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)

A 37 kDa protein that binds to diadenosine tetraphosphate (Ap4A) was purified from human HeLa cells and identified as uracil DNA glycosylase/glyceraldehyde-3-phosphate dehydrogenase (UDG/GAPDH). Utilizing photoaffinity labeling with [alpha-32P]8N3-Ap4A, an Ap4A binding protein of 37 kDa was identified from HeLa cell nuclear extracts. The 37 kDa protein was purified to homogeneity and subjected to trypsin digestion followed by amino acid sequence analysis. Two peptide sequences were determined and both had complete identity with the amino acid sequence of the 37 kDa polypeptide of UDG/GAPDH. Purified UDG/GAPDH binds to Ap4A with the same affinity as the HeLa cell nuclear 37 kDa Ap4A binding protein, and monoclonal antibodies to UDG/GAPDH cross-react with the 37 kDa Ap4A binding protein. UDG/GAPDH has been previously demonstrated to have numerous nonglycolytic activities. The UDG function is involved in DNA repair by excision of uracil from DNA. GAPDH is a RNA binding protein and binds to tRNA and AU-rich RNA. The AU-rich RNA binding has been implicated in the regulation of AU-rich element dependent mRNA turnover and translation. The identification of UDG/GAPDH as an Ap4A binding protein may be physiologically relevant to the proposed role of Ap4A as a regulatory nucleotide in cell growth.
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PMID:Uracil DNA-glycosylase/glyceraldehyde-3-phosphate dehydrogenase is an Ap4A binding protein. 762 40

Ecto-protein kinases (ecto-PK), primarily of the serine/threonine kinase type, have been previously described on the surface of various normal, transformed, and tumor cells. We have found that in the presence of ATP and Mg2+, exogenously added substrates such as phosvitin and poly(Glu4-Tyr) are phosphorylated by intact K562 erythroleukemia, HL60 promyelocytic leukemia, and U937 histiocytic leukemia human cells. Phosphoamino acid analysis indicated that phosvitin, histone H2B, casein, and protamine are phosphorylated on serine and threonine residues, whereas poly(Glu4-Tyr) is phosphorylated on tyrosine. We also present evidence showing that the C9 complement protein, a key component of the membranolytic protein complex of the complement system, is exclusively phosphorylated by the K562 cells on serine residues. Phosphorylation of poly(Glu4-Tyr) is markedly enhanced by Mn2+, whereas C9 phosphorylation is rather inhibited by Mn2+. It is concluded that human leukemic cells express on their surface two types of ecto-PK, one phosphorylating serines and threonines and one specific to tyrosines. The ecto-PKs are spontaneously shed from fully viable cells into the medium in a temperature-dependent manner. Upon sedimentation of cell supernatants at 100,000g, the ecto-PKs are found sedimented with small membrane vesicles. Treatment of intact K562 cells or of released membrane vesicles with bacterial phospholipase C, but not with trypsin or pronase, releases the two types of ecto-PK from the cell or vesicle membrane, respectively. This is accompanied by a marked increase in the released phosphorylating activity. It is, therefore, suggested that these ecto-PKs are either covalently linked to phospholipids or strongly attached to lipid-anchored molecules in the cell surface membrane. Several endogenous proteins in the released membranes are phosphorylated by the ecto-PKs on serines and to a lesser extend on threonines. Two proteins (PTP79 and PTP54) are phosphorylated in a manganese-dependent manner on tyrosines.
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PMID:Shedding of tyrosine and serine/threonine ecto-protein kinases from human leukemic cells. 786 34

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

The structural and functional properties of chloroplast glyceraldehyde-3-P-dehydrogenase I (D-Glyceraldehyde-3-phosphate: NADP oxidoreductase (phosphorylating) EC 1.2.1.13) from Spinacia oleracea were investigated by limited proteolysis. The enzyme is insensitive to trypsin and chymotrypsin, while Staphylococcus aureus V8 protease cleaves the C-terminal region of its subunits. Subunit A (36 kDa) is only partially cleaved at Glu 317. No intact subunit B (39 kDa) is found at the end of the proteolytic experiment: two forms are originated from this subunit which is cleaved at Glu 342 and Glu 320. Proteolytic cleavage at these sites does not significantly alter enzymatic activity, but leads to destabilization of the protein. Unlike the intact parent enzyme (600 kDa) the cleaved enzyme behaves as a 150-kDa species in size exclusion chromatography.
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PMID:Limited proteolysis of chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP) from Spinacia oleracea. 835 35

