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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To identify the genes involved in the partial resistance of sunflower (Helianthus annuus) to the necrotrophic fungus Phoma macdonaldii, we developed a 1000-element cDNA microarray containing carefully chosen genes putatively involved in primary metabolic pathways, signal transduction and biotic stress responses. A two-pass general linear model was used to normalize the data and then to detect differentially expressed genes. This method allowed us to identify 38 genes differentially expressed among genotypes, treatments and times, mainly belonging to plant defense, signaling pathways and amino acid metabolism. Based on a set of genes whose differential expression was highly significant, we propose a model in which negative regulation of a dual-specificity MAPK phosphatase could be implicated in sunflower defense mechanisms against the pathogen. The resulting activation of the MAP kinase cascade could subsequently trigger defense responses (e.g. thaumatin biosynthesis and phenylalanine ammonia lyase activation), under the control of transcription factors belonging to MYB and WRKY families. Concurrently, the activation of protein phosphatase 2A (PP2A), which is implicated in cell death inhibition, could limit pathogen development. The results reported here provide a valuable first step towards the understanding and analysis of the P. macdonaldii-sunflower interaction.
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PMID:A cDNA microarray approach to decipher sunflower (Helianthus annuus) responses to the necrotrophic fungus Phoma macdonaldii. 1662 74

Loss of fragile histidine triad (Fhit) expression is often associated with human malignancies, and Fhit functions as a tumor suppressor in controlling cell growth and apoptosis, although specific signal pathways are still undefined. We have used a proteomic approach to define proteins in the Fhit-mediated tumor suppression pathway. Because substitution of Tyr(114) (Y114) with phenylalanine (Y114F) diminishes Fhit functions, we did protein expression profiling to identify proteins differentially expressed in Fhit-negative H1299 lung cancer cells infected with wild-type (Ad-FHIT-wt) and Y114 mutant FHIT-expressing (Ad-FHIT-Y114F) adenoviruses. Among 12 distinct proteins that exhibited 4-fold differences in expression on comparison of the two infected cell lysates, cyclophilin A, the intracellular reporter of the immunosuppressive drug cyclosporine A, showed a remarkably decreased protein level in cells infected with Ad-FHIT-wt versus Ad-FHIT-Y114F. Conversely, loss of Fhit expression resulted in increased cyclophilin A expression in mouse tissues and cell lines. Restoration of Fhit expression led to down-regulated cyclophilin A protein expression and subsequently prevented cyclophilin A-induced up-regulation of cyclin D1, Cdk4, and resultant cell cycle progression (G(1)-S transition), which was independent of Ca(2+)/calmodulin-dependent kinase inhibitor, KN-93. Interestingly, Fhit down-modulation of phosphatase activity of calcineurin, which controls cyclin D1/Cdk4 activation, was reversed by cyclophilin A treatment in a concentration-dependent manner, a reversal that was inhibited by additional cyclosporine A treatment. Thus, cyclophilin A is a downstream target in Fhit-mediated cessation of cell cycle progression at late G(1) phase. Elucidation of the protein effectors of Fhit signaling may lead to identification of targets for lung cancer therapy.
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PMID:Protein expression profiling identifies cyclophilin A as a molecular target in Fhit-mediated tumor suppression. 1688 64

The present study demonstrates that acute activation with either beta-adrenergic receptor agonists or H(2)O(2) treatment increases protein phosphatase 2a (PP2a) activity in ventricular myocytes. PP2a activation occurs concomitant with an increase in methylation of PP2a, changes in localization of a PP2a targeting subunit PP2aB56alpha, and a decrease in phosphorylation of PP2a substrates, such as troponin I (TnI) and ERK in ventricular myocytes. Okadaic acid, a well-established pharmacological inhibitor of PP2a, and the peptide Thr-Pro-Asp-Tyr-Phe-Leu (TPDYFL) were used to block PP2a methylation, localization, and phosphorylations. TPDYFL is a highly conserved sequence of the PP2a catalytic subunit COOH-terminus. Specifically, both okadaic acid and the peptide increased beta-adrenergic-cAMP-dependent phosphorylation of TnI and blocked the beta-adrenergic-cAMP-dependent translocation of PP2aB56alpha. TPDYFL, but not a scrambled version of this sequence, blocked H(2)O(2)-induced changes in PP2a methylation and TnI dephosphorylation. Okadaic acid produces similar inhibition of H(2)O(2) effects. Thus we propose that the novel peptide TPDYFL acts as an inhibitor of PP2a activity and may be a useful tool to increase our understanding of how PP2a is regulated and the role of PP2a in a variety of physiological and pathological processes. In addition, the present study is consistent with acute beta-adrenergic receptor activation and H(2)O(2) exposure, simultaneously activating kinases and PP2a to work on common substrates, such as TnI. We hypothesize that dual activation of opposing enzymes provides for a tighter regulation of substrate phosphorylations in ventricular myocytes.
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PMID:Acute modulation of PP2a and troponin I phosphorylation in ventricular myocytes: studies with a novel PP2a peptide inhibitor. 1701 62

AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recently, LKB1 was identified as a kinase that regulates AMPK. Using immunoblot analysis, we confirmed high expression of LKB1 in isolated rat cardiac myocytes but show that, under basal conditions, LKB1 is primarily localized to the nucleus, where it is inactive. We examined the role of LKB1 in cardiac myocytes, using adenoviruses that express LKB1, and its binding partners Ste20-related adaptor protein (STRADalpha) and MO25alpha. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRADalpha/MO25alpha expression, LKB1 activity, and AMPKalpha phosphorylation at its activating phosphorylation site (threonine-172). Since activation of AMPK can inhibit hypertrophic growth and since LKB1 is upstream of AMPK, we hypothesized that expression of an active LKB1 complex would also inhibit protein synthesis associated with hypertrophic growth. Expression of the LKB1/STRADalpha/MO25alpha complex in neonatal rat cardiac myocytes inhibited the increase in protein synthesis observed in cells treated with phenylephrine (measured via [(3)H]phenylalanine incorporation). This was associated with a decreased phosphorylation of p70S6 kinase and its substrate S6 ribosomal protein, key regulators of protein synthesis. In addition, we show that the pathological cardiac hypertrophy in transgenic mice with cardiac-specific expression of activated calcineurin is associated with a significant decrease in LKB1 expression. Together, our data show that increased LKB1 activity in the cardiac myocyte can decrease hypertrophy-induced protein synthesis and suggest that LKB1 activation may be a method for the prevention of pathological cardiac hypertrophy.
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PMID:Expression of an active LKB1 complex in cardiac myocytes results in decreased protein synthesis associated with phenylephrine-induced hypertrophy. 1709 23

TIMAP (TGF-beta1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.
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PMID:Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1. 1760 1

The inhibition of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) by glycogen phosphorylase a prevents the dephosphorylation and activation of glycogen synthase, suppressing glycogen synthesis when glycogenolysis is activated. Here, we show that a peptide ((280)LGPYY(284)) comprising the last five amino acids of G(L) retains high-affinity interaction with phosphorylase a and that the two tyrosines play crucial roles. Tyr284 deletion abolishes binding of phosphorylase a to G(L) and replacement by phenylalanine is insufficient to restore high-affinity binding. We show that a phosphorylase inhibitor blocks the interaction of phosphorylase a with the G(L) C-terminus, suggesting that the latter interaction could be targeted to develop an anti-diabetic drug.
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PMID:The hepatic PP1 glycogen-targeting subunit interaction with phosphorylase a can be blocked by C-terminal tyrosine deletion or an indole drug. 1787 73

