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:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.
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PMID:Cyclin E, a redundant cyclin in breast cancer. 898 90

Actin-binding proteins such as profilin and gelsolin bind to phosphatidylinositol (PI) 4,5-bisphosphate (PI 4,5-P2) and regulate the concentration of monomeric actin. We report here that profilin and gelsolin stimulate PI 3-kinase-mediated phosphorylation of PI 4,5-P2 (lipid kinase activity) in a concentration-dependent manner. This effect is specific to profilin and gelsolin because other cytoskeletal proteins such as tau or actin do not affect PI 3-kinase activity. In addition to lipid kinase activity, PI 3-kinase also has protein kinase activity: it phosphorylates proteins (p85 subunit of PI 3-kinase). However, the protein kinase activity of PI 3-kinase was not affected in the presence of profilin. Kinetic analysis, as a function of varying concentrations of ATP and PI 4,5-P2, showed that profilin affects the Vmax of PI 3-kinase without affecting k(m). Profilin may also affect PI 3-kinase activity by its direct association to the enzyme because dot-blot analysis using antibody to glutathione S-transferase (GST) suggested that GST-85 kDa, a fusion protein of PI 3-kinase, binds to profilin. However, PI 3-kinase did not affect the actin-sequestering ability of profilin (determined by pyrene-labeled actin), which indicates that actin and p85 do not share a common binding site on profilin. These studies suggest that profilin and gelsolin may control the generation of 3-OH phosphorylated phosphoinositides, which in turn may regulate the actin polymerization.
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PMID:Profilin and gelsolin stimulate phosphatidylinositol 3-kinase activity. 898 88

TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators.
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PMID:A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein. 900 69

Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) is consistently found in Kaposi's sarcoma lesions and in body-cavity-based lymphomas. A 17-kb KSHV lambda clone was obtained directly from a Kaposi's sarcoma lesion. DNA sequence analysis of this clone identified an open reading frame which has 32% amino acid identity and 53% similarity to the virus-encoded cyclin (v-cyclin) of herpesvirus saimiri (HVS) and 31% identity and 53% similarity to human cellular cyclin D2. This KSHV open reading frame was shown to encode a 29- to 30-kDa protein with the properties of a v-cyclin. KSHV v-cyclin protein was found to associate predominantly with cdk6, a cellular cyclin-dependent kinase known to interact with cellular type D cyclins and HVS v-cyclin. The KSHV v-cyclin was also found to associate weakly with cdk4. KSHV v-cyclin-cdk6 complexes strongly phosphorylated glutathione S-transferase-Rb fusion protein and histone H1 as substrates in vitro. Thus, KSHV v-cyclin resembles the v-cyclin of the T-lymphocyte-transforming HVS in its specificity for association with cdk6 and in its ability to strongly activate cdk6 protein kinase activity.
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PMID:Kaposi's sarcoma-associated herpesvirus encodes a functional cyclin. 903 30

We previously compared the structure and motility suppressive capacity of nm23-H1 by transfection of wild type and site-directed mutant forms into breast carcinoma cells. Wild type nm23-H1 and an nm23-H1(S44A) (serine 44 to alanine) mutant suppressed motility, whereas the nm23-H1(P96S), nm23-H1(S120G), and to a lesser extent, nm23-H1(S120A) mutant forms failed to do so. In the present study wild type and mutant recombinant Nm23-H1 proteins have been produced, purified, and assayed for phosphorylation and phosphotransfer activities. We report the first association of Nm23-H1 mutations lacking motility suppressive capacity with decreased in vitro activity in histidine-dependent protein phosphotransferase assays. Nm23-H1(P96S), a Drosophila developmental mutation homolog, exhibited normal autophosphorylation and nucleoside-diphosphate kinase (NDPK) characteristics but deficient phosphotransfer activity in three histidine protein kinase assays, using succinic thiokinase, Nm23-H2, and GST-Nm23-H1 as substrates. Nm23-H1(S120G), found in advanced human neuroblastomas, exhibited deficient activity in several histidine-dependent protein phosphotransfer reactions, including histidine autophosphorylation, downstream phosphorylation on serines, and slightly decreased histidine protein kinase activity; significant NDPK activity was observed. The Nm23-H1(S120A) mutant was deficient in only histidine-dependent serine autophosphorylation. Nm23-H1 and Nm23-H1(S44A) exhibited normal activity in all assays conducted. Based on this correlation, we hypothesize that a histidine-dependent protein phosphotransfer activity of Nm23-H1 may be responsible for its biological suppressive effects.
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PMID:Site-directed mutation of Nm23-H1. Mutations lacking motility suppressive capacity upon transfection are deficient in histidine-dependent protein phosphotransferase pathways in vitro. 903 58

