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)

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.
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PMID:cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors. 632 45

Limited proteolysis and photoaffinity labeling of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase were studied. Proteolysis by trypsin proceeds in two stages in which the first cleavage yields a product, Mr about 53,000, which has lost 90% of fructose-6-P,2-kinase, but retains nearly 80% of fructose-2,6-bisphosphatase. Further digestion of this product yields a second cleavage product, Mr about 50,000, which is completely devoid of the kinase and most of the phosphatase activities. These results indicate that fructose-6-P,2-kinase resides only in the original ("native") enzyme (Mr = 55,000), but fructose-2,6-bisphosphatase activity is present in both the native enzyme and the cleavage product(s). All three activities of fructose-6-P,2-kinase including the forward, the reverse, and ATP-ADP exchange activities are lost to the same degree by the mild proteolysis. Ki of fructose-6-P for fructose-2,6-bisphosphatase is not altered by the proteolysis. Partial protection against the proteolysis is provided by ATP, fructose-6-P, and fructose-2,6-P2. When the tryptic digestion of fructose-6-P,2-kinase:fructose-2,6-bisphosphatase was performed before and after phosphorylation of the enzyme by cAMP-dependent protein kinase, both the first and the second cleavage products contained the phosphorylation site. 8-Azido-ATP serves as a substrate for fructose-6-P,2-kinase with a Km of about 1 mM. Exposure of the enzyme-8-azido-ATP complex results in covalent incorporation (0.7 mol/mol of subunit) and 90% inactivation of fructose-6-P,2-kinase without loss of fructose 2,6-bisphosphatase. When the native and the first cleavage product of tryptic digestion were photoaffinity labeled with [alpha-32P]8-azido-ATP, the radiolabel occurred only in the native enzyme. These results provide evidence in support of, although not conclusive, the idea that the active sites of this bifunctional enzyme are different and located in two distinct sites.
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PMID:Limited proteolysis and photoaffinity labeling with 8-azido-ATP of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase. 633 Jan 9

Postsynaptic membranes from the electric organ of Torpedo californica, rich in the nicotinic acetylcholine receptor, were shown to contain an endogenous tyrosine protein kinase. This endogenous kinase phosphorylated three major proteins with molecular masses corresponding to 50 kDa, 60 kDa, and 65 kDa. The phosphorylation of these three proteins occurred exclusively on tyrosine residues under the experimental conditions used and was abolished by 0.1% Nonidet P-40 and stimulated by Mn2+. The 50-kDa, and 60-kDa, and 65-kDa phosphoproteins were demonstrated to be the beta, gamma, and delta subunits, respectively, of the nicotinic acetylcholine receptor by purification of the phosphorylated receptor using affinity chromatography. The endogenous tyrosine kinase specifically phosphorylated the beta, gamma, and delta subunits rapidly to a final stoichiometry of approximately equal to 0.5 mol of phosphate per mol of sub-unit. Two-dimensional phosphopeptide mapping of the phosphorylated beta, gamma, and delta subunits, after limit proteolysis with trypsin or thermolysin, indicated that each subunit was phosphorylated on a single site. Locations are proposed for the amino acid residues phosphorylated on the receptor by the tyrosine-specific protein kinase and by two other protein kinases (cAMP-dependent protein kinase and protein kinase C) which phosphorylate the receptor.
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PMID:Phosphorylation of the nicotinic acetylcholine receptor by an endogenous tyrosine-specific protein kinase. 659 75

Certain metabolic properties of hormonally responsive osteogenic sarcoma cells derived from a transplantable rat tumor have been compared with those of related normal rat bone cells. All studies were carried out on cells grown in monolayer culture. Normal rat bone cells derived by repeated collagenase/trypsin digestion of newborn rat calvaria. Bone cells selected for comparison were thought to be osteoblast-like, as judged by enrichment of alkaline phosphatase and adenylate cyclase responsiveness to parathyroid hormone and prostaglandin E2. The adenylate cyclases of the two cell strains were similarly stimulated by a range of prostanoids and their metabolites and analogs. Morphology showed the two cell strains to be similar; the only obvious difference was a multilayering of cells in the sarcoma cultures, while the normal cultures showed abundant extracellular fibril formation which was not seen in the tumor cells. Investigation of the cAMP-dependent protein kinase isoenzymes showed the presence of two forms in both cell types, one eluting at a low salt concentration and the other at a high salt concentration. There was approximately twice the amount of the first isoenzyme compared to the second isoenzyme. The results indicate the usefulness of the two cell strains to elucidate further the molecular mechanisms of action of parathyroid hormone and prostaglandins.
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PMID:Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells. 693 60

