardiolipin- and protease-activated protein kinase (PAK) has been isolated from cytoplasmic extracts of rat liver. The enzyme (PAK-1) phosphorylates the ribosomal protein S6-(229-239) peptide analogue and can be activated by limited proteolysis. Partial amino acid sequences of tryptic peptides derived from both the purified 116-kDa PAK-1 holoenzyme and its active catalytic fragment reveal that the catalytic domain is most related (50-58% identity) to the protein kinase C family. PAK-1 has protein and peptide substrate specificities distinct from those of known protein kinase C isoforms and is insensitive to inhibition by the protein kinase C-alpha-(19-31) pseudosubstrate peptide. Phosphatidylserine, diacylglycerol, and phorbol ester do not activate PAK-1 toward the S6 peptide substrate. However, other acidic phospholipids, the most effective being cardiolipin, activate PAK-1 to a similar extent as trypsin. The PAK-1 catalytic activities generated through activation by cardiolipin or limited proteolysis were kinetically similar, with Km values of 3.6 and 3.4 microM, respectively, for the S6-(229-239) peptide substrate. However, differences were observed in the catalytic activities with protamine sulfate and the glycogen synthase-(1-12) peptide analogue as substrates. It was concluded that PAK-1 is a phospholipid-regulated protein kinase with a primary structure, substrate specificity, and mechanism of regulation in vitro distinct from those of any known member of the protein kinase C superfamily.
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PMID:A cardiolipin-activated protein kinase from rat liver structurally distinct from the protein kinases C. 805 Oct 89

The effects of amylin and insulin on the phosphorylation of glycogen synthase and phosphorylase were investigated using rat diaphragms incubated with 32Pi. Muscles were incubated with insulin (200 nM) or amylin (200 nM) for 30 min before extracts were prepared. The 32P contents of the enzymes were determined after immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Amylin increased both the activity ratio (-AMP/+AMP) and the 32P content of phosphorylase by approximately 2-fold. Insulin alone was without significant effect on phosphorylase, but insulin blocked the effect of amylin on increasing the phosphorylation of phosphorylase. Insulin increased the glycogen synthase activity ratio (low glucose-6-P/high glucose-6-P) by approximately 80%. Amylin decreased this ratio from 0.14 to 0.08 and increased the phosphorylation of synthase by approximately 40%. To investigate changes in phosphorylation of different sites in the synthase, the enzyme was subjected to exhaustive proteolysis with trypsin, and 32P-labeled fragments were separated by reverse phase high performance liquid chromatography. Insulin decreased the 32P contents of sites 3(a+b+c) and 2(a+b), which appears to account for the increase in synthase activity. Amylin increased phosphorylation of sites 1a, 1b, and 3(a+b+c), but not sites 2(a+b). With insulin plus amylin, phosphorylation of none of the sites was significantly changed. The results indicate that the effects of amylin on glycogen synthase must involve more than activation of cAMP-dependent protein kinase, as this kinase phosphorylates site 2 and does not phosphorylate sites 3(a+b+c).
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PMID:Control of glycogen synthase and phosphorylase by amylin in rat skeletal muscle. Hormonal effects on the phosphorylation of phosphorylase and on the distribution of phosphate in the synthase subunit. 815 93

In this study, we examined protein phosphatase 1 (PP-1) and protein phosphatase 2A (PP-2A) activities during various stages of myogenesis and their regulation by insulin in rat skeletal muscle cells. Protein phosphatase activities were measured using 32P-labeled phosphorylase a, glycogen synthase, and phosphorylase kinase as substrates. Spontaneous PP-1 activity increased progressively in cultures from 2 to 5 days, PP-2A activities remained constant in days 2-4 cultures and increased sharply on day 5. Most of the times in culture, a significant proportion (approximately 65%) of PP-1 was in a form that could be activated by trypsin. Insulin stimulated PP-1 activity (40-80% increase over basal) in a time (t1/2 approximately 5 min)- and dose (EC50 approximately 0.1 nM)-dependent manner. Insulin activation of PP-1 was accompanied by a corresponding inhibition in PP-2A activity. The effects of insulin on PP-1 and PP-2A were differentiation dependent and were observed only in cells at fusion (day 5) and post-fusion. The insulin's effect on PP-1 correlated with the gradual appearance of PP-1 G subunit in cells at fusion. Immunoprecipitation of PP-1 from 32P-labeled cells with an antibody directed against the site 1 sequence of rabbit skeletal muscle PP-1G detected a 160-kDa protein, phosphorylation of which was significantly increased by insulin. This correlated well with the increase observed in immunoprecipitated PP-1G activity. Treatment of cells with a cAMP agonist (SpcAMP) completely blocked activation of PP-1 by insulin and diminished insulin-stimulated phosphorylation of the 160-kDa protein. The likely identity of the 160-kDa band as the regulatory subunit of PP-1 was confirmed by assay of PP-1 activity in the immunoprecipitates and by competition studies with the site 1 peptide against which the antibody was made. From these studies, we conclude that insulin activates PP-1 in L6 cells by increasing the phosphorylation of its regulatory subunit.
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PMID:Regulation of protein phosphatase 1 and 2A activities by insulin during myogenesis in rat skeletal muscle cells in culture. 817 60

