Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of Ins P3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications. © 2015, Public Library of Science. All rights reserved

Piperine inhibits type II phosphatidylinositol 4-kinases: a key component in phosphoinositides turnover

Piperine has been shown to have anti-inflammatory properties. The molecular mechanisms by which it mediates anti-inflammatory activities remain elusive. Type II phosphatidylinositol 4-kinase(s) are key components in Fc epsilon RI receptor-mediated signaling leading to inflammatory mediators release in RBL-2H3 cells. The effects of piperine on IgE-mediated signaling and mast cell degranulation were investigated. Pretreatment of RBL-2H3 cells with piperine inhibited IgE-induced activation of type II PtdIns 4-kinase(s). In vitro lipid kinase assays showed piperine-inhibited type II PtdIns 4-kinase activity in a dose-dependent fashion with no effect on PtdIns 3-kinase activity. Concomitantly, pretreatment of RBL-2H3 cells with piperine also inhibited IgE-induced beta-hexosaminidase release in RBL-2H3 cells. These results suggest that type II PtdIns 4-kinases are part of piperine-mediated anti-inflammatory signaling mechanisms

Fyn kinase regulates type II PtdIns 4-kinases in RBL 2H3 cells

Type II phosphatidylinositol 4-kinases are implicated in Fc epsilon RI-mediated signaling cascades leading to release of inflammatory molecules. Cross-linking of Fc epsilon RI on RBL 2H3 cells results in protein tyrosine phosphorylation and activation of type II PtdIns 4-kinase activity. Protein tyrosine kinase(s) that phosphorylate type II PtdIns 4-kinase(s) in RBL 2H3 cells remains elusive and is being addressed in this manuscript. Anti-Fyn kinase antibodies co-immunoprecipitated type II PtdIns 4-kinase activity from Fc epsilon RI cross-linked RBL 2H3 cells. In reciprocal assays, His-tagged types II PtdIns 4-kinases were shown to pull down Fyn kinase. Further, anti-Fyn immunoprecipitates were shown to phosphorylate type II PtdIns 4-kinase alpha and beta in in vitro assays. Pull down studies with GST-Fyn-SH2 and GST-Fyn-SH3 domains showed that type II PtdIns 4-kinases associate with Fyn-SH2 domain. Knockdown of Fyn kinase in RBL 2H3 cells abrogated activation of type II PtdIns 4-kinase activity in response to Fc epsilon RI cross-linking and type II PtdIns 4-kinase activity in anti-phosphotyrosine immunoprecipitates. Knockdown of Fyn kinase was also strongly correlated with a reduction in beta-hexosaminidase release in response to Fc epsilon RI cross-linking. These results suggest that type II PtdIns 4-kinases act downstream of Fyn kinase in Fc epsilon RI signaling cascades and are regulated by Fyn kinase

Type II phosphatidylinositol 4-kinases interact with Fc epsilon RI gamma subunit in RBL-2H3 cells

Ligation of high-affinity IgE receptor I (Fc epsilon RI) on RBL-2H3 cells leads to recruitment of Fc epsilon RI and type II phosphatidylinositol 4-kinases (PtdIns 4-kinases) into lipid rafts. Lipid raft integrity is required for the activation of type II PtdIns 4-kinases and signal transduction through Fc epsilon RI gamma during RBL-2H3 cell activation. However, the molecular mechanism by which PtdIns 4-kinases are coupled to Fc epsilon RI signaling is elusive. Here, we report association of type II PtdIns 4-kinase activity with Fc epsilon RI gamma subunit in anti-Fc epsilon RI gamma immunoprecipitates. Fc epsilon RI gamma-associated PtdIns 4-kinase activity increases threefold upon Fc epsilon RI ligation in anti-Fc epsilon RI gamma immunoprecipitates. Biochemical characterization of PtdIns 4-kinase activity associated with Fc epsilon RI gamma reveals that it is a type II PtdIns 4-kinases. Canonical tyrosine residues mutation in Fc epsilon RI gamma ITAM (Y65 and Y76) reveals that these two tyrosine residues in gamma subunit are required for its interaction with type II PtdIns 4-kinases

Sanguinarine suppresses IgE induced inflammatory responses through inhibition of type II PtdIns 47kinase(s)

The effects of sanguinarine on IgE mediated early signaling mechanisms leading to inflammatory mediators release were investigated. Pretreatment of RBL 2H3 cells with sanguinarine inhibited IgE induced activation of type II PtdIns 4-kinase activity. Concomitant with type II PtdIns 4-kinase inhibition, sanguinarine also inhibited IgE induced degranulation and 0 hexosaminidase release in RBL 2H3 cells. In vitro assays showed sanguinarine inhibited type II Ptdlns 4-kinase activity in a dose dependent fashion with no effect on PtdIns 3-kinase activity. Fluorescence spectroscopic studies suggested that sanguinarine binds to type II PtdIns 4-kinases alpha and beta isoforms with a K-d of 2.4 and 1.8 mu M, respectively. Kinetic studies showed that sanguinarine competes with PtdIns binding site of type II PtdIns 4-kinase beta. These results suggest that the anti-inflammatory effects of sanguinarine on PtdIns 3-kinase signaling pathway are more likely an indirect effect and emphasize the importance of the cross talk between type II Ptdlns 4-kinases and Ptdlns 3-kinases. (C) 2013 Elsevier Inc. All rights reserved

Type II PtdIns 4-kinase beta associates with CD4-p56lck complex and is involved in CD4 receptor signaling

Type II phosphatidylinositol (PtdIns) 4-kinases are involved in the synthesis of PtdIns 4-phosphates and modulate various cell functions like, intracellular signaling, cytoskeletal rearrangements, vesicular trafficking, and pathogen invasion. In CD3 receptor activated T cells, a type II PtdIns 4-kinase beta is recruited to CD3 receptor zeta and plays a role in intracellular calcium release and probably in actin cytoskeleton reorganization. T cell receptor mediated activation is supported by CD4 receptor. The role of type II PtdIns 4-kinase beta in CD4 receptor-mediated signaling was addressed in the present manuscript. Crosslinking of CD4 receptors with monoclonal antibodies showed an increase in CD4-associated PtdIns 4-kinase activity and requires p56(lck) activity. Biochemical characterization suggests that it belongs to type II PtdIns 4-kinase family. shRNA mediated knockdown of type II PtdIns 4-kinase beta showed abrogation of CD4 receptor induced intracellular calcium release. These results suggest that type II PtdIns 4-kinase beta plays an integral part in CD4 receptor-mediated signaling