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)

Activation of protein kinase B (PKB) by growth factors has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). Here, we show that agents which raise intracellular cAMP can also stimulate PKB. However, this effect is not sensitive to wortmannin, indicating that it is PI3-kinase independent. This activation does not appear to result from direct phosphorylation by protein kinase A (PKA) since GST-PKB is not an effective PKA substrate. In addition, the activation pathway of PKB by cAMP seems to be linked to that of growth factors, albeit downstream of PI3-kinase. Evidence for this is that a constitutive active PKB, T308D, S473D, containing activating mutations in the serine and threonine residues which are phosphorylated subsequent to PI3-kinase activation, cannot be further stimulated by cAMP elevations. Hence, these data suggest that, in addition to growth factors, cAMP can also lead to activation of PKB. This cAMP stimulatory action appears to require phosphorylation of T308 and S473, and hence would indicate that cAMP modulates the phosphorylation event of these PKB regulatory sites.
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PMID:cAMP stimulates protein kinase B in a Wortmannin-insensitive manner. 920 56

Myosin II was identified as a binding protein to the pleckstrin homology (PH) domain of protein kinase B (PKB) in CHO cell extract by using the glutathione S-transferase-fusion protein as a probe. When myosin II purified from rabbit skeletal muscle was employed, myosin II was shown to bind almost exclusively to the PH domain of PKB among the PH domain fusion proteins examined. The purified myosin II bound to the PH domain of PKB with a Kd value of 1.1 x 10(-7) M. Studies with a series of truncated molecules indicated that the whole structure of the PH domain is required for the binding of myosin II, and the binding to the PH domain was inhibited by phosphatidylinositol 4,5-bisphosphate. These results suggest that myosin II is a specific binding protein to the PH domain of particular proteins including PKB.
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PMID:Identification of myosin II as a binding protein to the PH domain of protein kinase B. 1008 74

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.
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PMID:Substrate recognition by Ca2+/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain. 1033 83

The tumor suppressor gene PTEN/MMAC-1/TEP-1 (referred to hereafter as PTEN) maps to chromosome 10q23 and encodes a dual specificity phosphatase. The PTEN protein negatively regulates cell migration and cell survival and induces a G1 cell cycle block via negative regulation of the phosphatidylinositol 3'-kinase/protein kinase B/Akt signaling pathway. PTEN is frequently mutated or deleted in both prostate cancer cell lines and primary prostate cancers. A murine polyclonal antiserum was raised against a glutathione S-transferase fusion polypeptide of the COOH terninus of PTEN. Archival paraffin tissue sections from 109 cases of resected prostate cancer were immunostained with the antiserum, using DU145 and PC-3 cells as positive and negative controls, respectively. PTEN expression was seen in the secretory cells. Cases were considered positive when granular cytoplasmic staining was seen in all tumor cells, mixed when areas of both positive and negative tumor cell clones were seen, and negative when adjacent benign prostate tissue but not tumor tissue showed positive staining. Seventeen cases (15.6%) of prostate cancer were positive, 70 cases (64.2%) were mixed, and 22 cases (20.2%) were negative. Total absence of PTEN expression correlated with the Gleason score (P = 0.0081) and correlated more significantly with a Gleason score of 7 or higher (P = 0.0004) and with advanced pathological stage (American Joint Committee on Cancer stages T3b and T4; P = 0.0078). Thus, loss of PTEN protein is correlated with pathological markers of poor prognosis in prostate cancer.
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PMID:Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage. 1048 74

The plethora of extracellular stimuli modulating the status of a cell results in the engagement of relatively few pathways responsible for transducing signals to the interior of the cell. One such pathway is the activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of a membrane-restricted second messenger, polyphosphatidylinositide 3'-phosphate. Among the enzymes activated by 3'-phosphorylated inositol lipids is Akt/protein kinase B (PKB). Here we describe a protocol for the expression and one-step purification of human recombinant GST-PKB in Sf9 cells. This scheme allows generating large amounts of homogeneously purified GST-PKB with high specific activity to be employed in high-throughput screening or structural studies.
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PMID:Homogeneous purification of human recombinant GST-Akt/PKB from Sf9 cells. 1049 72

