Thapsigargin-induced mast cell degranulation requires PI3Kγ, but not GPCR signaling.

<p>(A) Granule release of wild type and p110γ^−/− BMMCs was determined detecting β-hexosaminidase (β-Hex) release into extracellular media. BMMC stimulation with IgE/antigen was initiated with the antigen (Ag, DNP-HSA at 10 ng/ml; 100 ng/ml IgE overnight). Alternatively, BMMCs were stimulated by the addition of thapsigargin (1 µM). Where indicated, BMMCs were preincubated for 15 min with 100 nM wortmannin. Released β-Hex was quantified 20 min after stimulation, and is expressed as mean ± standard error of the mean (SEM) (<i>n</i> = 3; <i>p</i>-values in all figures are * or &: <i>p</i><0.05, **: <i>p</i><0.005; ***: <i>p</i><0.0005; * depict here comparison with respective wild type control; & refer to comparison of untreated versus treated samples). (B) Granule release was assessed as above, but ADA (10 units/ml) was added to BMMC suspensions 1 min before stimulation where depicted. (C) Wild type or p110γ^−/− BMMCs were stimulated with adenosine (Ade; 1 µM) or thapsigargin (1 µM) for 2 min, and phosphorylation of PKB/Akt on Thr308 (pPKB), total PKB and p110γ was analyzed by Western blotting. BMMCs were incubated in starving medium (2% FCS, without IL-3) for 3 h before stimulation, and pretreated with ADA where indicated. (D) Heterotrimeric Gα_i proteins were inactivated by preincubation of wild type and p110γ^−/− BMMCs with 100 ng/ml <i>P</i>Tx for 4 h, before thapsigargin (Tg) or adenosine was added as in (C).</p

Thapsigargin-triggered PI3Kγ activation requires influx of extracellular Ca²⁺.

<p>(A) Where indicated, IL-3 starved BMMCs were incubated with EDTA (5 mM) for 5 min, before cells were stimulated with thapsigargin (1 µM) or ionomycin (1 µM). Cells were lysed 5 min after stimulation, and phosphorylation of PKB/Akt on Ser473 was analyzed. (B) BMMCs as in (A) were pretreated for 10 min with the cell-permeable Ca²⁺-chelator BAPTA/AM (10 µM) and stimulated either with IL-3 (10 ng/ml), adenosine (1 µM), or thapsigargin (1 µM). (C, D) BMMCs were loaded with the ratiometric low affinity Ca²⁺ probe Fura-4F/AM for 10 min in physiologic HEPES buffer at 1 mM Ca²⁺ (for details see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio.1001587.s013" target="_blank">Text S1</a>). After the loading, washed cells were resuspended in the presence of increasing Ca²⁺ concentrations (extracellular Ca²⁺, [Ca²⁺]_e) to modulate maximal stimulation-induced intracellular Ca²⁺ levels ([Ca²⁺]_i). Cells were then stimulated with 0.5 µM thapsigargin, and maximal [Ca²⁺]_i increase and phosphorylation of PKB/Akt were measured. pPKB S473 levels are displayed as a function of the individually determined [Ca²⁺]_i. Data points come from two independently performed experiments. (E) Representative anti-phospho-PKB/Akt immunoblot as used to determine pPKB/Akt levels in (D). (F) Intracellular Ca²⁺ concentrations were measured in wild type BMMCs following stimulation with the adenosine 3A receptor-selective agonist <i>N</i>⁶-(3-iodobenzyl)-adenosine-5′-<i>N</i>-methylcarbox-amide (IB-MECA) (10 nM) or thapsigargin (1 µM). <i>B. Pertussis</i> toxin (100 ng/ml) was added 4 h before stimulation where marked.</p

Phosphorylation of Ser582—loss of GPCR coupling of p110γ.

