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)

The polymerase chain reaction was used to amplify protein tyrosine phosphatase (PTPase)-related cDNA from a template of total RNA isolated from human skeletal muscle. A novel PTPase, which we term PTP-PEST, was detected by this method. The polymerase chain reaction fragment was used to screen two different HeLa cell libraries to obtain full length cDNA clones. The cDNA predicts a protein of 510 amino acids, approximately 60 kDa, that does not contain an obvious signal sequence or transmembrane segment suggesting it is a nonreceptor type enzyme. The PTPase domain is located in the N-terminal portion of the molecule and displays approximately 35% identity to other members of this family of enzymes. The C-terminal segment is rich in Pro, Glu, Asp, Ser, and Thr residues, possessing features of PEST motifs which have previously been identified in proteins with very short intracellular half-lives. The protein was expressed in Escherichia coli as a fusion product with glutathione S-transferase. Intrinsic activity was demonstrated in vitro against a variety of phosphotyrosine-containing substrates including BIRK, the autophosphorylated cytoplasmic kinase domain of the insulin receptor beta subunit. It did not dephosphorylate phosphoseryl-phosphorylase a. PTP-PEST mRNA is broadly distributed in a variety of cell lines. Stimulation of human rhabdomyosarcoma A204 cells, a transformed muscle line, with insulin led to an approximately 4-fold induction of PTP-PEST mRNA within 36 h.
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PMID:Cloning and expression of PTP-PEST. A novel, human, nontransmembrane protein tyrosine phosphatase. 834 45

The cDNA of a novel, ubiquitously expressed protein kinase (Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid sequence (763 amino acids) contains a catalytic domain that is only distantly related to that of other mammalian protein kinases. Its closest relative is the protein kinase Mnb of Drosophila, which is presumably involved in postembryonic neurogenesis (85% identical amino acids within the catalytic domain). Outside the catalytic domain, the sequence comprises several striking structural features: a bipartite nuclear translocation signal, a tyrosine-rich hydrophilic motif flanking the nuclear localization signal, a PEST region, a repeat of 13 histidines, a repeat of 17 serine/threonine residues, and an alternatively spliced insertion of nine codons. A recombinant glutathione S-transferase-Dyrk fusion protein catalyzed autophosphorylation and histone phosphorylation on tyrosine and serine/threonine residues with an apparent Km of approximately 3.4 microM. Exchange of two tyrosine residues in the "activation loop" between subdomains VII and VIII for phenylalanine almost completely suppressed the activity and tyrosine autophosphorylation of Dyrk. Tyrosine autophosphorylation was also reduced by exchange of the tyrosine (Tyr-219) in a tyrosine phosphorylation consensus motif. The data suggest that Dyrk is a dual specificity protein kinase that is regulated by tyrosine phosphorylation in the activation loop and might be a component of a signaling pathway regulating nuclear functions.
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PMID:Dyrk, a dual specificity protein kinase with unique structural features whose activity is dependent on tyrosine residues between subdomains VII and VIII. 863 52

The cDNA of a novel protein kinase (referred to as SNRK) was isolated from a rat fat cell cDNA library with a probe generated by a cloning approach based on the polymerase chain reaction. The encoded polypeptide (746 amino acids, Mr=81627) contains all conserved subdomains characteristic of the protein serine/threonine kinase family. A recombinant fusion protein with glutathione S-transferase catalysed autophosphorylation as well as phosphorylation of histone, confirming that SNRK has indeed protein kinase activity. By Northern blot hybridization, a 5-kb mRNA was detected in brain, heart, fat cells, intestine, testis, ovary, adrenal gland and thymus. In 3T3-L1 cells. SNRK was specifically expressed in the differentiated, adipocyte-like phenotype, whereas its mRNA was not detected in fibroblasts. Sequence comparisons of its catalytic domain relate SNRK to the SNF1 family of protein kinases. The noncatalytic domain comprises several intriguing structural features, including a glycine-rich region, two PEST sequences, and a bipartite nuclear localization signal which is preceded by a stretch of ten consecutive acidic residues. This part of the sequence exhibits no extended similarity with other proteins. In addition, we detected a high degree of sequence similarity with other SNF1-related proteinases in a small region (30-35 amino acids) flanking the C-terminus of the catalytic domain. This domain (designated the SNH domain) appears to define the subfamily of SNF1-related protein kinases and might represent a new type of regulatory domain of protein kinases.
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PMID:Molecular cloning and characterization of a novel mammalian protein kinase harboring a homology domain that defines a subfamily of serine/threonine kinases. 865 23

