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1.
J Immunol ; 192(6): 2846-56, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-24534530

ABSTRACT

The molecular mechanisms that fine tune TLRs responses need to be fully elucidated. Protein phosphatase-1 (PP1) has been shown to be important in cell death and differentiation. However, the roles of PP1 in TLR-triggered immune response remain unclear. In this study, we demonstrate that PP1 inhibits the activation of the MAPK and NF-κB pathway and the production of TNF-α, IL-6 in macrophages triggered by TLR3, TLR4, and TLR9 in a phosphatase-dependent manner. Conversely, PP1 knockdown increases TLRs-triggered signaling and proinflammatory cytokine production. Tautomycetin, a specific inhibitor of PP1, aggravates LPS-induced endotoxin shock in mice. We further demonstrate that PP1 negatively regulates TLR-triggered signaling by targeting TGF-ß-activated kinase 1 (TAK1) serine 412 (Ser412) phosphorylation, which is required for activation of TAK1-mediated IL-1R and TLR signaling. Mutation of TAK1 Serine 412 to alanine (S412A) significantly inhibits TLR/IL-1R-triggered NF-κB and MAPK activation and induction of proinflammatory cytokines in macrophage and murine embryonic fibroblast cells. DNA damage-inducible protein 34 (GADD34) specifies PP1 to dephosphorylate TAK1 at Ser412. GADD34 depletion abolished the interaction between TAK1 and PP1, and it relieved PP1 overexpression-induced inhibition of TLRs signaling and proinflammatory cytokine production. In addition, knockdown of GADD34 significantly promotes TLR-induced TAK1 Ser412 phosphorylation, downstream NF-κB and MAPK activation, and proinflammatory cytokine production. Therefore, PP1, as a physiologic inhibitor, together with its regulatory subunit GADD34, tightly controls TLR-induced TAK1 Ser412 phosphorylation, preventing excessive activation of TLRs and protecting the host from overwhelmed inflammatory immune responses.


Subject(s)
MAP Kinase Kinase Kinases/immunology , Protein Phosphatase 1/immunology , Signal Transduction/immunology , Toll-Like Receptors/immunology , Animals , Cell Line , Cells, Cultured , Furans/pharmacology , HEK293 Cells , HeLa Cells , Holoenzymes/genetics , Holoenzymes/immunology , Holoenzymes/metabolism , Humans , Interleukin-6/genetics , Interleukin-6/immunology , Interleukin-6/metabolism , Lipids/pharmacology , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinases/immunology , Mitogen-Activated Protein Kinases/metabolism , Models, Immunological , Mutation/immunology , NF-kappa B/immunology , NF-kappa B/metabolism , Phosphorylation/immunology , Protein Phosphatase 1/genetics , Protein Phosphatase 1/metabolism , RNA Interference , Serine/genetics , Serine/immunology , Serine/metabolism , Toll-Like Receptors/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism
2.
Immunity ; 37(4): 697-708, 2012 Oct 19.
Article in English | MEDLINE | ID: mdl-23084358

ABSTRACT

Signal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas.


Subject(s)
Phosphoprotein Phosphatases/immunology , Signal Transduction , T-Lymphocytes/immunology , Biocatalysis , Cell Differentiation , Cells, Cultured , Holoenzymes/immunology , Humans , I-kappa B Kinase/immunology , I-kappa B Kinase/metabolism , Lymphocyte Activation , NF-kappa B/immunology , NF-kappa B/metabolism , Phosphoprotein Phosphatases/genetics , RNA Interference
3.
Biochem Biophys Res Commun ; 391(1): 182-6, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19900413

ABSTRACT

F(o)F(1)-ATPase activity is regulated by external links on beta subunits with different molecular weight. It is inhibited when anti-beta subunit antibody, streptavidin and H9 antibody link on the beta subunits successively, but is activated when virus was binded. Western blotting indicated that the employed anti-beta antibody target was on the non-catalytic site of the beta subunit. Furthermore, an ESR study of spin-labeled ATP (SL-ATP) showed that the affinity of ATP to the holoenzyme increases with increasing external links on the beta subunits. This simple regulation method may have great potential in the design of rapid, free labeled, sensitive and selective biosensors.


Subject(s)
Biosensing Techniques , Proton-Translocating ATPases/chemistry , Adenosine Triphosphate/chemistry , Antibodies, Monoclonal/immunology , Catalytic Domain , Electron Spin Resonance Spectroscopy , Holoenzymes/chemistry , Holoenzymes/immunology , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/immunology , Proton-Translocating ATPases/immunology , Spin Labels
4.
Biochemistry ; 47(15): 4535-43, 2008 Apr 15.
Article in English | MEDLINE | ID: mdl-18361505

ABSTRACT

Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.


