The cyclic heptapeptide, microcystin‐LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with K i , values below 0.1 nM. Protein phosphatase 2B is inhibited 1000‐fold less potently, while six other eight kinases tested are unaffected. These results strikingly similar to those obtained the tumour promoter okadaic acid. We establish that acid prevents binding of microcystin‐LR PP2A, inhibitors 2 prevent PP1. discuss possibility inhibition PP1 PP2A accounts for extreme toxicity indicate its...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTInhibition of T cell signaling by immunophilin-ligand complexes correlates with loss calcineurin phosphatase activityJ. Liu, M. W. Albers, T. J. Wandless, S. Luan, D. G. Alberg, P. Belshaw, Cohen, C. MacKintosh, B. Klee, and L. SchreiberCite this: Biochemistry 1992, 31, 16, 3896–3901Publication Date (Print):April 28, 1992Publication History Published online1 May 2002Published inissue 28 April...

14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes targets for binding. The did not bind to (14-3-3s) after dephosphorylation protein phosphatase 2A (PP2A), indicating binding 14-3-3s requires their phosphorylation. identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP ATP), FAD, NADPH,...

More than 200 phosphorylated 14-3-3-binding sites in the literature were analysed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate, although +2 proline residue occurs less half, LX(R/K)SX(pS/pT)XP is prominent plant sites. Proline at +1 rarely reported, such did not stand up experimental reanalysis of human Ndel1. Instead, we discovered that interacts with two...

Abstract Motivation: The 14-3-3 family of phosphoprotein-binding proteins regulates many cellular processes by docking onto pairs phosphorylated Ser and Thr residues in a constellation intracellular targets. Therefore, there is pressing need to develop new prediction methods that use an updated set 14-3-3-binding motifs for the identification targets prioritize downstream analysis >2000 potential interactors identified high-throughput experiments. Results: Here, comprehensive from...

The interaction between protein phosphatase 1 (PP1) and microcystin (MC) was stable in 1% SDS or 70% formic acid indicative of a covalent interaction. Here we isolate the MC‐binding peptide demonstrate that Cys 273 PP1 binds covalently to methyl‐dehydroalanine (Mdha) residue toxin. Mutation Ala, Ser leu abolished binding MC, as did reduction Mdha toxin with ethanethiol. abolition increased IC 50 for inhibition by 5‐ 20‐fold. MC serine/threonine phosphatases explains failure detect this...

Summary Far‐Western overlays of soluble extracts cauliflower revealed many proteins that bound to digoxygenin (DIG)‐labelled 14‐3‐3 proteins. Binding DIG‐14‐3‐3s was prevented by prior dephosphorylation the extract or competition with 14‐3‐3‐binding phosphopeptides, indicating bind phosphorylated sites. The were also immunoprecipitated from anti‐14‐3‐3 antibodies, demonstrating they endogenous plant purified extracts, in sufficient quantity for amino acid sequence analysis, affinity...

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) is positively regulated by amino acids and insulin. PRAS40 associates with mTORC1 (which contains raptor) but not mTORC2. interacts raptor, this requires an intact TOR-signaling (TOS) motif in PRAS40. Like TOS motif-containing proteins such as eIF4E-binding protein (4E-BP1), a substrate for phosphorylation mTORC1. Consistent this, starvation cells or treatment alters binds 14-3-3 proteins, both Binding to inhibited TSC1/2...

AS160 (Akt substrate of 160 kDa) and TBC1D1 are related RabGAPs (Rab GTPase-activating proteins) implicated in regulating the trafficking GLUT4 (glucose transporter 4) storage vesicles to cell surface. All animal species examined contain TBC1D1, whereas evolved with vertebrates. has two clusters phosphorylated residues, either side second PTB (phosphotyrosine-binding domain). Each cluster contains a 14-3-3-binding site. When AMPK (AMP-activated protein kinase) is activated HEK (human...

