Protein kinase Cδ binds TIRAP/Mal to participate in TLR signaling

Toll-like receptor (TLR) family members recognize specific molecular patterns within pathogens. Signaling through TLRs results in a proximal event that involves direct binding of adaptor proteins to the receptors. We observed that TIRAP/Mal, an adaptor protein for TLR2 and TLR4, binds protein kinase Cδ (PKCδ). TIRAP/Mal GST-fusion protein and a TIRAP/Mal antibody were able to precipitate PKCδ from rat peritoneal macrophage and THP1 cell lysates. Truncation mutants of TIRAP/Mal showed that the TIR domain of TIRAP/Mal is responsible for binding. TLR2- and TLR4-mediated phosphorylation of p38 MAPK, IKK, and IκB in RAW264.7 cells were abolished by depletion of PKCδ. These results suggest that PKCδ binding to TIRAP/Mal promotes TLR signaling events

Phosphoinositide 3-Kinaseγ Controls the Intracellular Localization of CpG to Limit DNA-PKcs-Dependent IL-10 Production in Macrophages

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG) stimulate innate immune responses. Phosphoinositide 3-kinase (PI3K) has been implicated in CpG-induced immune activation; however, its precise role has not yet been clarified. CpG-induced production of IL-10 was dramatically increased in macrophages deficient in PI3Kγ (p110γ−/−). By contrast, LPS-induced production of IL-10 was unchanged in the cells. CpG-induced, but not LPS-induced, IL-10 production was almost completely abolished in SCID mice having mutations in DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Furthermore, wortmannin, an inhibitor of DNA-PKcs, completely inhibited CpG-induced IL-10 production, both in wild type and p110γ−/− cells. Microscopic analyses revealed that CpG preferentially localized with DNA-PKcs in p110γ−/− cells than in wild type cells. In addition, CpG was preferentially co-localized with the acidic lysosomal marker, LysoTracker, in p110γ−/− cells, and with an early endosome marker, EEA1, in wild type cells. Over-expression of p110γ in Cos7 cells resulted in decreased acidification of CpG containing endosome. A similar effect was reproduced using kinase-dead mutants, but not with a ras-binding site mutant, of p110γ. Thus, it is likely that p110γ, in a manner independent of its kinase activity, inhibits the acidification of CpG-containing endosomes. It is considered that increased acidification of CpG-containing endosomes in p110γ−/− cells enforces endosomal escape of CpG, which results in increased association of CpG with DNA-PKcs to up-regulate IL-10 production in macrophages

Negative Regulation of Class IA Phosphoinositide 3-kinase by Protein Kinase Cδ Limits Fcγ Receptor-mediated Phagocytosis in Macrophages

Stimulation of macrophages by various ligands results in the activation of both phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC). Here, we showed that PKCδ selectively inhibits class IA PI3K. Prior exposure of macrophages to a PKC activator, phorbol 12-myristate 13-acetate (PMA) inhibited the PI3K activation induced by the Fcγ receptor (FcγR) ligation but not that induced by C5a. Prolonged PKC inhibition by GF109203X increased the basal PI3K activity of quiescent macrophages. The effect of the PKC inhibitor can be observed in macrophages from mice lacking class IB PI3K (p110γ). Thus PKC was suggested to selectively attenuate the class IA activity. Chronic PKC activation by PMA induced PKCδ degradation and Akt activation. Enhancement of the basal Akt actvity was also observed in cells stably deficient in PKCδ prepared by shRNA technique. FcγR-mediated phagocytosis was dramatically increased in these cells. Thus it is suggested that inactivation of class IA PI3K by PKCδ is functioning in regulation of FcγR-mediated phagocytosis

Inhibition of PTEN and activation of Akt by menadione

Menadione (vitamin K3) has been shown to activate Erk in several cell lines. This effect has been shown to be due to the activation of EGF receptors (EGFR) as a result of inhibition of some protein tyrosine phosphatases. In the present study, we examined the effects of menadione on Akt in Chinese hamster ovary cells. The phosphorylation of Akt by menadione was not inhibited by AG1478, an inhibitor of EGFR. Menadione inhibited the lipid phosphatase activity of PTEN in a cell-free system. In an intact cell system, menadione inhibited the effect of transfected PTEN on Akt. Thus, one mechanism of its action was considered the accelerated activation of Akt through inhibition of PTEN. This was not the sole mechanism responsible for the EGFR-independent activation of Akt, because menadione attenuated the rate of Akt dephosphorylation even in PTEN-null PC3 cells. The decelerated inactivation of Akt, probably through inhibition of some tyrosine phosphatases, was considered another mechanism of its action

Involvement of Class II Phosphoinositide 3-Kinase α-Isoform in Antigen-Induced Degranulation in RBL-2H3 Cells

<div><p>In this study, we present findings that suggest that PI3K-C2α, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of FcεRI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2α. The knockdown impaired the FcεRI-induced release of a lysosome enzyme, β-hexosaminidase, without affecting the intracellular Ca²⁺ mobilization. The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells. The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α. In wild-type cells, FcεRI stimulation induced the formation of large vesicles, which were associated with CD63, a marker protein of secretory granules. On the vesicles, the existence of PI3K-C2α and PtdIns(3,4)P₂ was observed. These results indicated that PI3K-C2α and its product PtdIns(3,4)P₂ may play roles in the secretory process.</p></div