<p>In a resting mast cell, the PI3Kγ complex is responsive to GPCR-mediated dissociation of trimeric G proteins. An adapter protein (here p84) is required for a productive relay of the GPCR signal to PI3Kγ. When FcεRI receptors are clustered via IgE/antigen complexes, a signaling cascade is initiated, which triggers the depletion of intracellular Ca²⁺ stores and the opening of store-operated Ca²⁺ channels. The resulting increase in [Ca²⁺]_i and PLCγ-derived diacylglycerol activate PKCβ, which binds to p110γ and subsequently phosphorylates Ser582 (→pp110γ). Phosphorylated p110γ cannot interact with p84, and is therefore unresponsive to GPCR inputs. GPCR input to PI3Kγ coincides with migration and adhesion, while Ca²⁺/PKCβ activation of p110γ occurs when mast cells degranulate. The phosphorylation of PKB/Akt occurs downstream of PtdIns(3,4,5)<i>P</i>₃, which originates from G protein-activated p84-p110γ complex or PKCβ-activated pp110γ. The phosphorylated residues Thr308 and Ser473 of PKB/Akt are used to monitor PI3K activation. More detailed effector signaling event schemes can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio.1001587-Wymann1" target="_blank">[52]</a>.</p

PKCβ interacts with and phosphorylates the catalytic subunit of PI3Kγ.

<p>(A) Schematic representation of the PKCβ-p110γ interaction: full-length (fl) PKCβ is in a closed conformation due to the interaction of the pseudo-substrate domain with the catalytic pocket of PKCβ, while the truncated catalytic domain (cat; amino acids 302–673) and pseudo-substrate deletion mutant (Δps; deletion of aa 19–31) give access to p110γ. (B) HEK293 cells were co-transfected with p110γ and HA-tagged PKCβ2 constructs. Protein complexes were immunoprecipitated with anti-p110γ or anti-HA antibodies, before HA-PKCβ2 and p110γ was detected by immunoblotting. Ig: immunoglobulin heavy chain signals of mouse anti-p110γ and anti-HA antibodies. (C) Recombinant GST-p110γ wild type (wt) or a catalytically inactive p110γ mutant (KR, Lys833Arg mutant) were incubated with recombinant PKCβ2 and [γ³²P]-ATP in kinase buffer for 30 min, before proteins were denatured and separated by SDS-PAGE. Phosphatidylserine (PS) lipid vesicles containing 1-oleoyl-2-acetyl-sn-glycerol (OAG) were present during the reaction where marked. Protein-bound ³²P was determined by radioisotope imaging, and recombinant proteins were stained with Coomassie blue (mean ± standard error of the mean [SEM], <i>n</i> = 3; * point to comparison with respective sample without PKC). (D) In vitro and in vivo phosphorylation of PI3Kγ on S582, analyzed by LC-MRM. S582 non-phospho- and phospho-peptides were detected in the MRM mode, quantifying the transition 501.1 to 709.3 for the non-modified peptide (blue) and 541.3 to 492.1 for the phospho-peptide (red). Data were normalized to the transition of the non-modified peptide, which was set to 1. Upper part: recombinant catalytically inactive human GST-PI3Kγ (2 µg) was incubated alone, together with PKCβ2 or with PKCβ2 and PKC-inhibitor (Ro318425, 2 µM) as in (C). After SDS-PAGE and Coomassie staining, PI3Kγ was excised from the gel and prepared for LC-MRM. Lower part: wild type BMMCs (300 M cells/stimulation) were starved for 4 h in IL-3 free medium/2% FCS, and were left unstimulated or were treated for 2 min with 50 nM PMA or for 4 min with 10 ng/ml antigen (cells preloaded with 100 ng/ml IgE overnight). Endogenous PI3Kγ was immunoprecipitated from cell lysates, resolved by SDS-PAGE and analyzed with LC-MRM.</p

PKCβ relays thapsigargin-induced PI3Kγ activation.