The native form of ATP citrate lyase (2 mol of phosphate/tetramer) and the dephospho-ATP citrate lyase (phosphate-free) purified to homogeneity from rat liver, are phosphorylated by ATP and by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle. A total of 2 mol of phosphate/tetramer were incorporated into native enzyme, while with the dephospho form, 4 mol of phosphate were incorporated. The phosphopeptides resulting from trypsin treatment which were isolated from phosphorylated forms of both native enzyme and the dephospho enzyme were similar. The ATP citrate lyase, phosphorylated to an extent of 4 mol of phosphate/tetramer, has the same Vmax as the native enzyme (2 mol of phosphate/tetramer). Native ATP citrate lyase, trypsin-treated to remove the phosphopeptide, could not be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle, suggesting a common trypsin-sensitive specific phosphorylation site. The phosphorylation rate varied with pH in potassium phosphate, imidazole/HCl, and Tris/HCl buffers. Divalent cations were essential for the activity of the protein kinase. The apparent Km value for ATP was found to be 50 microM.
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PMID:Phosphorylation of dephospho-ATP citrate lyase by the catalytic subunit of cAMP-dependent protein kinase. 705 76

The regulatory subunit of cAMP-dependent protein kinase I has been cleaved proteolytically into two structurally independent domains. The larger domain (35K with trypsin or thermolysin and 31K with chymotrypsin) corresponded to the COOH-terminal end of the polypeptide chain and retained the cAMP binding site(s). The smaller domain (11 to 12K with trypsin), corresponding to the NH2-terminal region of the regulatory subunit, contained the region of dimer interaction. In the absence of reducing reagent, the two protomers of the native regulatory subunit and of the smaller domain could be covalently cross-linked by a disulfide bond. In addition to the two major domains, a 15-residue peptide that links the two domains has been isolated and partially characterized. Two major sites on the type I regulatory subunit were susceptible to proteolytic degradation. Site 1, susceptible to cleavage by both trypsin and thermolysin, has the following sequence: LysArg-Arg-Gly-Ala-Ile-Ser-Ala-. Cleavage at this site generated a 35K cAMP-binding fragment. Site 2 contained a chymotryptic cleavage site as well as a secondary tryptic site. The sequence at Site 2 was Val-Arg-Arg-Val-Ile-Ala. Cleavage here generated a 31K cAMP-binding fragment. Both sites contained 2 consecutive basic amino acid residues similar to the corresponding sequence in the type II regulatory subunit; however, in the case of the type I regulatory subunit, the serine at Site 1 does not serve as a site of autophosphorylation. In contrast to the dissociated regulatory subunit, the holoenzyme is partially protected from proteolytic degradation.
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PMID:The structural domains of cAMP-dependent protein kinase I. Characterization of two sites of proteolytic cleavage and homologies to cAMP-dependent protein kinase II. 743 94

