Microcystin affinity purification of plant protein phosphatases: PP1C, PP5 and a regulatory A-subunit of PP2A. (41/1760)

Proteins of approximately 35, 55 and 65kDa were purified from cauliflower extracts by microcystin-Sepharose chromatography and identified by amino acid sequencing as plant forms of protein (serine/threonine) phosphatase 1 (PP1) catalytic subunit, PP5 and a regulatory A-subunit of PP2A, respectively. Peptides that corresponded both to the tetratricopeptide (TPR) repeat and catalytic domains of PP5 were identified. Similar to mammalian PP5,the casein phosphatase activity of plant PP5 was activated >10-fold by arachidonic acid, with half-maximal stimulation occurring at approximately 100 microM lipid.  (+info)

Protein phosphatase 2C inactivates F-actin binding of human platelet moesin. (42/1760)

During activation of platelets by thrombin phosphorylation of Thr(558) in the C-terminal domain of the membrane-F-actin linking protein moesin increases transiently, and this correlates with protrusion of filopodial structures. Calyculin A enhances phosphorylation of moesin by inhibition of phosphatases. To measure this moesin-specific activity, a nonradioactive enzyme-linked immunosorbent assay method was developed with the synthetic peptide Cys-Lys(555)-Tyr-Lys-Thr(P)-Leu-Arg(560) coupled to bovine serum albumin as the substrate and moesin phosphorylation state-specific polyclonal antibodies for the detection and quantitation of dephosphorylation. Calyculin A-sensitive and -insensitive protein-threonine phosphatase activities were detected in platelet lysates and separated by DEAE-cellulose chromatography. The calyculin A-sensitive enzyme was identified as a type 1 protein phosphatase. The calyculin A-insensitive enzyme activity was purified to homogeneity by phenyl- Sepharose, protamine-, and phosphonic acid peptide-agarose chromatography and characterized biochemically and immunologically as a 53-kDa protein(s) and a type 2C protein phosphatase (PP2C). Phosphorylation of Thr(558) is necessary for F-actin binding of moesin in vitro. The purified enzyme, as well as bacterially made PP2Calpha and PP2Cbeta, efficiently dephosphorylate(s) highly purified platelet phospho-moesin. This reverses the activating effect of phosphorylation, and moesin no longer co-sediments with actin filaments. In vivo, regulation of these phosphatase activities are likely to influence dynamic interactions between the actin cytoskeleton and membrane constituents linked to moesin.  (+info)

Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3. (43/1760)

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine/threonine phosphatases in STAT3 signaling in human antigen-specific CD4(+) T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.  (+info)

Biochemical characterization of a Dictyostelium myosin II heavy-chain phosphatase that promotes filament assembly. (44/1760)

In Dictyostelium cells, myosin II is found as cytosolic nonassembled monomers and cytoskeletal bipolar filaments. It is thought that the phosphorylation state of three threonine residues in the tail of myosin II heavy chain regulates the molecular motor's assembly state and localization. Phosphorylation of the myosin heavy chain at threonine residues 1823, 1833 and 2029 is responsible for maintaining myosin in the nonassembled state, and subsequent dephosphorylation of these residues is a prerequisite for assembly into the cytoskeleton. We report here the characterization of myosin heavy-chain phosphatase activities in Dictyostelium utilizing myosin II phosphorylated by myosin heavy-chain kinase A as a substrate. One of the myosin heavy-chain phosphatase activities was identified as protein phosphatase 2A and the purified holoenzyme was composed of a 37-kDa catalytic subunit, a 65-kDa A subunit and a 55-kDa B subunit. The protein phosphatase 2A holoenzyme displays two orders of magnitude higher activity towards myosin phosphorylated on the heavy chains than it does towards myosin phosphorylated on the regulatory light chains, consistent with a role in the control of filament assembly. The purified myosin heavy-chain phosphatase activity promotes bipolar filament assembly in vitro via dephosphorylation of the myosin heavy chain. This system should provide a valuable model for studying the regulation and localization of protein phosphatase 2A in the context of cytoskeletal reorganization.  (+info)

A new member of the alpha4-related molecule (alpha4-b) that binds to the protein phosphatase 2A is expressed selectively in the brain and testis. (45/1760)