In this study, we isolated and characterized a human cyclin A-like gene that we named cyclin A1. Cyclin A1 has 48% identity with human cyclin A and is more related to the recently cloned murine cyclin A1 (84% identity). The human cyclin A1 is specifically expressed in testis and brain among all of the normal tissues that we studied by Northern blot analysis; in addition, it is expressed in several myeloid leukemia cell lines, including ML-1, U937, NB4, KG-1, and THP1. A sensitive reverse transcription-PCR-Southern blot method also detected low-level expression of this gene in many other hematopoietic and nonhematopoietic cell lines. The expression of cyclin A1 mRNA is differentiation- and cell cycle-regulated in the ML-1 cells. We raised polyclonal antibodies against a glutathione S-transferase-cyclin A1 fusion protein produced in Escherichia coli. In immunoblot analyses, the antibodies recognized the Mr 65,000 cyclin A1 protein in ML-1 cells. The anti-cyclin A1 also immunoprecipitated the Mr 65,000 cyclin A1, along with the Mr 33,000 cyclin-dependent kinase (CDK) 2 and other proteins at Mr 39,000, 42,000, 45,000, 95,000, and 110,000. In an in vitro kinase assay, the CDK2-cyclin A1 complex precipitated by anti-cyclin A1 showed kinase activities against histone H1. In a yeast two-hybrid assay, cyclin A1 can bind to CDK2 but not to CDC2, CDK4, and CDK5. We mapped the human cyclin A1 gene to chromosome 13q12.3-q13, approximately 1000 kb from the sequence-tagged site marker WI-3374.
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PMID:Characterization of a second human cyclin A that is highly expressed in testis and in several leukemic cell lines. 904 Nov 94

The human immunodeficiency virus type 1 Nef protein was expressed in Escherichia coli as a C-terminal fusion with glutathione S-transferase (GST). The ability of GST-Nef to act as a substrate for cellular kinases in vitro was examined by incubation of purified GST-Nef fusion proteins, immobilized on glutathione-agarose beads, with cytoplasmic extracts from a number of human cell lines. In the presence of [gamma32P]ATP, phosphorylation of Nef occurred predominantly on serine residues. Studies with protein kinase inhibitors suggested that protein kinase C (PKC) was involved in Nef phosphorylation. This was supported further by the demonstration that purified PKC was also able to phosphorylate Nef in the absence of cell extract. Serine/threonine phosphorylation of Nef was also observed in vivo when Nef was expressed with a C-terminal GST or 6-histidine tag in Spodoptera frugiperda insect cells by recombinant baculoviruses. In extracts from Jurkat T cells and U937 monocyte/macrophages Nef also associated with a 57 kDa cellular protein that was itself phosphorylated in vitro. Phosphorylation of this Nef-associated protein was inhibited by heparin and is thus likely to be mediated by casein kinase II. The observation that PKC can phosphorylate Nef in vitro raises the possibility that PKC might play a role in regulating both Nef function and the physical interactions between Nef and cellular components.
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PMID:The human immunodeficiency virus type 1 Nef protein functions as a protein kinase C substrate in vitro. 904 29

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 ((His)p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKIIbeta) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53wt) undergoes better phosphorylation by CKIIbeta and by WCE kinases than mutant forms of p53 at amino acid 249 (p53(249)) or 273 (p53(273)). Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53wt. Comparing truncated forms of p53 revealed that the ability of CKIIbeta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97-117 that increase p53 phosphorylation by CKIIbeta while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 within amino acids 97-117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.
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PMID:Conformation-dependent phosphorylation of p53. 905 Aug 39

Protein phosphorylation is one of several representative post-translational modifications. Cyclic AMP-dependent protein kinase (PKA) plays the crucial and varying role of signal transduction. On the other hand, ras proteins plays an important role in cell proliferation and growth. Although a previous report showed that H-ras protein was phosphorylated by PKA, the stoichiometry was not determined, so we investigated the stoichiometry of phosphorylation of the protein by PKA. H-ras cDNA inserted into a pGEX-2T expressing vector produced high levels of recombinant H-ras (rH-ras) in a fusion protein with glutathione S-transferase. rH-ras was obtained after cleavage by thrombin. Phosphorylation of ras protein by the catalytic subunit of PKA was performed, and the radioactivity was counted after SDS-PAGE and autoradiography. The results indicate that less than 0.1 mol of phosphate was incorporated per mol of H-ras protein, and suggest that H-ras protein could not be a physiologically meaningful substrate for PKA.
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PMID:Phosphorylation of H-ras proteins by protein kinase A. 906 27

All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (delta 1-14) as well as the entire N-terminal segment (delta 1 -39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(delta 1-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decrease in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(delta 1-39) as a fusion protein with glutathione-S-transferase (GST-rC(delta 1-39), although its thermostability (Tm) was decreased by 12.5 degrees C, was catalytically similar to wild-type C and was inhibited by both the type I and II R-subunits and the heat-stable protein kinase inhibitor (PKI). The Tm for holoenzyme II formed with GST-rC(delta 1-39) was 16.5 degrees C greater than the Tm for free GST-rC(delta 1-39), and the Ka(cAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.
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PMID:Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface. 907 Apr 39


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