Calu-3, a cell line derived from a lung adenocarcinoma, forms tight junctions, expresses cystic fibrosis transmembrane conductance regulator (CFTR), and secretes Cl- in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Anion conductance of Calu-3 cells was assessed with isotopic flux and patch-clamp methods at 22 degrees C. Iodide efflux was increased by cAMP-elevating agents and brief trypsin treatment. A 7.1 +/- 0.4-pS voltage-independent Cl- channel with linear current-voltage relation was the most common channel observed in cell-attached recordings and was identified as CFTR on the basis of shared features with recombinant CFTR. In unstimulated cells, the mean minimum number of active CFTR channels per patch was 1 +/- 1 (n = 12), increasing to 6 +/- 8 (n = 40) after stimulation with cAMP-elevating agents or after brief trypsin treatment. Channel closure after excision was biexponential with tau 1 approximately 4 s and tau 2 approximately 79 s; typically channels were open continuously until closing permanently. In 11 of 12 excised patches, channels were reactivated by exposure to cAMP-dependent protein kinase (PKA) plus ATP. Efficacy of reactivation was inversely related to the duration from excision to addition of PKA. Channels were blocked by 20-40 microM 5-nitro-2-(3-phenylpropylamino)benzoate on cytosolic but not external side. Active CFTR channels were recorded in 83% of total patches. Other types of Cl- channels were observed in 5 of 52 (10%) cell-attached patches and in 17 of 34 (50%) excised patches, including an outwardly rectifying channel in 2 patches. CFTR channels are the predominant pathway for cAMP-stimulated Cl- conductance in Calu-3 cells; the long open times in the absence of ATP are not explained by present models of CFTR activation.
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PMID:CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance. 751 79

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

Ribonucleotide reductase is responsible for supplying the deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two dissimilar protein components called R1 and R2. Immunoprecipitation of R1 and R2 proteins from [32P]orthophosphate-labeled exponentially growing mouse L cells showed that the R2 protein but not the R1 protein of ribonucleotide reductase could be phosphorylated in vivo. Two-dimensional phosphopeptide mapping experiments of trypsin-digested R2 protein showed a major spot containing more than 90% of the total radioactivity and a minor spot with the remaining radioactivity. Phosphoamino acid analysis of R2 phosphorylated protein indicated that phosphorylation occurred exclusively on serine. Protein kinase C, cAMP-dependent protein kinase, p34cdc2, and CDK2 were capable of phosphorylating the R2 protein in vitro, whereas casein kinase II was not. To determine whether any of these enzymes could phosphorylate peptides observed to be phosphorylated in actively growing cells, tryptic phosphopeptide maps of R2 that had been phosphorylated in vitro were compared with maps of R2 that had been isolated from [32P]-labeled cells. Only the phosphopeptide maps obtained with p34cdc2 and CDK2 matched the pattern found in [32P]-labeled cells. Experiments in which tryptic digests from different samples were mixed prior to two-dimensional separation demonstrated comigration of phosphopeptides obtained by in vivo phosphorylation with phosphopeptides derived from p34cdc2 or CDK2 obtained by in vitro phosphorylations. These studies indicate that protein R2 phosphorylation may play an important role in the regulation of ribonucleotide reduction, DNA synthesis, and cell cycle progression, and suggest a potentially important p34cdc2 and/or CDK2 regulation point in DNA replication.
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PMID:Phosphorylation of ribonucleotide reductase R2 protein: in vivo and in vitro evidence of a role for p34cdc2 and CDK2 protein kinases. 825 5

A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, exhibited a molecular mass of 60 kDa as estimated by SDS-polyacrylamide gel electrophoresis. The 60-kDa protein underwent autophosphorylation, was labeled by 8-azido-[alpha-32P]ATP, and reacted with an antibody to the conserved APE domain of the cAMP-dependent protein kinase. The protein did not cochromatograph with p70 S6 kinase and did not cross-react with an anti-p70 kinase antibody. The synthetic peptide S6-21, histone H4, and myelin basic protein were phosphorylated by the purified S6/H4 kinase. Mild digestion of the inactive S6/H4 kinase with trypsin generated a 40-kDa fragment, as determined by SDS-polyacrylamide gel electrophoresis. The trypsin treatment was necessary, but not sufficient, to fully activate the kinase. Subsequent incubation of the trypsin-treated S6 kinase with MgATP resulted in the rapid autophosphorylation of the 40-kDa fragment along with a coordinate increase in kinase activity. The autophosphorylation of the 40-kDa protein was positively correlated with MgATP incubation time and an increase in activity toward the S6-21 peptide, histone H4, and myelin basic protein. Taken together, these data support the hypothesis that this previously uncharacterized S6 kinase belongs to a unique family of protein kinases which utilize autophosphorylation as part of their in vivo activation mechanism.
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PMID:Activation of an S6 kinase from human placenta by autophosphorylation. 836 21


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