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)

Ste5 is a scaffold for the mitogen-activated protein kinase (MAPK) cascade components in a yeast pheromone response pathway. Ste5 also associates with Ste4, the beta subunit of a heterotrimeric guanine nucleotide-binding protein, potentially linking receptor activation to stimulation of the MAPK cascade. A RING-H2 motif at the Ste5 amino terminus is apparently essential for function because Ste5(C177S) and Ste5(C177A C180A) mutants did not rescue the mating defect of a ste5Delta cell. In vitro Ste5(C177A C180A) bound each component of the MAPK cascade, but not Ste4. Unlike wild-type Ste5, the mutant did not appear to oligomerize; however, when fused to a heterologous dimerization domain (glutathione S-transferase), the chimeric protein restored mating in an ste5Delta cell and an ste4Delta ste5Delta double mutant. Thus, the RING-H2 domain mediates Ste4-Ste5 interaction, which is a prerequisite for Ste5-Ste5 self-association and signaling.
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PMID:Ste5 RING-H2 domain: role in Ste4-promoted oligomerization for yeast pheromone signaling. 931 11

Ste5 is essential for pheromone response and binds components of a mitogen-activated protein kinase (MAPK) cascade: Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK). Pheromone stimulation releases Gbetagamma (Ste4-Ste18), which recruits Ste5 and Ste20 (p21-activated kinase) to the plasma membrane, activating the MAPK cascade. A RING-H2 domain in Ste5 (residues 177-229) negatively regulates Ste5 function and mediates its interaction with Gbetagamma. Ste5(C177A C180A), carrying a mutated RING-H2 domain, cannot complement a ste5Delta mutation, yet supports mating even in ste4Delta ste5Delta cells when artificially dimerized by fusion to glutathione S-transferase (GST). In contrast, wild-type Ste5 fused to GST permits mating of ste5Delta cells, but does not allow mating of ste4Delta ste5Delta cells. This differential behavior provided the basis of a genetic selection for STE5 gain-of-function mutations. MATa ste4Delta ste5Delta cells expressing Ste5-GST were mutagenized chemically and plasmids conferring the capacity to mate were selected. Three independent single-substitution mutations were isolated. These constitutive STE5 alleles induce cell cycle arrest, transcriptional activation, and morphological changes normally triggered by pheromone, even when Gbetagamma is absent. The first, Ste5(C226Y), alters the seventh conserved position in the RING-H2 motif, confirming that perturbation of this domain constitutively activates Ste5 function. The second, Ste5(P44L), lies upstream of a basic segment, whereas the third, Ste5(S770K), is situated within an acidic segment in a region that contacts Ste7. None of the mutations increased the affinity of Ste5 for Ste11, Ste7, or Fus3. However, the positions of these novel-activating mutations suggested that, in normal Ste5, the N terminus may interact with the C terminus. Indeed, in vitro, GST-Ste5(1-518) was able to associate specifically with radiolabeled Ste5(520-917). Furthermore, both the P44L and S770K mutations enhanced binding of full-length Ste5 to GST-Ste5(1-518), whereas they did not affect Ste5 dimerization. Thus, binding of Gbetagamma to the RING-H2 domain may induce a conformational change that promotes association of the N- and C-terminal ends of Ste5, stimulating activation of the MAPK cascade by optimizing orientation of the bound kinases and/or by increasing their accessibility to Ste20-dependent phosphorylation (or both). In accord with this model, the novel Ste5 mutants copurified with Ste7 and Fus3 in their activated state and their activation required Ste20.
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PMID:Mutational analysis suggests that activation of the yeast pheromone response mitogen-activated protein kinase pathway involves conformational changes in the Ste5 scaffold protein. 1107 25

VHL is part of an SCF related E3-ubiquitin ligase complex with 'gatekeeper' function in renal carcinoma. However, no mutations have been identified in VHL interacting proteins in wild type VHL tumors. We previously reported that the TRC8 gene was interrupted by a t(3;8) translocation in a family with hereditary renal and non-medullary thyroid cancer. TRC8 encodes a multi-membrane spanning protein containing a RING-H2 finger with in vitro ubiquitin ligase activity. We isolated the Drosophila homologue, DTrc8, and studied its function by genetic manipulations and a yeast 2-hybrid screen. Human and Drosophila TRC8 proteins localize to the endoplasmic reticulum. Loss of either DTrc8 or DVhl resulted in an identical ventral midline defect. Direct interaction between DTrc8 and DVhl was confirmed by GST-pulldown and co-immunoprecipitation experiments. CSN-5/JAB1 is a component of the COP9 signalosome, recently shown to regulate SCF function. We found that DTrc8 physically interacts with CSN-5 and that human JAB1 localization is dependent on VHL mutant status. Lastly, overexpression of DTrc8 inhibited growth consistent with its presumed role as a tumor suppressor gene. Thus, VHL, TRC8, and JAB1 appear to be linked both physically and functionally and all three may participate in the development of kidney cancer.
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PMID:The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. 1203 52

