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)

We have performed comparative studies on the E7 proteins from malignant and non-malignant Human Papillomavirus types HPV 1, 6, 11, 16, 18, 33). GST/E7 fusion proteins from all these HPV types associate with Rb1, p107 and the cyclin A/CDK2 complex. As has been shown for Rb1, the association with p107 and Cyclin A was weaker for the 'low risk' HPV6 and 11 E7 proteins as compared to 'high risk' HPV16, 18 and 33 E7 proteins. In contrast the E7 protein of the benign type HPV1 bound Rb1, p107 and cyclin A with the same affinity as the 'high risk' E7 proteins. The affinities of the E7/Rb1 interaction have been confirmed in vivo by the 'two hybrid' method in the yeast Saccharomyces cerevisiae. Although HPV1 E7 showed the same affinity in vitro and in vivo for Rb1 as the high risk HPV E7s, it did not have the ability to activate the E2F-1 transcription factor inhibited by Rb1, nor did it have any transforming activity when coexpressed with activated ras in primary rodent cells.
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PMID:Functional studies of E7 proteins from different HPV types. 805 27

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

Cyclin E, a G(1) cyclin, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In this study we have examined the possibility that the shortened mRNA splice variants could give rise to tumor-specific cyclin E LMW proteins. We used the Splice Capture method to identify, enumerate and isolate known spliced mRNAs and to look for previously undetected mRNA forms of cyclin E that might be translated into the LMW proteins. We show that a new splice variant of cyclin E found in tumor cells isolated by the Splice Capture strategy, named Delta48, activates CDK2 more robustly than full-length cyclin E when assayed from transiently transfected cells with the natural substrate GST-Rb. We also found the Splice Capture method to be superior to the conventional RNase protection assay in analyzing the cyclin E mRNA present in normal and tumor cells. Splice Capture enumerated the relative abundance of known forms of cyclin E mRNA and easily discovered new splice variants in both normal and tumor cells. We conclude that the abundance of cyclin E splice variants in cells may represent a novel form of regulation of cyclin E, and if translated they show altered substrate specificity compared to the full length form of cyclin E.
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PMID:Novel splice variants of cyclin E with altered substrate specificity. 1109 97

The cyclin-dependent kinase (CDK)-activating kinase (CAK) phosphorylates a conserved threonine residue on CDKs and activates them. Two known classes of CAKs are represented by monomeric Cak1p in budding yeast Saccharomyces cerevisiae and by heterotrimeric CDK7-cyclin H-Mat1 in human and other metazoa. We report here the identification of p42, a novel CAK activity in human cells. p42 has sequence homology to both Cak1p and CDK7 groups of CAKs. p42 is essential for the phosphorylation of Thr-160 and activation of CDK2. A dominant-negative p42 mutant, T161A, and posttranscriptional gene silencing of p42 with RNA(i)-impaired Thr-160 phosphorylation and activity of CDK2. Purified p42 phosphorylated glutathione S-transferase-CDK2 at Thr-160 within the T-loop and activated its histone H1 kinase activity. Finally, p42 is indispensable for cell growth. Cells lacking p42 were incapable of growing and forming colonies whereas cells with a reduced level of p42 grew at significantly slower rates than control cells. Our findings suggest that p42 represents a novel CAK activity in mammalian cells.
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PMID:p42, a novel cyclin-dependent kinase-activating kinase in mammalian cells. 1459 12

Human DOC-1/CDK2AP1 gene encodes a growth suppressor protein of 12kDa (p12(DOC-1/CDK2AP1)). Recently, p12(DOC-1/CDK2AP1) has been shown to associate with cell cycle proteins including CDK2 and DNA polymerase alpha/primase. It negatively regulates CDK2 activities and suppresses DNA replication. Therefore, identification of other p12(DOC-1/CDK2AP1) interacting proteins might clarify its role in the cell cycle regulation and carcinogenesis. The purpose of this study was to identify additional p12(DOC-1/CDK2AP1) interacting proteins using the yeast two-hybrid system. Using human p12(DOC-1/CDK2AP1) as a bait in a liver cDNA library screening, cDNA clones identical to human DOC-1R transcript were identified. The interaction between p12(DOC-1/CDK2AP1) and p14(DOC-1R) was verified in vitro and in cells. GST pull-down assay and immunoprecipitation experiments confirmed the interaction between the two proteins. The critical region for p12(DOC-1/CDK2AP1)'s interaction with p14(DOC-1R) was defined to amino acids 20-25 by using a series of deletion mutants as baits in the yeast two-hybrid system. Our data indicated that p12(DOC-1/CDK2AP1) could associate with its homologous protein, p14(DOC-1R).
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PMID:Interaction of the CDK2-associated protein-1, p12(DOC-1/CDK2AP1), with its homolog, p14(DOC-1R). 1498 11