The epithelial Na+ channel (ENaC) constitutes the rate-limiting step for Na+ transport across tight epithelia and is the principal target of hormonal regulation, particularly by insulin and mineralocorticoids. Recently, the serine-threonine kinase (SGK) was identified as a rapidly mineralocorticoid-responsive gene, the product of which stimulates ENaC-mediated Na+ transport. Like its close relative, protein kinase B (also called Akt), SGK's kinase activity is dependent on phosphatidylinositol 3-kinase (PI3K), a key mediator of insulin signaling. In our study we show that PI3K is required for SGK-dependent stimulation of ENaC-mediated Na+ transport as well as for the production of the phosphorylated form of SGK. In A6 kidney cells, mineralocorticoid induction of the phosphorylated form of SGK preceded the increase in Na+ transport, and specific inhibition of PI3K inhibited both phosphorylation of SGK and mineralocorticoid-induced Na+ transport. Insulin both augmented SGK phosphorylation and synergized with mineralocorticoids in stimulating Na+ transport. In a Xenopus laevis oocyte coexpression assay, SGK-stimulated ENaC activity was also markedly reduced by PI3K inhibition. Finally, in vitro-translated SGK specifically interacted with the ENaC subunits expressed in Escherichia coli as glutathione S-transferase fusion proteins. These data suggest that SGK is a PI3K-dependent integrator of insulin and mineralocorticoid actions that interacts with ENaC subunits to control Na+ entry into kidney collecting duct cells.
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PMID:SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport. 1120 6

The prolactin receptor (PrlR) is a member of the cytokine receptor superfamily that lacks an intrinsic kinase domain and relies on the cytoplasmic Jak tyrosine kinases to transduce signals. Prolactin-induced Jak2 activation and consequent tyrosine phosphorylation of the receptor and downstream signaling molecules have been studied, but phosphorylation of the PrlR on serine or threonine residues has not been reported. Here we describe a novel interaction between the PrlR and the phosphoserine/phosphothreonine-binding 14-3-3 proteins. This association is mediated by the KCST391WP motif, which occurs in the major functional isoform of the human receptor and is conserved among a wide variety of species. Mutagenesis of threonine 391 to alanine significantly impaired 14-3-3 binding to the PrlR in both glutathione S-transferase pulldown and coimmunoprecipitation assays. In breast carcinoma and mouse mammary epithelial cell lines, the endogenous receptor was found to associate with glutathione S-transferase-14-3-3 proteins independent of prolactin stimulation. A phospho-specific peptide antibody was generated and used to demonstrate phosphorylation of Thr391 in vivo. Phosphorylation of this site was found to be sensitive to okadaic acid, a specific inhibitor of serine/threonine protein phosphatases. Interestingly, the T391A PrlR mutant exhibited increased basal and prolactin-induced tyrosine phosphorylation compared with the wild-type receptor. This was accompanied by a ligand-induced increase in protein kinase B and Erk activation but not that of Stat5a. Phosphorylation of the receptor on Thr391 may therefore provide a new mechanism by which prolactin signaling is attenuated.
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PMID:Threonine 391 phosphorylation of the human prolactin receptor mediates a novel interaction with 14-3-3 proteins. 1281 9

Activation of the phosphatidylinositol 3-kinase (PI3K)-AKT/protein kinase B signaling pathway has been associated with multiple human cancers. Recently we showed that AKT is activated in both the thyroid and metastatic lesions of a mouse model of follicular thyroid carcinoma [thyroid hormone beta receptor (TRbeta)(PV/PV) mice]. This TRbeta(PV/PV) mouse harbors a knock-in mutant TRbeta gene (TRbetaPV mutant) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. Here we show that in thyroid tumors, PV mutant bound significantly more to the PI3K-regulatory subunit p85alpha, resulting in a greater increase in the kinase activity than did TRbeta1 in wild-type mice. By GST pull-down assays, the ligand-binding domain of TR was identified as the interaction site with p85alpha. By confocal fluorescence microscopy, p85alpha was shown to colocalize with TRbeta1 or PV mainly in the nuclear compartment of cultured tumor cells from TRbeta(PV/PV) mice, but cytoplasmic p85alpha/PV or p85alpha/TRbeta1 complexes were also detectable. Further biochemical analysis revealed that the activation of the PI3K-AKT-mammalian target of the rapamycin-p70(S6K) pathway was observed in both the cytoplasmic and nuclear compartments, whereas the activation of the PI3K-integrin-linked kinase-matrix metalloproteinase 2 pathway was detected mainly in the extranuclear compartments. These results suggest that PV, via the activation of p85alpha, could act to affect PI3K downstream signaling in both the nuclear and extranuclear compartments, thereby contributing to thyroid carcinogenesis. Importantly, the present study unveils a mechanism by which a mutant TR acts to activate PI3K activity via protein-protein interactions.
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PMID:Activation of phosphatidylinositol 3-kinase signaling by a mutant thyroid hormone beta receptor. 1644 24

