Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.
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PMID:Measurement of cdk4 kinase activity using an affinity peptide-tagging technology. 1070 11

Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucleotide protein (G protein) betagamma subunits (Gbetagamma), while Phd-like orphan proteins (PhLOPs) lack the major functional domain for the binding of Gbetagamma. A retina- and pineal gland-specific transcription factor, cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen using PhLOP1 as the bait. Direct protein-protein interactions between Phd or PhLOP1 and CRX were demonstrated using a beta-galactosidase quantitative assay in the yeast two-hybrid system and were confirmed by an in vitro binding assay and a glutathione S-transferase (GST) pull-down assay. To determine if the interaction with Phd or PhLOP1 affected CRX transactivation, a 120-bp interphotoreceptor retinoid binding protein (IRBP) promoter-luciferase reporter construct containing a CRX consensus element (GATTAA) was cotransfected into either COS-7 or retinoblastoma Weri-Rb-1 cells with expression constructs for CRX and either Phd or PhLOP1. Phd and PhLOP1 inhibited the transcriptional activation activity of CRX by 50% during transient cotransfection in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably transfected with CRX. Phd inhibited CRX transactivation in a dose-dependent manner. Whereas Phd is a cytoplasmic phosphoprotein, coexpression of Phd with CRX results in Phd being localized both in the cytoplasm and nucleus. By contrast, PhLOP1 is found in the nucleus even without CRX coexpression. To address the physiological relevance of these potential protein interacting partners, we identified immunoreactive proteins for Phd and CRX in retinal cytosolic and nuclear fractions. Immunohistochemical analysis of bovine retinas reveals colocalization of Phd isoforms with CRX predominantly in the inner segment of cone cells, with additional costaining in the outer nuclear layer and the synaptic region. Our findings demonstrate that both Phd and PhLOP1 interact directly with CRX and that each diminishes the transactivation activity of CRX on the IRBP promoter. A domain that interacts with CRX is found in the carboxyl terminus of the Phd isoforms. Phd antibody-immunoreactive peptides are seen in light-adapted mouse retinal cytosolic and nuclear extracts. Neither Phd nor PhLOP1 affected CRX binding to its consensus DNA element in electrophoretic mobility shift assays. A model that illustrates separate functional roles for interactions between Phd and either SUG1 or CRX is proposed. The model suggests further a mechanism by which Phd isoforms could inhibit CRX transcriptional activation.
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PMID:Modulation of CRX transactivation activity by phosducin isoforms. 1086 77

The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) can inhibit the growth and induce apoptosis of tumor cells. In this study we analysed the growth suppressive effect of HPR on human breast cancer cell lines in vitro and the role of the retinoblastoma protein (pRb) in this response. Treatment of MCF7, T47D and SKBR3 for 24 - 48 h with 3 microM HPR, a concentration attainable in vivo, resulted in growth inhibition and marked dephosphorylation of pRb involving Ser612, Thr821, Ser795 and Ser780, target residues for cyclin-dependent kinase 2 (Cdk2) the former two, and Cdk4 the latter two. Interestingly, this dephosphorylation of pRb occurred in S-G2-M phase cells, as revealed by experiments on cells fractionated by FACS according to the cell cycle phase, hence suggesting that the retinoid interferes with the regulation of pRb phosphorylation. The in vitro phosphorylation of a GST-pRb recombinant substrate by Cdk2 immunocomplexes from MCF7, T47D and SKBR3 was markedly suppressed after HPR treatment, whereas that by Cdk4 complexes was suppressed in T47D and SKBR3 but not in MCF7. The steady-state levels of Cdk2, Cdk4 and Cyclin A proteins were unaffected by HPR, while those of Cyclin D1 were significantly reduced in all three cell lines. Interestingly, Cyclin D1 downregulation by HPR correlated with transcriptional repression, but not with enhanced proteolysis of Cyclin D1 typically elicited by other retinoids. Collectively, our data suggest that the antiproliferative activity of HPR arises from its capacity to maintain pRb in a de-phosphorylated growth-suppressive status in S-G2/M, possibly through Cyclin D1 downregulation and inhibition of pRb-targeting Cdks. Oncogene (2000) 19, 4035 - 41.
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PMID:pRb and Cdk regulation by N-(4-hydroxyphenyl)retinamide. 1096 60