Tautomycin inhibited the catalytic subunits of protein phosphatase-1 (Kiapp = 0.16 nM) more potently than phosphatase 2A 0.4 nM), and native forms these enzymes in mammalian, protozoan plant extracts were a similar manner. Protein 2B was 10,000-fold less potently, while two other phosphatases six kinases unaffected at 10 microM. Okadaic acid prevented binding tautomycin to 2A, indicating common site for both inhibitors. The different relative potencies okadaic 1 suggest that parallel use...

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated by 14-3-3-affinity chromatography, we found that binding to 14-3-3 isoforms HEK (human embryonic kidney)-293 cells was induced IGF-1 (insulin-like growth factor-1), EGF (epidermal factor), PMA and, a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside). AS160-14-3-3 interactions...

Summary Trehalose‐6‐phosphate is a ‘sugar signal’ that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose‐6‐phosphate synthase (TPS) trehalose‐6‐phosphatase (TPP) enzymes. It also class II proteins (TPS isoforms 5–11) contain both TPS‐like TPP‐like domains, although whether these have enzymatic activity unknown. In this paper, we show TPS5, 6 7 are phosphoproteins bind to 14‐3‐3 proteins, by using affinity chromatography, overlay assays,...

TBC1D1 is a Rab-GTPase-activating protein (GAP) known to be phosphorylated in response insulin, growth factors, pharmacological agonists that activate 5'-AMP-activated kinase (AMPK), and muscle contraction. Silencing L6 cells by siRNA increases insulin-stimulated GLUT4 translocation, overexpression of 3T3-L1 adipocytes with low endogenous expression inhibits suggesting role regulating translocation. Aiming unravel the regulation during contraction potential AMPK intact skeletal muscle, we...

Fusarium head blight (FHB; scab), primarily caused by graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing germplasm pools for resistance low may not provide sufficient to develop cultivars through traditional breeding approaches. Thus, engineering provides an additional approach enhance resistance. objectives this study were transgenic...

AS160 has emerged as a key player in insulin-mediated glucose transport through controlling GLUT4 trafficking, which is thought to be regulated by insulin-stimulated phosphorylation of sites including the 14-3-3 binding phospho-Thr649 (equivalent Thr642 human AS160). To define physiological roles AS160-Thr649 and homeostasis, we substituted this residue nonphosphorylatable alanine knockin mutation mice. The mutant protein was expressed at normal levels, while 14-3-3s abolished homozygous...

A microcystin (MC)-Sepharose column was prepared by addition of 2-aminoethanethiol to the alpha, beta-unsaturated carbonyl N-methyldehydroalanine residue MC-LR, followed reaction introduced amino group with N-hydroxysuccinimide-activated CH-Sepharose. The MC-Sepharose bound protein phosphatase-1 (PP1) high capacity and purified human PP1 gamma in one step from E. coli extracts. It also used purify forms myofibrils skeletal muscle. Two these comprised complexed N-terminal fragments M-subunit...

The low-activity, phosphorylated form of nitrate reductase (NR) became activated during purification from spinach (Spinacia oleracea) leaves harvested in the dark. This activation resulted its separation an approximately 110-kd inhibitor protein (NIP). Readdition NIP inactivated purified NR, but not active dephosphorylated indicating that inactivation NR requires interaction with as well phosphorylation. Consistent this hypothesis, had been vitro presence kinase, ATP-Mg, and could be...

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The activity dephosphorylated beta-subunit phosphorylase kinase selectively was inhibited by same concentrations okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], inhibitor (IC50 = 0.6 nM) 2 2.0 as rabbit muscle phosphatase. plant alpha-subunit preferentially, exquisitely sensitive...

Protein phosphatases 1 and 2A (PP1 PP2A) were identified in a variety of plant cells found to be particulate or soluble depending on the species. In extracts prepared from oilseed-rape seeds these enzymes associated with microsomes more rapidly sedimenting fractions, whereas wheat leaf they largely microsomal, remainder being present fraction. pea carrot cell PP1 PP2A almost entirely soluble. No activity was membranes stroma chloroplasts seeds, leaves leaves. An Mg2(+)-dependent okadaic...