Extracellular adenosine triphosphate (ATPo) has been suggested to play a role in lymphocyte effector functions. Recently, it has been suggested that MgATP2- may be the molecular species which is involved in modulating the lytic interaction between cytotoxic T-lymphocytes (CTL) and their target cells. In this study, we provide evidence that ATPo mediates the phosphorylation of extracellular proteins on T-lymphocytes through the action of ectoprotein kinases. The ectophosphorylation is temperature-dependent, supported by Mg2+ and Mn2+, and both ATP and GTP, whereas kinase activity and/or substrates were removed by pretreatment of intact lymphocytes with trypsin. We show the presence of extracellular ATP/GTP-binding sites, indicating the presence of ectoenzymes on intact lymphocytes. The major ectoprotein kinase was identified as a casein kinase II-like protein kinase and could be inhibited by heparin, whereas its activity was enhanced by spermine. The ectoprotein kinase showed remarkable substrate specificity, phosphorylating the serum protein vitronectin, but not fibronectin. In experiments with the cell-impermeable protein kinase inhibitor K-252b, we demonstrate the possible functional importance of ectoprotein kinase in CTL-mediated cytotoxicity, i.e., target cell death was completely blocked by K-252b without affecting intracellular phosphorylation. These results suggest that ectoprotein phosphorylation may possibly be an important event in immunologically relevant cell-cell interactions.
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PMID:Phosphorylation of T-lymphocyte plasma membrane-associated proteins by ectoprotein kinases: implications for a possible role for ectophosphorylation in T-cell effector functions. 931 12

We have previously shown that protein kinase C-beta (PKC-beta) is required for activation of tyrosinase (Park, H. Y., Russakovsky, V., Ohno, S., and Gilchrest, B. A. (1993) J. Biol. Chem. 268, 11742-11749), the rate-limiting enzyme in melanogenesis. We now examine its mechanism of activation in human melanocytes. In vivo phosphorylation experiments revealed that tyrosinase is phosphorylated through the PKC-dependent pathway and that introduction of PKC-beta into nonpigmented human melanoma cells lacking PKC-beta lead to the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-beta in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-beta but not PKC-alpha was closely associated with tyrosinase on the outer surface of melanosomes. Western blot analysis confirmed the association of PKC-beta with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which contains two serines but no threonines, was phosphorylated by the serine/threonine kinase PKC-beta. These two serines at positions 505 and 509 both are present in the C-terminal peptide generated by trypsin digestion of tyrosinase. Co-migration experiments comparing synthetic peptide standards of all three possible phosphorylated tryptic peptides, a diphosphopeptide and two monophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC-beta and then subjected to trypsin digestion revealed that both serine residues are phosphorylated by PKC-beta. We conclude that PKC-beta activates tyrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein.
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PMID:Protein kinase C-beta activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain. 1034 9

Expression of the porin genes of Escherichia coli is regulated in part by the osmolarity of the growth medium. The process is controlled by the histidine kinase EnvZ and the response regulator OmpR. We have previously shown that phosphorylation of OmpR increases its affinity for the upstream regulatory regions of ompF and ompC. We now report that, in the presence of DNA, there is a dramatic stimulation in the level of phospho-OmpR. This effect is independent of the source of phosphorylation, i.e., stimulation of phosphorylation is observed with a small phosphorylating agent such as acetyl phosphate or with protein-catalyzed phosphorylation by the kinase EnvZ. The dephosphorylation rate of phospho-OmpR is affected only slightly by the presence of DNA; thus, the increased level is largely caused by an increased rate of phosphorylation. Stimulation of phosphorylation requires specific binding of DNA by OmpR. Occupancy of the DNA binding domain exposes a trypsin cleavage site in the linker, which connects the phosphorylation domain with the DNA binding domain. Our results indicate that when DNA binds in the C terminus, it enhances phosphorylation in the N terminus, and the linker undergoes a conformational change. A generalized mechanism involving a four-state model for response regulators is proposed.
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PMID:C-terminal DNA binding stimulates N-terminal phosphorylation of the outer membrane protein regulator OmpR from Escherichia coli. 1051 29