The androgen receptor (AR) N-terminal domain plays a critical role in androgen-responsive gene regulation. A novel AR N-terminal-interacting protein (ARNIP) was isolated using the yeast two-hybrid system and its interaction with amino acids 11-172 of the normal or corresponding region of the polyglutamine-expanded human AR confirmed by glutathione S-transferase pulldown assays. ARNIP cDNAs cloned from NSC-34 (mouse neuroblastoma/spinal cord) or PC-3 (human prostate adenocarcinoma) mRNA encoded highly homologous 30 kDa (261 amino acids) cysteine-rich proteins with a RING-H2 (C3H2C3 zinc finger) domain; this motif is highly conserved in predicted ARNIP-homologous proteins from several other species. Expression of the approximately 1.7 kb ARNIP mRNA was detected in various tissues by Northern blotting, but was highest in mouse testes, kidney and several neuronal cell lines. In addition, the human ARNIP protein was found to be encoded by nine exons spanning 32 kb on chromosome 4q21. In COS-1 cells, coexpression of ARNIP and AR did not affect AR ligand-binding kinetics, nor did ARNIP act as a coactivator or corepressor in transactivation assays. However, AR N-terminal:C-terminal interaction was reduced in the presence of ARNIP. Intriguingly, ARNIP, and in particular its RING-H2 domain, functioned as a ubiquitin-protein ligase in vitro in the presence of a specific ubiquitin-conjugating enzyme, Ubc4-1. Mutation of a single cysteine residue in the ARNIP RING-H2 domain (Cys145Ala) abolished this E3 ubiquitin ligase activity. Fluorescent protein tagging studies revealed that AR-ARNIP interaction was hormone-independent in COS-1 cells, and suggest that colocalization of both AR and ARNIP to the nucleus upon androgen addition may allow ARNIP to play a role in nuclear processes. Thus, identification of a novel AR-interacting protein with ubiquitin ligase activity will stimulate further investigation into the role of ubiquitination and the ubiquitin-proteasome system in AR-mediated cellular functions.
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PMID:Cloning and characterization of an androgen receptor N-terminal-interacting protein with ubiquitin-protein ligase activity. 1220 Feb 28

A breast cancer-associated mRNA originally cloned as a 475-bp partial cDNA from a library enriched for tumour cDNAs [Oncogene 16 (1998) 327] is expressed at high levels in breast and prostate cancer cells. Immunohistochemical analysis indicates that the protein is expressed in primary breast tumours. We used RT-PCR to generate a full-length 2852 nt mRNA sequence that includes the hypothetical open reading frame (ORF) for human RNF11. Our analysis shows that RNF11 encodes modular domains and motifs likely to interact with other proteins involved in oncogenesis. Chief among these are the RING-H2 finger domain that could facilitate the degradation of specific substrate(s) involved in oncogenesis and the PY motif which binds to WW-domain proteins, several of which are known to be E3 ubiquitin ligases. Our GST-pulldown and immunoprecipitation results indicate that RNF11 interacts with the E3 ligase AIP4 when coexpressed with RNF11 in mammalian cells.
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PMID:The RING-H2 protein RNF11 is differentially expressed in breast tumours and interacts with HECT-type E3 ligases. 1455 17

The breast cancer-associated T2A10 clone was originally isolated from a cDNA library enriched for tumour messenger ribonucleic acids. Our survey of 125 microarrayed primary tumour tissues using affinity purified polyclonal antibodies has revealed that corresponding protein is overexpressed in invasive breast cancer and is weakly expressed in kidney and prostate tumours. Now known as RNF11, the gene encodes a RING-H2 domain and a PY motif, both of which mediate protein-protein interactions. In particular, the PPPPY sequence of RNF11 PY motif is identical to that of Smad7, which has been shown to bind to WW domains of Smurf2, an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the TGFbeta receptor complex. Using various mutants of RNF11 in GST pulldown and immunoprecipitation assays, we found that RNF11 interacts with Smurf2 through the PY motif, leading to ubiquitination of both proteins. Smurf2 plays an active role in the repression of TGFbeta signalling, and our data indicate that overexpression of RNF11, through its interaction with Smurf2, can restore TGFbeta responsiveness in transfected cells.
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PMID:The RING-H2 protein RNF11 is overexpressed in breast cancer and is a target of Smurf2 E3 ligase. 1456 29

Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally co-immunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by co-immunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.
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PMID:Inhibition of ubiquitination and stabilization of human ubiquitin E3 ligase PIRH2 by measles virus phosphoprotein. 1614 Jul 59

TRPCs function as cation channels in non-excitable cells. The N-terminal tails of all TRPCs contain an ankyrin-like repeat domain, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid screening approach, we found that RNF24, a new membrane RING-H2 protein, interacted with the ankyrin-like repeat domain of TRPC6. GST pull-down and co-immunoprecipitation assays showed that RNF24 interacted with all TRPCs. Cell surface-labelling assays showed that the expression of TRPC6 at the surface of HEK 293T cells was greatly reduced when it was transiently co-transfected with RNF24. Confocal microscopy showed that TRPC3 and TRPC6 co-localized with RNF24 in a perinuclear compartment and that RNF24 co-localized with mannosidase II, a marker of the Golgi cisternae. Using a pulse-chase approach, we showed that RNF24 did not alter the maturation process of TRPC6. Moreover, in HEK 293T cells, RNF24 did not alter carbachol-induced Ca(2+) entry via endogenous channels or TRPC6. These results indicate that RNF24 interacts with TRPCs in the Golgi apparatus and affects TRPC intracellular trafficking without affecting their activity.
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PMID:RNF24, a new TRPC interacting protein, causes the intracellular retention of TRPC. 1785 Aug 65