sq-23377 and rottlerin

Fertilization of mammalian eggs is characterized by a series of Ca(2+) oscillations triggered by a phospholipase C activity. These Ca(2+) increases and the parallel generation of diacylglycerol (DAG) stimulate protein kinase C (PKC). However, the dynamics of PKC activity have not been directly measured in living eggs. Here, we have monitored the dynamics of PKC-induced phosphorylation in mouse eggs, alongside Ca(2+) oscillations, using fluorescent C-kinase activity reporter (CKAR) probes. Ca(2+) oscillations triggered either by sperm, phospholipase C zeta (PLCζ) or Sr(2+) all caused repetitive increases in PKC-induced phosphorylation, as detected by CKAR in the cytoplasm or plasma membrane. The CKAR responses lasted for several minutes in both the cytoplasm and plasma membrane then returned to baseline values before subsequent Ca(2+) transients. High frequency oscillations caused by PLCζ led to an integration of PKC-induced phosphorylation. The conventional PKC inhibitor, Gö6976, could inhibit CKAR increases in response to thapsigargin or ionomycin, but not the repetitive responses seen at fertilization. Repetitive increases in PKCδ activity were also detected during Ca(2+) oscillations using an isoform-specific δCKAR. However, PKCδ may already be mostly active in unfertilized eggs, since phorbol esters were effective at stimulating δCKAR only after fertilization, and the PKCδ-specific inhibitor, rottlerin, decreased the CKAR signals in unfertilized eggs. These data show that PKC-induced phosphorylation outlasts each Ca(2+) increase in mouse eggs but that signal integration only occurs at a non-physiological, high Ca(2+) oscillation frequency. The results also suggest that Ca(2+) -induced DAG formation on intracellular membranes may stimulate PKC activity oscillations at fertilization.

Topics: Acetophenones; Animals; Benzopyrans; Calcium; Calcium Ionophores; Carbazoles; Diglycerides; Enzyme Inhibitors; Female; Fertilization in Vitro; Gene Expression Regulation, Enzymologic; Ionomycin; Male; Mice; Ovum; Phosphorylation; Protein Kinase C; RNA, Complementary; Spermatozoa; Thapsigargin

The function of mast cells is modified by the phosphoinositol-3 (PI3)-kinase pathway. The kinase signals partially through the phosphoinositide-dependent kinase PDK1, which on the one hand activates the serum- and glucocorticoid- inducible kinase SGK1 and on the other hand activates protein kinase PKCδ. SGK1 participates in the stimulation of Ca(2+) entry and degranulation, PKCδ inhibits degranulation. The present experiments explored the role of PDK1 in mast cell function. As mice completely lacking PDK1 are not viable, experiments have been performed in mast cells isolated from bone marrow (BMMCs) of PDK1 hypomorphic mice (pdk1(hm)) and their wild-type littermates (pdk1(wt)). Antigen stimulation via the FceRI receptor was followed by Ca(2+) entry leading to increase of cytosolic Ca(2+) activity in pdk1(wt) BMMCs, an effect significantly blunted in pdk1(hm) BMMCs. In contrast, Ca(2+) release from intracellular stores was not different between BMMCs of the two genotypes. The currents through Ca(2+)-activated K(+) channels following antigen exposure were again significantly larger in pdk1(wt) than in pdk1(hm) cells. The Ca(2+) ionophore ionomycin (1 μM) increased the K(+) channel conductance to similar values in both genotypes. β-hexosaminidase and histamine release were similar in pdk1(wt) BMMCs and pdk1(hm) BMMCs. PKCδ inhibitor rottlerin increased β-hexosaminidase release in pdk1(wt) BMMCs but not in pdk1(hm) BMMCs. Phosphorylation of PKCδ and of the SGK1 target NDRG1, was stimulated by the antigen in pdk1(wt) but not in pdk1(hm) cells. The observations reveal a role for PDK1 in the regulation of Ca(2+) entry into and degranulation of murine mast cells.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Acetophenones; Animals; Benzopyrans; beta-N-Acetylhexosaminidases; Calcium; Cell Cycle Proteins; Enzyme Inhibitors; Histamine; Immediate-Early Proteins; Intracellular Signaling Peptides and Proteins; Ionomycin; Ionophores; Mast Cells; Mice; Patch-Clamp Techniques; Phosphorylation; Potassium Channels; Protein Kinase C-delta; Protein Serine-Threonine Kinases

Maturation of dendritic cells (DCs) regulates protein sorting in endosomal compartments to promote the surface expression of molecules involved in T cell activation. MHC Class II complexes are mobilized to the surface via intracellular effector molecules that remain largely unknown. We here show that protein kinase C (PKC) stimulates Class II antigen surface expression, using knock-in mice that express a Class II-green fluorescent protein fusion protein as a read out. Selective inhibition of PKCdelta counteracts the ability of DCs to stimulate Class II MHC-restricted antigen-specific T cells. Activation of PKC does not affect antigen uptake, peptide loading and surface display of Class I MHC and transferrin receptor in DCs. We show that activation-induced Class II MHC surface expression is dependent on activation of PKCdelta and conclude that this event is pivotal for optimal CD4 T cell activation.