In identified B6 neurons of Aplysia buccal ganglia under voltage-clamp, application of quisqualic acid (QA) induces a unique slow K(+)-current response independent of G-protein. The response was augmented by raising the temperature in a similar fashion to the Phe-Met-Arg-Phe-NH(2)-induced K(+)-current response mediated by Gi/o. The QA-induced K(+)-current response markedly increased during the perfusion with Ca(2+)-free solution or after the application of W-7, a calmodulin (CaM) inhibitor. It was also enhanced by intracellular application either of H-7, a serine/threonine protein kinase inhibitor, or of KN-93, a Ca(2+)/CaM-dependent kinase II (CaMKII) inhibitor. Furthermore, the QA-induced response was markedly augmented by pre-treatment with 2,3-butanedione monoxime, an inorganic phosphatase. Intracellular application of protein phosphatase 2A (PP2A) significantly augmented the QA-induced response although neither protein phosphatase 1 nor protein phosphatase 2B altered the response. Application of either okadaic acid or calyculin A, protein phosphatase inhibitors, only slightly depressed the QA-induced response. Surprisingly, W-7 had no augmenting effect on the QA-induced response when examined after the application of either okadaic acid or calyculin A. These results suggest that the K(+)-current response is reciprocally but sequentially regulated by PP2A and CaMKII, the response of which the former is facilitating and the latter is inhibiting.
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PMID:Regulatory roles of Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 2A on the quisqualic acid-induced K+-current response in identified neurons of Aplysia. 1798 Apr 50

Red light, acting via phytochrome, stimulates phenylalanine ammonia lyase (PAL) activity in cotyledons and hypocotyls of tomato seedlings. The time course of photoinduction of PAL activity has a peak level at 4 h after which activity declines significantly. In tomato seedlings PAL activity comprised of three isoforms and light stimulated activity of all three isoforms. A polyclonal antibody raised against PAL purified from tomato leaves recognized PAL protein belonging to PAL-II and PAL-III isoforms. The mode of increase in PAL activity was investigated by immunochemical techniques. The photostimulated increase in PAL activity appeared to be dependent on de novo synthesis of protein and nucleic acid. However, inhibition of protein phosphatase activity blocked increase in PAL activity without affecting the increase in PAL protein levels. The results indicate that in addition to de novo synthesis, the photostimulation of PAL activity likely requires dephosphorylation by a type 2C protein phosphatase.
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PMID:Differential regulation of phenylalanine ammonia lyase activity and protein level by light in tomato seedlings. 1834 45

The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25-residue peptide taken from the CaMBD, joined by a 5-glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25-residue CaMBD peptide without the glycine linker at a resolution of 2.1 A. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N-lobe from one molecule and C-lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully-designed CaM-sepharose pull-down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg-408, Val-409, and Phe-410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N- and C-lobes, ultimately keeping these lobes apart and forcing "head-to-tail" dimerization to attain the requisite N- and C-lobe pairing for CaMBD binding.
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PMID:The complex structure of calmodulin bound to a calcineurin peptide. 1838 83

Responses to transforming growth factor beta and multiple cytokines involve activation of transforming growth factor beta-activated kinase-1 (TAK1) kinase, which activates kinases IkappaB kinase (IKK) and MKK3/6, leading to the parallel activation of NF-kappaB and p38 MAPK. Activation of TAK1 by autophosphorylation is known to involve three different TAK1-binding proteins (TABs). Here we report a protein phosphatase subunit known as type 2A phosphatase-interacting protein (TIP) that also acts as a TAB because it co-precipitates with and directly binds to TAK1, enhances TAK1 autophosphorylation at unique sites, and promotes TAK1 phosphorylation of IKKbeta and signaling to NF-kappaB. Mass spectrometry demonstrated that co-expression of TAB4 protein significantly increased phosphorylation of four sites in TAK1, in a linker region between the kinase and TAB2/3 binding domains, and two sites in TAB1. Recombinant GST-TAB4 bound in an overlay assay directly to inactive TAK1 and activated TAK1 but not TAK1 phosphorylated in the linker sites, suggesting a bind and release mechanism. In kinase assays using TAK1 immune complexes, added GST-TAB4 selectively stimulated IKK phosphorylation. TAB4 co-precipitated polyubiquitinated proteins dependent on a Phe-Pro motif that was required to enhance phosphorylation of TAK1. TAB4 mutated at Phe-Pro dominantly interfered with IL-1beta activation of NF-kappaB involving IKK-dependent but not p38 MAPK-dependent signaling. The results show that TAB4 binds TAK1 and polyubiquitin chains to promote specific sites of phosphorylation in TAK1-TAB1, which activates IKK signaling to NF-kappaB.
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PMID:TAB4 stimulates TAK1-TAB1 phosphorylation and binds polyubiquitin to direct signaling to NF-kappaB. 1845 59


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