The CDK2-associated cyclin A1 is essential for spermatogenesis and contributes to leukemogenesis. The detailed molecular functions of cyclin A1 remain unclear, since the molecular networks involving cyclin A1-CDK2 have not been elucidated. Here, we identified novel cyclin A1/CDK2 interaction partners in a yeast triple-hybrid approach. Several novel proteins (INCA1, KARCA1, and PROCA1) as well as the known proteins GPS2 (G-protein pathway suppressor 2), Ku70, receptor for activated protein kinase C1/guanine nucleotide-binding protein beta-2-like-1, and mRNA-binding motif protein 4 were identified as interaction partners. These proteins link the cyclin A1-CDK2 complex to diverse cellular processes such as DNA repair, signaling, and splicing. Interactions were confirmed by GST pull-down assays and co-immunoprecipitation. We cloned and characterized the most frequently isolated unknown gene, which we named INCA1 (inhibitor of CDK interacting with cyclin A1). The nuclear INCA1 protein is evolutionarily conserved and lacks homology to any known gene. This novel protein and two other interacting partners served as substrates for the cyclin A1-CDK2 kinase complex. Cyclin A1 and all interaction partners were highly expressed in testis with varying degrees of tissue specificity. The highest expression levels were observed at different time points during testis maturation, whereas expression levels in germ cell cancers and infertile testes decreased. Taken together, we identified testicular interaction partners of the cyclin A1-CDK2 complex and studied their expression pattern in normal organs, testis development, and testicular malignancies. Thereby, we establish a new basis for future functional analyses of cyclin A1. We provide evidence that the cyclin A1-CDK2 complex plays a role in several signaling pathways important for cell cycle control and meiosis.
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PMID:Identification of interaction partners and substrates of the cyclin A1-CDK2 complex. 1515 2

Using a yeast two-hybrid screening technique and the p50 subunit of human DNA polymerase delta (pol delta) as a bait, p21 was found to interact with the p50 subunit of pol delta. A direct interaction between p21 and p50 was confirmed by using ELISA and pull-down assays with purified proteins. The interaction sites between p50 and p21 were mapped by pull down assays with GST deletion mutants. Residues 127-193 constitute the primary interaction region on p50 to which p21 binds, while p50 binds to the C-terminal 26 residues of p21. A histone kinase activity was associated with the highly purified calf thymus pol delta and addition of purified recombinant p21 inhibited the kinase activity in a dose dependent manner. p50 is phosphorylated in vivo and can be phosphorylated by CDK2/cyclinA in vitro. In vivo evidence of p21 association with p50 was obtained by coimmunoprecipitation using MCF7 cells. It was also shown that the association of p21 with p50 and other components of the pol delta complex increased in MCF7 cells treated with adriamycin. Our results suggested that p50 might target or anchor p21 to pol delta complex upon certain DNA damage such as adriamycin treatment.
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PMID:Direct interaction of p21 with p50, the small subunit of human DNA polymerase delta. 1647 63

We present an NMR-based antagonist induced dissociation assay (AIDA) for validation of inhibitor action on protein-protein interactions. As opposed to many standard NMR methods, AIDA directly validates the inhibitor potency in an in vitro NMR competition binding experiment. AIDA requires a large protein fragment (larger than 30 kDa) to bind to a small reporter protein (less than 20 kDa). We show here that a small fragment of a protein fused to glutathione S-transferase (GST) can effectively substitute the large protein component. We successfully used a GST-tagged N-terminal 73-residue p53 domain for binding studies with the human MDM2 protein. Other interactions we studied involved complexes of CDK2, cyclin A, p27, and the retinoblastoma protein. All these proteins play a key role in the cell division cycle, are associated with tumorigenesis, and are thus the subject of anticancer therapy strategies.
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PMID:An NMR-based antagonist induced dissociation assay for targeting the ligand-protein and protein-protein interactions in competition binding experiments. 1769 13

Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.
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PMID:Expanding the substantial interactome of NEMO using protein microarrays. 2009 47

DOC-1R (deleted in oral cancer-1 related) is a novel putative tumor suppressor. This study investigated DOC-1R antitumor activity and the underlying molecular mechanisms. Cell phenotypes were assessed using flow cytometry, BrdU incorporation and CDK2 kinase assays in DOC-1R overexpressing HeLa cells. In addition, RT-PCR and Western blot assays were used to detect underlying molecular changes in these cells. The interaction between DOC-1R and CDK2 proteins was assayed by GST pull-down and immunoprecipitation-Western blot assays. The data showed that DOC-1R overexpression inhibited G1/S phase transition, DNA replication and suppressed CDK2 activity. Molecularly, DOC-1R inhibited CDK2 expression at the mRNA and protein levels, and there were decreased levels of G1-phase cyclins (cyclin D1 and E) and elevated levels of p21, p27, and p53 proteins. Meanwhile, DOC-1R associated with CDK2 and inhibited CDK2 activation by obstructing its association with cyclin E and A. In conclusion, the antitumor effects of DOC-1R may be mediated by negatively regulating G1 phase progression and G1/S transition through inhibiting CDK2 expression and activation.
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PMID:Overexpression of DOC-1R inhibits cell cycle G1/S transition by repressing CDK2 expression and activation. 2378 Nov 48


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