Topics: Acetophenones; Animals; Antigen Presentation; Benzopyrans; Blotting, Western; Cell Membrane; Dendritic Cells; Endocytosis; Enzyme Inhibitors; Flow Cytometry; Gene Expression; Green Fluorescent Proteins; Histocompatibility Antigens Class II; Ionomycin; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Tetradecanoylphorbol Acetate

Murine nuclear factor of activated T cells (NFAT)2.alpha/beta differ by 42 and 28 unique amino-terminal amino acids and are differentially expressed. Both isoforms share conserved domains that regulate DNA-binding and subcellular localization. A genetic "one-hybrid" assay was used to define two distinct transactivation (TA) domains: in addition to a conserved TAD present in both isoforms, a second, novel TAD exists within the beta-specific amino terminus. Pharmacologic inhibitors Gö6976 and rottlerin demonstrate that both conventional and novel protein kinase C (PKC) family members regulate endogenous mast cell NFAT activity, and NFAT2 TA. Overexpression of dominant active PKC (which has been implicated in immune receptor signaling) induces NFAT2.alpha/beta TA. Mutations within the smallest PKC-responsive transactivation domain demonstrate that the PKC effect is at least partially indirect. Significantly, the beta-specific domain confers greater ability to TA in response to treatment with phorbol 12-myristate 13-acetate/ionomycin or lipopolysaccharide, and unique sensitivity to FcepsilonRI signaling. Accordingly, overexpression of NFAT2.beta results in significantly greater NFAT- and interleukin-4 reporter activity than NFAT2.alpha. These results suggest that whereas NFAT2 isoforms may share redundant DNA-binding preferences, there are specialized functional consequences of their isoform-specific domains.

Topics: Acetophenones; Amino Acid Sequence; Animals; Base Sequence; Benzopyrans; Binding Sites; Carbazoles; Cell Line; DNA; DNA-Binding Proteins; Enzyme Inhibitors; In Vitro Techniques; Indoles; Ionomycin; Mast Cells; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; NFATC Transcription Factors; Nuclear Proteins; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Protein Structure, Tertiary; Receptors, IgE; Recombinant Proteins; Tetradecanoylphorbol Acetate; Transcription Factors; Transcriptional Activation; Two-Hybrid System Techniques

CD8(+) cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKCtheta. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKCepsilon, and especially of PKCtheta, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKCtheta prevented the induction of the reporter. Finally, transfection with PKCalpha A/E, but not with PKCtheta A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKCtheta activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.

Topics: Acetophenones; Androstadienes; Animals; Antibodies, Monoclonal; Benzopyrans; Carbazoles; Cell Degranulation; Cell Line; Chromones; Cycloheximide; Cytotoxicity, Immunologic; Dactinomycin; Fas Ligand Protein; Humans; Indoles; Ionomycin; Isoenzymes; Jurkat Cells; Luciferases; Lymphocyte Activation; Membrane Glycoproteins; Mice; Morpholines; Perforin; Phorbol Esters; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pore Forming Cytotoxic Proteins; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-epsilon; Protein Kinase C-theta; Receptor-CD3 Complex, Antigen, T-Cell; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes, Cytotoxic; Transfection; Wortmannin

Activation-induced cell death is mediated by the TCR-induced expression of the Fas ligand (FasL) on the surface of T cells, followed by binding to its receptor Fas. FasL expression is induced by stimulating T cells with a combination of phorbol ester and Ca2+ ionophore, implicating a role for protein kinase C (PKC) in this process. However, the precise mechanisms that regulate FasL expression, including the contribution of distinct T cell-expressed PKC isoforms, are poorly understood. Herein, we report that PKCtheta, a Ca2+-independent PKC isoform that we have previously isolated as a PKC enzyme selectively expressed in T cells, plays an important role in these processes. A constitutively active PKCtheta mutant preferentially induced FasL expression and activated the corresponding gene promoter; conversely, a dominant-negative PKCtheta mutant blocked FasL expression induced by anti-CD3 or PMA plus ionomycin stimulation. Furthermore, PKCtheta synergized with calcineurin to provide a potent stimulus for FasL promoter activation. Full activation of the promoter required its binding sites for the transcription factors NF-AT, AP-1, and NF-kappaB. The biological significance of these findings is implicated by the finding that rottlerin, a selective PKCtheta inhibitor, blocked FasL induction by anti-CD3 or PMA plus ionomycin stimulation and, consequently, protected human Jurkat T cells and the mouse T cell hybridoma A1.1 from activation-induced cell death.

Topics: Acetophenones; Animals; Apoptosis; Benzopyrans; Binding Sites; Calcineurin; Carbazoles; DNA-Binding Proteins; Drug Interactions; Fas Ligand Protein; Gene Expression Regulation; Humans; Indoles; Ionomycin; Isoenzymes; Jurkat Cells; Membrane Glycoproteins; Mice; Mutation; NFATC Transcription Factors; Nuclear Proteins; Promoter Regions, Genetic; Protein Kinase C; Protein Kinase C-theta; Signal Transduction; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription Factors