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)

We report that albumin is translocated to the nucleus in response to oxidative stress. Prior measurements have demonstrated that in concert with known transcription factors albumin binds to an antioxidant response element, which controls the expression of glutathione S-transferase and other antioxidant enzymes that function to mediate adaptive cellular responses [Holderman, M. T., Miller, K. P., Dangott, L. J., and Ramos, K. S. (2002) Mol. Pharmacol. 61, 1174-1183]. To investigate the mechanisms underlying this adaptive cell response, we have identified linkages between calcium signaling and the nuclear translocation of albumin in JB6 epithelial cells. Under resting conditions, albumin and the calcium regulatory protein calmodulin (CaM) co-immunoprecipitate using antibodies against either protein, indicating a tight association. Calcium activation of CaM disrupts the association between CaM and albumin, suggesting that transient increases in cytosolic calcium levels function to mobilize intracellular albumin to facilitate its translocation into the nucleus. Likewise, nuclear translocation of albumin is induced by exposure of cells to hydrogen peroxide or a phorbol ester, indicating a functional linkage between reactive oxygen species, calcium, and PKC-signaling pathways. Inclusion of an antioxidant enzyme (i.e., superoxide dismutase) blocks nuclear translocation, suggesting that the oxidation of sensitive proteins functions to coordinate the adaptive cellular response. These results suggest that elevated calcium transients and associated increases in reactive oxygen species contribute to adaptive cellular responses through the mobilization and nuclear translocation of cellular albumin.
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PMID:Calmodulin involvement in stress-activated nuclear localization of albumin in JB6 epithelial cells. 1518 87

Intermediate-conductance (IK) Ca(2+)-activated K(+) channels are expressed in many different cell types where they perform a variety of functions including cell volume regulation, transepithelial secretion, lymphocyte activation and cell cycle progression. IK channels are thought to be regulated by phosphorylation; however, whether kinases act directly on the channel is unclear. Using IK channels heterologously expressed in Xenopus oocytes, we demonstrate that IK channels are potently inhibited (60%) by the catalytic subunit of protein kinase A (PKA). Inhibition of IK channel current by PKA is abolished by mutation of four phosphorylation residues (S312, T327, S332, and T348) in the putative calmodulin-binding region of the channel. Evidence for direct modulation of the IK channel by PKA was further demonstrated using GST fusion proteins. The major site of phosphorylation was found to be serine 332; however, other residues were also phosphorylated. We conclude that IK channels can be directly regulated by the cAMP second-messenger system. The mechanism appears to involve direct phosphorylation by PKA of a modulatory locus in the cytoplasmic region of the channel, the site at which calmodulin is thought to interact. Modulation of IK channels by protein kinases may be an important mechanism regulating cell function.
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PMID:Protein kinase A inhibits intermediate conductance Ca2+-activated K+ channels expressed in Xenopus oocytes. 1524 42

A novel CaM-binding protein was isolated through protein-protein interaction based screening of an Arabidopsis cDNA expression library using a 35S calmodulin (CaM) probe. There are four additional homologs in the Arabidopsis genome with similar structures: a BTB domain in the N-terminus and a Zf-TAZ domain in the C-terminus. Hence, they were designated as AtBT1-5 (Arabidopsis thaliana BTB and TAZ domain protein). CaM-binding experiments revealed that all five AtBTs are CaM-binding proteins, and their CaM-binding domains were mapped to the C-terminus. AtBT homologs are also present in rice, but are not present in human, animal, yeast or other organisms, suggesting that the BTB and TAZ domain proteins are plant-specific. The AtBT1-smGFP fusion protein expressed in tobacco BY-2 cells showed that AtBT1 targets the nucleus. Yeast two-hybrid screening using an AtBT1 fragment as bait identified two interacting proteins (AtBET10 and AtBET9) belonging to the family of fsh/Ring3 class transcription regulators. The BTB domain of the AtBTs is required for the interaction, and this protein-protein interaction was confirmed by GST pull-down. AtBET10 also interacts with AtBT2 and AtBT4, and exhibited a transcriptional activation function in yeast cells. AtBTs exhibit varying responses to different stress stimuli, but all five genes responded rapidly to H2O2 and salicylic acid (SA) treatments. These results suggest that AtBTs play a role in transcriptional regulation, and signal molecules such as Ca2+, H2O2, and SA affect transcriptional machinery by altering the expression and conformation of AtBTs which interact with transcriptional activators such as AtBET10.
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PMID:A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. 1531 89

Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin-resistant subline, A431/Pt, were used as a model system. The experimental set-up involved not just a two-way comparison of the control vs. the drug-resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four-way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat-shock proteins HSP60, HSP90, HSC71, heat-shock cognate 71 kDa protein), Ca2+-binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione-S-transferase, GST), anti-apoptotic proteins (such as 14-3-3 switched on in cisplatin-exposed cells) and ion channels (such as VDAC-1, voltage-dependent anion-selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up-regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up-regulated the anti-apoptotic 14-3-3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug-induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.
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PMID:A proteomic approach to cisplatin resistance in the cervix squamous cell carcinoma cell line A431. 1537 90

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP-glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP was responsible for the binding to GAPDH. Further, anti-CaMKP antibody coimmunoprecipitated GAPDH in a rat brain extract. GAPDH was phosphorylated by CaMKI or CaMKIV in vitro; however, when CaMKP coexisted, the phosphorylation was markedly attenuated. Under these conditions, CaMKP significantly dephosphorylated CaMKI and CaMKIV, which had been phosphorylated by CaMK kinase, whereas it did not dephosphorylate the previously phosphorylated GAPDH. The results suggest that CaMKP regulates the phosphorylation level of GAPDH in the CaMKP-GAPDH complex by dephosphorylating and deactivating CaMKs that are responsible for the phosphorylation of GAPDH.
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PMID:Identification of major Ca(2+)/calmodulin-dependent protein kinase phosphatase-binding proteins in brain: biochemical analysis of the interaction. 1568 Sep 15

In this work, we report that the recombinant glutathione S-transferase (GST)-human L-glutamic acid decarboxylase (HGAD) isoforms, 65-kDa L-glutamic acid decarboxylase (GAD) (GST-HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Delta1-70) and HGAD67(Delta1-90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST-HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the V(max) of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin-agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti-GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.
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PMID:Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase. 1568 75

The endothelial cell Ca2+/calmodulin (CaM)-dependent myosin light chain kinase isoform (EC MLCK) is a multifunctional contractile effector involved in vascular barrier regulation, leukocyte diapedesis, apoptosis, and angiogenesis. The EC MLCK isoform and its splice variants contain a unique N-terminal sequence not present in the smooth muscle MLCK isoform (SM MLCK), which allows novel upregulation of MLCK activation by signaling cascades including p60src. The yeast two-hybrid assay system using the entire EC MLCK1 N-terminus (922 aa) as bait, identified additional stable MLCK binding partners including the 12 KDa macrophage migration inhibitory factor (MIF). This finding was confirmed by cross immunoprecipitation assays under non-denaturing conditions and by GST pull down experiments using GST-N-terminal MLCK (#1-923) and MLCK N-terminal deletion mutants in TNFalpha- and thrombin-stimulated endothelium. This EC MLCK-MIF interaction was shown biochemically and by immunofluorescent microscopy to be enhanced in TNFalpha- and thrombin-stimulated endothelium, both of which induce increased MLCK activity. Thrombin induced the colocalization of an epitope-tagged, full-length MIF fusion protein with phosphorylated MLC along peripheral actin stress fibers. Together these studies suggest that the novel interaction between MIF and MLCK may have important implications for the regulation of both non-muscle cytoskeletal dynamics as well as pathobiologic vascular events that involve MLCK.
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PMID:Intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (MIF) in endothelium. 1583 79

Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca(2+) receptor, modulates both osteoclastogenesis and bone resorption. The calmodulin antagonist, trifluoperazine, rescues bone loss in ovariectomized mice (Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536-4543). We show here that a 3-h treatment of mouse osteoclasts with either of the calmodulin antagonists, tamoxifen or trifluoperazine, induces osteoclast apoptosis dose-dependently. Tamoxifen, 10 microm, and trifluoperazine, 10 microm, induce 7.3 +/- 1.8-fold and 5.3 +/- 0.9-fold increases in osteoclast apoptosis, respectively. In Jurkat cells, calmodulin binds to Fas, the death receptor, and this binding is regulated during Fas-mediated apoptosis (Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661-5666). In osteoclasts, calmodulin also binds Fas. When osteoclasts are treated with 10 microm trifluoperazine, the binding between Fas and calmodulin is dramatically decreased at 15 min and gradually recovers by 60 min. A point mutation of the Fas death domain in the Lpr(-cg) mouse renders Fas inactive. Using glutathione S-transferase fusion proteins, the human Fas cytoplasmic domain is shown to bind calmodulin, whereas a point mutation (V254N) comparable with the Lpr(-cg) mutation in mice has markedly reduced calmodulin binding. Osteoclasts derived from Lpr(-cg) mice have diminished calmodulin/Fas binding and are more sensitive to calmodulin antagonist-induced apoptosis than those from wild-type mice. Both tamoxifen- and trifluoperazine-induced apoptosis are increased 1.6 +/- 0.2-fold in Lpr(-cg)-derived osteoclasts compared with osteoclasts derived from wild-type mice. In summary, calmodulin antagonists induce apoptosis in osteoclasts by a mechanism involving interference with calmodulin binding to Fas. The effects of calmodulin/Fas binding on calmodulin antagonist-induced apoptosis may open a new avenue for therapy for osteoporosis.
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PMID:Fas binding to calmodulin regulates apoptosis in osteoclasts. 1596 36

To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, we observed the phosphorylation of Numb family proteins in various rat tissue extracts, and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl-blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein.
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PMID:Phosphorylation of Numb family proteins. Possible involvement of Ca2+/calmodulin-dependent protein kinases. 1610 44

Using protein sequence data obtained from a calcium- and phospholipid-regulated protein kinase purified from maize (Zea mays), we isolated a cDNA encoding a calcium-dependent protein kinase (CDPK), which we designated ZmCPK11. The deduced amino acid sequence of ZmCPK11 includes the sequences of all the peptides obtained from the native protein. The ZmCPK11 sequence contains the kinase, autoregulatory, and calmodulin-like domains typical of CDPKs. Transcripts for ZmCPK11 were present in every tested organ of the plant, relatively high in seeds and seedlings and lower in stems, roots, and leaves. In leaves, kinase activity and ZmCPK11 mRNA accumulation were stimulated by wounding. The level of ZmCPK11 is also increased in noninjured neighboring leaves. The results suggest that the maize protein kinase is involved in a systemic response to wounding. Bacterially expressed glutathione S-transferase (GST)-ZmCPK11 was catalytically active in a calcium-dependent manner. Like the native enzyme, GST-ZmCPK11 was able to phosphorylate histone III-S and Syntide 2. Phosphorylation of histone was stimulated by phosphatidylserine, phosphatidylinositol, and phosphatidic acid, whereas phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, diolein, and cardiolipin did not increase the enzymatic activity. Autophosphorylation of GST-ZmCPK11 was stimulated by calcium and by phosphatidic acid and, to a lesser extent, by phosphatidylserine. Phosphatidylcholine did not affect autophosphorylation. These data unequivocally identify the maize phospholipid- and calcium-regulated protein kinase, which has protein kinase C-like activity, as a CDPK, and emphasize the potential that other CDPKs are regulated by phospholipids in addition to calcium.
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PMID:A wound-responsive and phospholipid-regulated maize calcium-dependent protein kinase. 1629 85


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