Subject(s)
Kinesins/chemistry , Animals , Antibodies/immunology , Dimerization , Holoenzymes/analysis , Holoenzymes/chemistry , Holoenzymes/immunology , Intracellular Membranes/chemistry , Kinesins/analysis , Kinesins/immunology , Mice , Microsomes/chemistry , Protein Subunits/analysis , Protein Subunits/chemistry , Protein Subunits/immunology
5.
Mol Cell Biol ; 27(6): 2074-83, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17220281

ABSTRACT

Telomerase replenishes the telomeric repeats that cap eukaryotic chromosome ends. To perform DNA synthesis, the active site of telomerase reverse transcriptase (TERT) copies a template within the integral telomerase RNA (TER). In vivo, TERT and TER and additional subunits form a telomerase holoenzyme capable of telomere elongation. We previously purified epitope-tagged Tetrahymena thermophila TERT and characterized two of the associated proteins. Here we characterize the remaining two proteins that were enriched by TERT purification. The primary sequence of the p75 polypeptide lacks evident homology with other proteins, whereas the p20 polypeptide is the Tetrahymena ortholog of a conserved multifunctional protein, Skp1. Genetic depletion of p75 induced telomere shortening without affecting the accumulation of TER or TERT, suggesting that p75 promotes telomerase function at the telomere. Affinity purification of p75 coenriched telomerase activity and each other known telomerase holoenzyme protein. On the other hand, genetic depletion of Skp1p induced telomere elongation, suggesting that this protein plays a negative regulatory role in the maintenance of telomere length homeostasis. Affinity purification of Skp1p did not detectably enrich active telomerase but did copurify ubiquitin ligase machinery. These studies reveal additional complexity in the positive and negative regulation of Tetrahymena telomerase function.


Subject(s)
Gene Expression Regulation, Enzymologic , Telomerase/metabolism , Tetrahymena/enzymology , Animals , Antibodies/immunology , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Gene Deletion , Holoenzymes/genetics , Holoenzymes/immunology , Holoenzymes/isolation & purification , Holoenzymes/metabolism , Peptides/genetics , Peptides/isolation & purification , Peptides/metabolism , Phenotype , Protein Binding , S-Phase Kinase-Associated Proteins/metabolism , Telomerase/genetics , Telomerase/immunology , Telomerase/isolation & purification , Tetrahymena/genetics
6.
FEBS J ; 272(7): 1559-67, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15794744

ABSTRACT

Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T-cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T-cell RNA using C subunit-specific primers revealed expression of two catalytically active PKA C subunits C alpha1 (40 kDa) and C beta2 (47 kDa) in these cells. Anti-RI alpha and Anti-RII alpha immunoprecipitations demonstrated that both C alpha1 and C beta2 associate with RI alpha and RII alpha to form PKAI and PKAII holoenzymes. Moreover, Anti-C beta2 immunoprecipitation revealed that C alpha1 coimmunoprecipitates with C beta2. Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme, released C alpha1 but not C beta2 from the Anti-C beta2 precipitate, indicating that C beta2 and C alpha1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP-responsive enzymes that may mediate specific effects of cAMP.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , T-Lymphocytes/enzymology , Antibodies/immunology , Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/immunology , Cyclic AMP-Dependent Protein Kinases/isolation & purification , Enzyme Activation , Holoenzymes/immunology , Holoenzymes/isolation & purification , Holoenzymes/metabolism , Humans , T-Lymphocytes/immunology
7.
Immunity ; 13(3): 313-22, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11021529

ABSTRACT

CD28 and CTLA-4 are related members of a family of T lymphocyte cell surface receptors that function to regulate T cell activation. We have found that the cytoplasmic domains of both CTLA-4 and CD28 can associate with members of the PP2A family of serine/threonine phosphatases. The association of PP2A with CD28 was negatively regulated by tyrosine phosphorylation of the CD28 cytoplasmic domain. Inhibition of PP2A activity in Jurkat leukemia T cells by treatment with okadaic acid or by expression of a dominant-negative mutant enhanced T cell activation induced by CD28 engagement. Interactions between cell surface receptors such as CTLA-4 and CD28 and serine/threonine phosphatases may represent a novel mechanism for modulating the intracellular signal transduction pathways associated with cell activation.


Subject(s)
Antigens, Differentiation/metabolism , CD28 Antigens/metabolism , Immunoconjugates , Phosphoprotein Phosphatases/metabolism , Receptors, Cell Surface/metabolism , Abatacept , Amino Acid Sequence , Antigens, CD , CD28 Antigens/physiology , CTLA-4 Antigen , Cell Line , Cytoplasm/immunology , Cytoplasm/metabolism , Down-Regulation , Enzyme Activation/drug effects , Enzyme Activation/immunology , Holoenzymes/immunology , Holoenzymes/metabolism , Humans , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Jurkat Cells/drug effects , Jurkat Cells/enzymology , Jurkat Cells/immunology , Jurkat Cells/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/physiology , Molecular Sequence Data , Okadaic Acid/pharmacology , Peptide Fragments/immunology , Peptide Fragments/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Protein Structure, Tertiary , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , T-Lymphocytes/metabolism
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