The involvement of CK1 (casein kinase 1) delta in the regulation of multiple cellular processes implies a tight regulation of its activity on many different levels. At the protein level, reversible phosphorylation plays an important role in modulating the activity of CK1delta. In the present study, we show that PKA (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and PKC (protein kinase C) alpha all phosphorylate CK1delta. PKA was identified as the major cellular CK1deltaCK (CK1delta C-terminal-targeted protein kinase) for the phosphorylation of CK1delta in vitro and in vivo. This was implied by the following evidence: PKA was detectable in the CK1deltaCK peak fraction of fractionated MiaPaCa-2 cell extracts, PKA shared nearly identical kinetic properties with those of CK1deltaCK, and both PKA and CK1deltaCK phosphorylated CK1delta at Ser370 in vitro. Furthermore, phosphorylation of CK1delta by PKA decreased substrate phosphorylation of CK1delta in vitro. Mutation of Ser370 to alanine increased the phosphorylation affinity of CK1delta for beta-casein and the GST (gluthatione S-transferase)-p53 1-64 fusion protein in vitro and enhanced the formation of an ectopic dorsal axis during Xenopus laevis development. Anchoring of PKA and CK1delta to centrosomes was mediated by AKAP (A-kinase-anchoring protein) 450. Interestingly, pre-incubation of MiaPaCa-2 cells with the synthetic peptide St-Ht31, which prevents binding between AKAP450 and the regulatory subunit RII of PKA, resulted in a 6-fold increase in the activity of CK1delta. In summary, we conclude that PKA phosphorylates CK1delta, predominantly at Ser370 in vitro and in vivo, and that site-specific phosphorylation of CK1delta by PKA plays an important role in modulating CK1delta-dependent processes.
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PMID:Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo. 3100 Jun 25

Sarcolemmal Na(+)/H(+) exchanger (NHE) activity is mediated by NHE isoform 1 (NHE1), which is subject to regulation by protein kinases. Our objectives were to determine whether NHE1 is phosphorylated by protein kinase B (PKB), identify any pertinent phosphorylation site(s), and delineate the functional consequences of such phosphorylation. Active PKBalpha phosphorylated in vitro a glutathione S-transferase (GST)-NHE1 fusion protein comprising amino acids 516 to 815 of the NHE1 carboxyl-terminal regulatory domain. PKBalpha-mediated phosphorylation of GST-NHE1 fusion proteins containing overlapping segments of this region localized the targeted residues to the carboxyl-terminal 190 amino acids (625 to 815) of NHE1. Mass spectrometry and phosphorylation analysis of mutated (Ser-->Ala) GST-NHE1 fusion proteins revealed that PKBalpha-mediated phosphorylation of NHE1 occurred principally at Ser648. Far-Western assays demonstrated that PKBalpha-mediated Ser648 phosphorylation abrogated calcium-activated calmodulin (CaM) binding to the regulatory domain of NHE1. In adult rat ventricular myocytes, adenovirus-mediated expression of myristoylated PKBalpha (myr-PKBalpha) increased cellular PKB activity, as confirmed by increased glycogen synthase kinase 3beta phosphorylation. Heterologously expressed myr-PKBalpha was present in the sarcolemma, colocalized with NHE1 at the intercalated disc regions, increased NHE1 phosphorylation, and reduced NHE1 activity following intracellular acidosis. Conversely, pharmacological inhibition of endogenous PKB increased NHE1 activity following intracellular acidosis. Our data suggest that NHE1 is a novel PKB substrate and that its PKB-mediated phosphorylation at Ser648 inhibits sarcolemmal NHE activity during intracellular acidosis, most likely by interfering with CaM binding and reducing affinity for intracellular H(+).
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PMID:Protein kinase B/Akt phosphorylates and inhibits the cardiac Na+/H+ exchanger NHE1. 1884 14


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