We have studied the role of core histone tails in the assembly of mitotic chromosomes using Xenopus egg extracts. Incubation of sperm nuclei in the extracts led to the formation of mitotic chromosomes, a process we found to be correlated with phosphorylation of the N-terminal tail of histone H3 at Ser10. When the extracts were supplemented with H1-depleted oligosomes, they were not able to assemble chromosomes. Selective elimination of oligosome histone tails by trypsin digestion resulted in a dramatic decrease in their ability to inhibit chromosome condensation. The chromosome assembly was also inhibited by each of the histone tails with differing efficiency. In addition, we found that nucleosomes were recruiting through the flexible histone tails some chromosome assembly factors, different from topoisomerase II and 13S condensin. These findings demonstrate that histone tails play an essential role in chromosome assembly. We also present evidence that the nucleosomes, through physical association, were able to deplete the extracts from the kinase phosphorylating histone H3 at Ser10, suggesting that this kinase could be important for chromosome condensation.
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PMID:Core histone N-termini play an essential role in mitotic chromosome condensation. 1065 37

Phosphorylation of eIF4E is required for protein synthesis during starfish oocyte maturation. The activity of protein kinase C-related kinase 2 (PRK2) increases prior to the phosphorylation of eIF4E (G. Stapleton et al., 1998, Dev. Biol. 193, 34-46). We investigate here whether eIF4E is activated by PRK2. A 3.5-kb eIF4E clone isolated from starfish cDNA is 57% identical to human eIF4E and contains the putative phosphorylation site serine-209. The serine-209 environment (SKTGS(209)MAKSRF) is similar to the consensus sequence of the phosphorylation site of protein kinase C and related kinases. A starfish eIF4E fusion protein (GST-4E) was phosphorylated in vitro by PRK2 in the presence of 1,2-diolyl-sn-glycerol 3-phosphate. In contrast, replacing the GST-4E serine-209 with an alanine significantly reduced this phosphorylation. Analysis by two-dimensional phosphopeptide mapping reveals a major phosphopeptide in trypsin-digested GST-4E, but not in its serine-209 mutant. Importantly, this major phosphopeptide in GST-4E corresponds to a major phosphopeptide of eIF4E isolated from (32)P-labeled oocytes. Thus, PRK2 may regulate translation initiation during oocyte maturation by phosphorylating the serine-209 residue of eIF4E in starfish. We also demonstrate that high levels of cAMP inhibit the activation of PRK2, eIF4E, and the eIF4E binding protein during starfish oocyte maturation, while PI3 kinase activates these proteins.
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PMID:Protein kinase C-related kinase 2 phosphorylates the protein synthesis initiation factor eIF4E in starfish oocytes. 1111 22

Heat shock (HS) induces a wide variety of biological processes, including inhibition of protein synthesis, elevated expression of heat shock proteins, induction of thermotolerance, and apoptotic cell death in a dose-dependent manner. We compared phosphorylated proteins in heat-shocked and thermotolerant cells using proteome analysis. After HS treatment of control RIF-1 and their thermotolerant derivatives, TR-RIF-1 cells, cellular proteins were separated by two-dimensional gel electrophoresis and the phosphorylated proteins were detected with the anti-phosphotyrosine antibodies. We found that 93 proteins showed significant changes in phosphorylation between control and thermotolerant cells as a function of recovery time after HS; we identified 81 of these proteins with peptide mass fingerprinting using MALDI-TOF MS after in-gel trypsin digestion. These phosphorylated proteins exhibit various cellular functions, including chaperones, ion channels, signaling molecules, in transcription and translation processes, in amino acid biosynthesis, oxidoreduction, energy metabolism, and cell motility or structure, suggesting that HS turns on the various signaling pathways by activating protein-tyrosine kinases (PTKs). Of these, 20 proteins were previously identified phosphorylated proteins and 64 were newly identified. These proteins can be grouped into three families: 1) proteins highly phosphorylated in TR-RIF-1 cells at basal level and phosphorylated more significantly by HS in RIF-1 than TR-RIF-1; 2) proteins highly phosphorylated in control RIF-1 cells at basal level and phosphorylated more easily by HS in TR-RIF-1 than in RIF-1 cells; and 3) proteins with a similar basal phosphorylation level in both RIF-1 and TR-RIF-1 cells and responding to HS similarly in both cells. Most of the phosphorylated proteins are presumably involved in HS signaling in different ways, with the first and second families of proteins influencing thermotolerance. The possible tyrosine phosphorylation sites, the possible PTKs phosphorylating these proteins, and the proteins binding to these phosphorylated sites were predicted by the Netphos, ScanProsite, and Scansite programs. These results suggest that HS can activate various PTKs and HS responses can be regulated by phosphorylations of proteins having various functions.
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PMID:Proteomic analysis of protein phosphorylations in heat shock response and thermotolerance. 1188 68


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