A murine alpha4, identified in lymphocytes, binds to protein phosphatase 2A (PP2A). We found another murine alpha4-related gene (named alpha4-b) expressed selectively in the brain and testis. The alpha4-b transcript is expressed in the brain and testis, but is not detected in the spleen, thymus, bone marrow, liver, kidney, lung, heart or muscle. In-situ RNA hybridization analysis suggested that alpha4-b is expressed in most neuronal cells in the brain, but it is not expressed in the glial cells. The alpha4-b cDNA encodes a putative protein that is highly homologous (66% identity in amino-acid sequence) to the alpha4 molecule. The alpha4-b protein associates with the catalytic subunit of PP2A (PP2Ac), suggesting that the alpha4-b protein is involved in the regulation of phosphatase activity in neuronal cells.  (+info)

Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor alpha leads to intercellular adhesion molecule-1 expression. (46/1760)

Tumor necrosis factor alpha (TNF-alpha) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. We have previously shown that mouse Sertoli cells respond to TNF-alpha by increasing interleukin-6 production and intercellular adhesion molecule-1 (ICAM-1) expression (1). In this cell type TNF-alpha activates the mitogen-activated protein kinase (MAPK) pathways p42/p44 MAPK, JNK/SAPK, and p38, the last of which is responsible for interleukin-6 production (1). To determine which MAPK signaling pathway is required for TNF-alpha induction of ICAM-1 expression, we have utilized the protein kinase inhibitor dimethylaminopurine, demonstrating that treatment of Sertoli cells with such compound significantly reduced ICAM-1 expression and JNK/SAPK activation. Moreover, dimethylaminopurine treatment increased the expression of MAPK phosphatase-2, providing a possible mechanism of action of this compound. By using agonist antibodies to p55 and to p75 TNF-alpha receptors and both human and mouse TNF-alpha, we demonstrate that both TNF receptors are expressed and that only the p55 receptor is involved in ICAM-1 expression. The p55 receptor activates all of the three pathways, whereas p75 failed to activate any of the MAPKs. Altogether our results demonstrate that TNF-alpha up-regulates ICAM-1 expression through the activation of the JNK/SAPK transduction pathway mediated by the p55 receptor.  (+info)

Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C. (47/1760)

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are rapidly deactivated by a membrane-bound phosphatase activity. The efficiency of this regulation suggests CFTR and protein phosphatases may be associated within a regulatory complex. In this paper we test that possibility using co-immunoprecipitation and cross-linking experiments. A monoclonal anti-CFTR antibody co-precipitated type 2C protein phosphatase (PP2C) from baby hamster kidney cells stably expressing CFTR but did not co-precipitate PP1, PP2A, or PP2B. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from baby hamster kidney membrane extracts. Exposing baby hamster kidney cell lysates to dithiobis (sulfosuccinimidyl propionate) caused the cross-linking of histidine-tagged CFTR (CFTR(His10)) and PP2C into high molecular weight complexes that were isolated by chromatography on Ni(2+)-nitrilotriacetic acid-agarose. Chemical cross-linking was specific for PP2C, because PP1, PP2A, and PP2B did not co-purify with CFTR(His10) after dithiobis (sulfosuccinimidyl propionate) exposure. These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel.  (+info)

Structure-based functional motif identifies a potential disulfide oxidoreductase active site in the serine/threonine protein phosphatase-1 subfamily. (48/1760)

In previous work, 3-dimensional descriptors of protein function ('fuzzy functional forms') were used to identify disulfide oxidoreductase active sites in high-resolution protein structures. During this analysis, a potential disulfide oxidoreductase active site in the serine/threonine protein phosphatase-1 (PP1) crystal structure was discovered. In PP1, the potential redox active site is located in close proximity to the phosphatase active site. This result is interesting in view of literature suggesting that serine/threonine phosphatases could be subject to redox control mechanisms within the cell; however, the actual source of this control is unknown. Additional analysis presented here shows that the putative oxidoreductase active site is highly conserved in the serine/threonine phosphatase-1 subfamily, but not in the serine/threonine phosphatase-2A or -2B subfamilies. These results demonstrate the significant advantages of using structure-based motifs for protein functional site identification. First, a putative disulfide oxidoreductase active site has been identified in serine-threonine phosphatases using a descriptor built from the glutaredoxin/thioredoxin family, proteins that have no apparent evolutionary relationship whatsoever to the PP1 proteins. Second, the proximity of the putative disulfide oxidoreductase active site to the phosphatase active site provides evidence toward a regulatory control mechanism. No sequence-based method could provide either piece of information.  (+info)