The existence of genetic alterations affecting genes involved in cellular proliferation and death, such as TP53 and K-ras, is one of the most common features of tumour cells. Recently, gene inactivation by promoter hypermethylation has been demonstrated. Methylation is the main epigenetic modification in mammals and abnormal methylation of the CpG islands located in the promoter region of the genes leads to transcriptional silencing. Examples include the p16INK4a, p15INK4B, p14ARF, Von Hippel-Lindau (VHL), the oestrogen and progesterone receptors, E-cadherin, death associated protein (DAP) kinase and the first tumour suppressor gene described, retinoblastoma (Rb) gene. In most cases, methylation involves loss of expression, absence of a coding mutation and restoration of transcription by the use of demethylating agents. However, is there a linkage between genetic and epigenetic alterations? Our results show one side of this puzzle demonstrating that epigenetic lesions drive genetic lesions in cancer. Four specific epigenetic lesions, promoter hypermethylation of the DNA mismatch repair gene hMLH1, the DNA alkyl-repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), the detoxifier glutathione S-transferase P1 (GSTP1) and the familial breast cancer gene BRCA1 may lead to four specific genetic lesions, microsatellite instability, G to A transitions, steroid-related adducts and double-strand breaks in DNA. This is probably only the beginning of an extensive list of epigenetic events that change and make the genetic environment of the transformed cell unstable.
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PMID:Epigenetic lesions causing genetic lesions in human cancer: promoter hypermethylation of DNA repair genes. 1109 2

The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, differentiation, and apoptosis. Several pRb binding proteins complex with pRb through an amino acid sequence called the LXCXE motif. The catalytic subunit of DNA polymerase delta (p125) contains a LXCXE motif. To further study the biochemical function of this polymerase, we sought to determine if p125 interacts with pRb. Experiments using GST-pRb fusion proteins showed that p125 from breast epithelial (MCF10A) cell extracts associates with pRb. In addition, GST-p125 fusion proteins bound pRb from the same cell extracts. The pRb that associated with GST-p125 was largely unphosphorylated. Coimmunoprecipitation experiments using cell cycle synchronized cells revealed that p125 and pRb form a complex predominantly during G1 phase, the phase during which pRb is mostly unphosphorylated. In vitro phosphorylation of GST-pRb by the cyclin dependent kinases reduced the ability of p125 to associate with GST-pRh. Addition of the LXCXE containing protein SV40 large T antigen to GST-pRb blocks the ability of p125 to associate with pRb, suggesting that it may be through a LXCXE sequence by which p125 interacts with pRb. Finally, in vitro polymerase assays demonstrate that GST-pRb fusion protein stimulates DNA polymerase delta activity.
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PMID:Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase delta (p125). 1111 23

GCIP, a newly identified cyclin D-interacting protein, was found to reduce the phosphorylation of retinoblastoma protein and inhibit E2F1-mediated transcriptional activity. To explore more GCIP interacting proteins, the yeast two-hybrid screening using GCIP as a bait protein was performed. One novel gene, p29, was demonstrated to associate with GCIP in the yeast two-hybrid method and in vitro GST pull-down assay. Multiple tissue Northern blot analysis showed that p29 was abundantly expressed in the heart, skeletal muscle, and kidney relative to other tissues. The transient expression of HA-tagged p29 in HeLa cells localized in the nucleus. Taken together, we have isolated a novel protein, p29, which may be involved in the functional regulation of GCIP.
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PMID:p29, a novel GCIP-interacting protein, localizes in the nucleus. 1111 53

Protein Ser/Thr phosphatase-1 (PP1) controls the retinoblastoma protein (pRb) function, including its dephosphorylation at mitotic exit. Since PP1delta was found to coimmunoprecipitate with pRb from mitotic and early G1 cells, we further investigated the PP1delta-pRb association using GST-full length and GST-deletion mutants of delta. GST-delta pulled-down pRb from G2, mitotic and G1 HeLa cells, thus confirming the coimmunoprecipitation results. Among the delta deletion mutants tested, pRb was pulled down by mutant 159-295, which reproduces the C-terminal domain of delta without the C-terminus, whereas the C-terminus alone did not pull-down pRb. Further fragmentation of the 159-295 mutant indicated that pRb was pulled down by fragment 195-260, which includes several residues involved in substrate binding, and by fragment 159-212, which contains the putative pRb-binding motif LxSxE. Altogether the results supported the hypothesis that PP1delta may contribute to the dephosphorylation of pRb at mitotic exit and that the PP1delta-pRb interaction may be at multiple sites.
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PMID:Binding of phosphatase-1 delta to the retinoblastoma protein pRb involves domains that include substrate recognition residues and a pRB binding motif. 1116 67