Reduced type 1 protein phosphatase (PP-1) activity in human muscle extracts may contribute to the reduced insulin-stimulated glycogen synthase activity associated with insulin resistance for glucose disposal in humans. Because inactive forms of PP-1 can be activated with trypsin plus Mn2+, these reagents were used to compare the PP-1 activities in skeletal muscle extracts before and after separation into cytosolic and glycogen microsomal (GM) fractions. PP-1 activities were reduced in the GM fraction from insulin-resistant subjects (54 +/- 2 vs. 61 +/- 1, P < 0.01) but, in contrast to our previously published results, were elevated in the extract (33 +/- 6 vs. 18 +/- 3, P < 0.05). Recombination of the cytosol and GM fractions (reconstituted extract) demonstrated that the low extract PP-1 activities could only be regenerated when the GM fraction from insulin-sensitive subjects was recombined with cytosol from either group. The results indicate that the elevated PP-1 activity observed in extracts of insulin-resistant compared with insulin-sensitive subjects is caused by an inhibitor of extract PP-1 activity that sediments with the GM pellet and is more active in the insulin-sensitive subjects.
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PMID:Trypsin-Mn(2+)-resistant form of type 1 protein phosphatase in human muscle. 817 78

The budding yeast Saccharomyces cerevisiae expresses two isoforms of glycogen synthase, of which glycogen synthase-2 (GS-2) appears to be the most important determinant of glycogen accumulation (Farkas, I., Hardy, T. A., Goebl, M. G., and Roach, P. J. (1991) J. Biol. Chem. 266, 15602-15607). Partial proteolysis of purified yeast glycogen synthase activated the enzyme, mimicking the effects of dephosphorylation. The cleavage was localized to the COOH terminus of the molecule and trypsin treatment released 32P from enzyme labeled in vivo with 32P or in vitro by cyclic AMP-dependent protein kinase. Similarly, when cells were labeled with 32P, no radioactivity was incorporated into a mutant form of GS-2 truncated at residue 643 while the wild type enzyme was phosphorylated at both Ser and Thr residues. The 9 Ser and Thr residues COOH-terminal to position 643 were mutated individually to Ala, and the GS-2 mutants were expressed from a low copy plasmid in yeast that lacked functional chromosomal copies of the two glycogen synthase genes. Mutations at Ser-650, Ser-654, and Thr-667 resulted in significant activation of yeast glycogen synthase and elevation in the level of accumulated glycogen as compared with wild type. Likewise, expression of the truncated GS-2 resulted in hyperactive enzyme and the overaccumulation of glycogen. None of the other Ser or Thr mutations substantially affected glycogen synthase activity and glycogen storage. We conclude that Ser-650, Ser-654, and Thr-667 are regulatory phosphorylation sites in vivo. However, in vitro, cyclic AMP-dependent protein kinase modified Ser residue(s) COOH-terminal to position 659, and so the identity of the physiological GS-2 kinases is unclear. Yeast strains bearing glc7 and gac1 mutations are defective in genes encoding type 1 protein phosphatase components and are impaired in their ability to accumulate glycogen. Expression of the truncated GS-2 in these strains restored glycogen accumulation, as did the presence of GS-2 mutated at Ser-650, Ser-654, or Thr-667. These data are consistent with the hypothesis that type 1 phosphatase regulates GS-2 by controlling its phosphorylation state.
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PMID:Control of yeast glycogen synthase-2 by COOH-terminal phosphorylation. 822 15

The skeletal muscle glycogen-binding subunit (GM) of protein phosphatase-1 (PP1) is the founding member of a family of proteins that tether the PP1 catalytic subunit (PP1C) to glycogen and promote the dephosphorylation of glycogen synthase. A hydrophobic sequence (called here the VFV motif) is conserved among GM, the liver subunit GL, and the widely expressed subunits, PTG, R5 and U5. This study analyzed the role of this VFV motif in binding to glycogen and PP1C. Glutathione S-transferase (GST) fusions with the N-terminal domain of GM (GST-GM(1-240)) and with the full length R5 protein (GST-R5) both bound to glycogen in a co-sedimentation assay. In contrast, GST itself did not bind to glycogen. A single residue substitution in GST-GM(1-240), F155A, reduced glycogen binding by 40%. Double residue substitutions V150A/F155A and F155A/V159A resulted in greater reductions (60-70%) in glycogen binding, showing these hydrophobic residues influenced the protein-glycogen interaction. The wild type and V150A/ F155A fusion proteins were digested by trypsin into the same sized fragments at the same rate. Furthermore, the wild type and mutated GST-GM proteins as well as GST-R5 bound equivalent amounts of PP1C, in either pull-down or far-Western assays. These results demonstrated retention of overall tertiary structure by the mutated fusion proteins, and indicated that glycogen and PP1C binding are independent of one another. A 68 residue segment of R5 encompassing the VFV motif was sufficient to produce glycogen binding when fused to GST. This motif, that is in bacterial and fungal starch metabolizing enzymes, probably has been conserved during evolution as a functional domain for binding glycogen and starch.
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PMID:A conserved domain for glycogen binding in protein phosphatase-1 targeting subunits. 984 3


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