The Interferon Regulatory Factors (IRFS) play an important role in the transcriptional control of growth regulatory and immunoregulatory genes. The inducibility and availability of IRF-1 and IRF-2 are influenced by external stimuli, such as virus infection or interferon treatment. In the present study, we sought to examine the potential modulatory role of phosphorylation on IRF-1 transcriptional activity. During the purification of IRF recombinant proteins, a kinase activity copurified with IRF-1 (and IRF-2) from baculovirus infected Sf9 insect cell extracts, but not from E. coli extracts. The kinase activity was also identified in Jurkat T cells, specifically interacted with IRF proteins in GST affinity chromatography, and phosphorylated IRF-1 with high specificity in vitro. Using an in gel kinase assay with recombinant IRF-1 as substrate, two molecular weight forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Furthermore, far western analysis of protein-protein interactions demonstrated that casein kinase II directly interacted with IRF-1 protein. Deletion mutation analysis of IRF-1 revealed that IRF-1 was phosphorylated at two clustered sites, one located between amino acids 138-150, the other in the C-terminal acidic activation domain between amino acids 219-231. Cotransfection studies comparing wild type and point mutated forms of IRF-1 demonstrated that mutations of the four phosphoaceptor residues in the C-terminal transactivation domain, significantly decreased transactivation by IRF-1, indicating that casein kinase II may be involved in the regulation of IRF-1 function. Strikingly, the casein kinase II clusters in IRF-1 resemble the sites identified in the C-terminal PEST domain of IkappaBalpha. The present experiments, together with previously published studies with IkappaBalpha, c-Jun and other proteins, indicate a broad role for casein kinase II phosphorylation in the regulation of transcription factor activity.
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PMID:A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity. 1009 6

The adeno-associated virus type 2 (AAV-2) Rep proteins are essential for AAV DNA replication and regulation of AAV gene expression. We have identified a cellular protein interacting with Rep78 and Rep68 in yeast two-hybrid analysis and in GST pull-down assays. This protein has recently been described as both a p53 (p53BP3) and a topoisomerase I interacting protein (Topors). It contains an arginine/serine-rich domain, a RING finger domain and five PEST sequences. A minimal sequence sufficient for interaction with Rep was mapped to Topors amino acids 871 to 917. We show that the same region is also involved in the interaction with p53. Rep sequences involved in interaction with Topors were mapped to Rep amino acids 172 to 481. Overexpression of Topors stimulated AAV gene expression in the absence of helper virus, suggesting a function of Topors as a transcriptional regulator.
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PMID:Topors, a p53 and topoisomerase I binding protein, interacts with the adeno-associated virus (AAV-2) Rep78/68 proteins and enhances AAV-2 gene expression. 1184 45

To study the biological role of p73 alpha, a member of the p53 tumor suppressor family, we performed a yeast two-hybrid screen of a human cDNA library. Using a p73 alpha fragment consisting of amino acids 49-636 as bait, we found that p73 alpha is functionally associated with the human homologue of mouse and hamster homeodomain-interacting protein kinase 2 (HIPK2). The hamster homologue, also known as haHIPK2 or PKM, was used for further characterization of interactions between HIPK2 and members of the p53 protein family. Systematic yeast two-hybrid assays indicated a physical interaction between the oligomerization domains of p73 alpha and p53 (amino acid regions 345-380 and 319-360, respectively) and amino acid region 812-907 of haHIPK2. This region of haHIPK2 includes a PEST sequence, an Ubc9-binding domain, and a partial speckle retention sequence and is identical to amino acid residues 846-941 of human HIPK2 (hHIPK2). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro and immunoprecipitation assays in vivo. HIPK2 colocalized with p73 and p53 in nuclear bodies, as shown by confocal microscopy. Overexpression of HIPK2 stabilized the p53 protein and greatly increased the p73- and p53-induced transcriptional repression of multidrug-resistant and collagenase promoters in Saos2 cells but had little effect on the p73- or p53-mediated transcriptional activation of synthetic p53-responsive and p21WAF1 promoters. Stable expression of HIPK2 in U2OS cells enhanced the cisplatin response of sub-G(1) and G(2)/M populations, and it also increased the apoptotic response to cisplatin and adriamycin as demonstrated by fluorescence-activated cell sorter and 4',6-diamidino-2-phenylindole-staining analyses. HIPK2 potentiated the inhibition of colony formation by p73 and p53. These results suggest that physical interactions between HIPK2 and members of the p53 family may determine the roles of these proteins in cell cycle regulation and apoptosis.
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PMID:Identification and characterization of HIPK2 interacting with p73 and modulating functions of the p53 family in vivo. 1192 30

NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105(758-967) fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.
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PMID:betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932. 1248 91

The activities of different kinases have been correlated to the phosphorylation of Wiscott-Aldrich syndrome protein (WASP) by studies in multiple cell systems. The purpose of this study was to elucidate the regulatory mechanisms involved in WASP phosphorylation and the resulting sealing ring formation in osteoclasts. The phosphorylation state of WASP and WASP-interacting proteins was determined in osteoclasts treated with osteopontin or expressing either constitutively active or kinase-defective Src by adenovirus-mediated delivery. In vitro kinase analysis of WASP immunoprecipitates exhibited phosphorylation of c-Src, PYK2, WASP, protein-tyrosine phosphatase (PTP)-PEST, and Pro-Ser-Thr phosphatase-interacting protein (PSTPIP). Phosphorylation of these proteins was increased in osteopontin-treated and constitutively active Src-expressing osteoclasts. Pulldown analysis with glutathione S-transferase-fused proline-rich regions of PTP-PEST revealed coprecipitation of WASP, PYK2, c-Src, and PSTPIP proteins with the N-terminal region (amino acids 294-497) of PTP-PEST. Similarly, interaction of the same signaling proteins, as well as PTP-PEST, was observed with glutathione S-transferase-fused proline-rich regions of WASP. Furthermore, osteopontin stimulation or constitutively active Src expression resulted in serine phosphorylation and inhibition of WASP-associated PTP-PEST. The inhibition of PTP-PEST was accompanied by an increase in tyrosine phosphorylation of WASP and other associated signaling proteins. Experiments with an inhibitor to phosphatase and small interference RNA to PTP-PEST confirmed the involvement of PTP-PEST in sealing ring formation and bone resorption. WASP, which is identified in the sealing ring of resorbing osteoclasts, also demonstrates colocalization with c-Src, PYK2, PSTPIP, and PTP-PEST in immunostaining analyses. Our findings suggest that both tyrosine kinase(s) and the tyrosine phosphatase PTP-PEST coordinate the formation of the sealing ring and thus the bone-resorbing function of osteoclasts.
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PMID:Phosphorylation of a Wiscott-Aldrich syndrome protein-associated signal complex is critical in osteoclast bone resorption. 1728 76

Tumor protein D52 (TPD52) is overexpressed in multiple cancers including prostate cancer due to gene amplification and investigations to understand its role in the pathophysiology of different cancers are continuing. GST pull-down assays and Tandem affinity purification of TPD52 as bait identified novel prey Peroxiredoxin 1 (PRDX1) in prostate cancer (PCa) cells. PRDX1 interaction with TPD52 was confirmed in immunoprecipitation and affinity interaction assays. Mapping of interaction domain indicated that PRDX1 interacts with C-terminal region of TPD52 containing PEST domain between 152 and 179 amino acids, a new binding region of TPD52. Here we show that TPD52 interaction with PRDX1 increased its peroxidase activity and ectopic expression of TPD52 induced dimerization of PRDX1 in PCa cells. Moreover, H₂O₂ exposure evoked the interaction between TPD52 and PRDX1 while depletion of both proteins led to the accumulation of H₂O₂ suggesting peroxidase activity is important to maintain oxidative capacity in PCa cells. We also observed that overexpression or downregulation of TPD52 and PRDX1 individually or together affecting PCa cells growth, survival, and migration. Altogether, our results show a novel interaction partner of TPD52 providing new insights of its functions and ascertain the role of TPD52-PRDX1 interaction in PCa progression.
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PMID:Tumor protein D52 (isoform 3) interacts with and promotes peroxidase activity of Peroxiredoxin 1 in prostate cancer cells implicated in cell growth and migration. 3098 92

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