Ebp1, an ErbB-3 binding protein, inhibits the proliferation and induces the differentiation of human breast cancer cells. The mechanisms of these effects are unknown. Rb, the product of the retinoblastoma gene, is an important modulator of cell cycle progression and cellular differentiation. We report that Rb is a binding target for Ebp1. Ebp1 was localized to both the nucleus and the cytoplasm of logarithmically growing AU565 breast cancer cells and HeLa cells as determined by confocal immunofluorescent microscopy. Ebp1 was present in Rb immunoprecipitates derived from AU565 breast cancer cells. GST-Rb also bound endogenous Ebp1. Using GST-Ebp1 constructs, we determined that the 72 C-terminal amino acids of Ebp1 were sufficient to bind Rb. Dephosphorylation of Ebp1 enhanced the interaction of Ebp1 with Rb. The overexpression of Ebp1 in MCF-7 and AU565 (Rb(+)) cells inhibited the activity of the E2F1 regulated cyclin-E promoter. Ebp1 bound E2F1 indirectly via Rb in lysates of MCF-7 cells. The interaction of Ebp1 with Rb may prove to be an important mechanism of Ebp1 induced changes in cell proliferation and differentiation.
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PMID:Ebp1, an ErbB-3 binding protein, interacts with Rb and affects Rb transcriptional regulation. 1126

It is well established that prolactin (PRL) sustains, while prostaglandin F(2 alpha) (PGF(2 alpha)) curtails, progesterone production by the rat corpus luteum (CL). We have previously shown that the actions of both molecules converge on the 20 alpha-HSD gene and control its expression in a dramatically opposed manner. In this investigation, we have found twelve more genes that are inversely regulated by PRL and PGF(2 alpha). In addition to 20 alpha-HSD, PGF(2 alpha) stimulated and PRL inhibited PGF(2 alpha)-receptor, phospholipase C delta(1) and TGF beta(1) expression. In contrast PRL stimulated and PGF(2 alpha) inhibited the LH receptor, 11 beta-HSD2, sterol carrier protein 2, mitochondrial glutathione S-transferase (GST), GST mu(2), inhibitory DNA-binding proteins 1, 2, and 3, and calcium binding protein 2. We have also identified new target genes for PRL and PGF(2 alpha). PGF(2 alpha) stimulated the expression of genes involved in cell signaling such as cell adhesion kinase-beta, ERK3, FRA2, IL-2 receptor, and 14-3-3 proteins. PGF(2 alpha) also up-regulated the expression of the sodium channel beta(1), Na/K ATPase, annexin IV, GST7pi, and P450 reductase. In contrast PGF(2 alpha) inhibited the expression of two genes involved in cell cycle: cyclin D2 and retinoblastoma related protein (Rb2/p130). It also inhibited genes involved in estradiol (P-450(AROM)) and cholesterol biosynthesis (HMG-CoA synthase), as well as genes involved in tissue remodeling: VEGF and TIMP3. PRL had a profound inhibitory effect on the expression of genes encoding the ADP-ribosylation factor 3, annexin V and c-jun, yet increased the expression of P450scc, 3beta-HSD, and SR-B1 (HDL-receptor), all genes involved in steroidogenesis. PRL also stimulated the expression of beta(2)-microglobulin, TIMP2, cytochrome c oxidase IV, cathepsin H and L, and copper-zinc superoxide dismutase as well as elongation factor SIII, heat shock protein-60 and mitochondrial ATP synthase-D. In conclusion, this investigation has revealed a "yin-yang" relationship between PRL and PGF(2 alpha) in regulating certain critical genes in the rodent CL, and has demonstrated novel regulation by these factors of other important genes involved in luteal function.
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PMID:Opposite effect of prolactin and prostaglandin F(2 alpha) on the expression of luteal genes as revealed by rat cDNA expression array. 1151 96

Retinoblastoma (RB) protein and antibody against RB protein in sera from 45 lung cancer patients and 30 healthy volunteers were examined using bacterially synthesized glutathione S-transferase (GST) RB fusion protein and immunoblot analysis. RB protein was not detected in sera from any individuals with lung cancer or in any healthy volunteers. Sera from 6 patients, including 4 with non-small cell carcinoma and 2 with small cell carcinoma, reacted to a GST-RB fusion protein but not with a GST protein. Sera from 30 normal volunteers reacted to neither GST-RB fusion protein nor GST protein. The backgrounds such as age, gender, performance status, histology, stage, smoking history, and prior treatment were not significantly different between the patients with and without anti-RB antibodies. This is the first report describing the presence of anti-RB antibody in patients with malignant tumors. Further studies are needed to establish clinical significance for anti-RB antibody.
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PMID:Presence of antibodies against retinoblastoma tumor suppressor protein in patients with lung cancer. 1160 6


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