<p>(A) Effect of PKC inhibitors on thapsigargin-induced PKB phosphorylation on Ser473 (S473). IL-3 starved BMMCs were preincubated with the indicated compounds for 20 min before stimulation (pan-PKC: Ro318425, Gö6983; classical PKC: PKC412 (CPG41251); classical and atypical PKC: Gö6976; Rotterlin: broad band inhibitor; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio.1001587.s013" target="_blank">Text S1</a>; & refers to comparison with untreated control; <i>p</i>-values see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio-1001587-g001" target="_blank">Figure 1</a>). (B) PKB/Akt activation in response to 100 nM PMA or 1 µM thapsigargin was analyzed in wild type and PKCβ^−/− BMMCs. Cells were IL-3 deprived as in (A), and were pretreated with wortmannin (Wm, 100 nM) for 15 min before stimulation where indicated. Cells were lysed 2 min after stimulation, and analyzed for phosphorylation of PKB/Akt (T308 and S473) and MAPK (T183/Y185). (C) Wild type and PKCβ^−/− BMMCs were stimulated with 1 µM adenosine, 10 ng/ml IL3, or 10 ng/ml SCF, and processed as in (B). (D–F) PtdIns(3,4,5)<i>P</i>₃ (PI<i>P</i>₃) levels were determined in untreated (Ctrl) and classical PKC-inhibitor (PKC412)-treated wild type BMMCs and PKCβ^−/− BMMCs after stimulation with 0.5 µM thapsigargin, 200 ng/ml PMA, or 5 µM adenosine (30 s). BMMCs were metabolically labeled with [³²P]-orthophosphate, lipids were extracted, deacylated, and applied to high-pressure liquid chromatography (HPLC). (D) shows representative elution peaks of PI<i>P</i>₃ of the HPLC chromatograms. (E) Levels of PI<i>P</i>₃ in relation to PtdIns(4,5)<i>P</i>₂ (PI<i>P</i>₂) were quantified by integration of the peak areas of PI<i>P</i>₃ and PI<i>P</i>₂ and expressed as ratio of PI<i>P</i>₃/PI<i>P</i>₂ (data shown as mean ± standard error of the mean [SEM], <i>n</i>≥4–6). (F) Cellular PI<i>P</i>₃ production was measured over time in wild type BMMCs in response to PMA (200 nM) stimulation in the presence or absence of the classical PKC inhibitor PKC412 (mean ± SEM, <i>n</i> = 3). (G) Granule release and PKB activation (S473) in response to thapsigargin (1 µM) or IgE/antigen (100 ng/ml IgE overnight, 10 ng/ml DNP) was measured in the presence of increasing concentrations of the classical PKC inhibitor PKC412. Cells starved as in (A) were stimulated with IgE/antigen (IgE/Ag) or thapsigargin (Tg), and PKB phosphorylation and β-hexosaminidase release assays were performed in parallel (mean ± SEM, <i>n</i> = 3). (H) β-hexosaminidase release determined in wild type, PKCβ^−/−, and p110γ^−/− BMMCs incubated with IgE, and stimulated with the indicated antigen (Ag) concentrations (mean ± SEM, <i>n</i> = 5; * refer to comparison with wild type control. Only the higher <i>p</i>-values of the overlapping data points are indicated).</p

Phosphorylation of PI3Kγ requires Ca²⁺ and is PKCβ-dependent.

<p>(A) Stimulus-induced phosphorylation of endogenous p110γ on Ser582 in wild type BMMCs. IL-3 deprived cells were stimulated with 100 nM PMA, 1 µM thapsigargin, 1 µM adenosine, or 20 ng/ml DNP for 2 min. Where indicated (IgE), BMMCs were loaded with IgE (100 ng/ml) overnight. PI3Kγ was immunoprecipitated from cell lysates with an anti-PI3Kγ antibody, before precipitated protein was probed for phosphorylated p110γ (pp110γ) with a phospho-specific anti-pSer582 antibody (validation of antibody see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio.1001587.s004" target="_blank">Figure S4</a>). PI3Kγ phosphorylation is shown normalized to total PI3Kγ levels (mean ± standard error of the mean [SEM], <i>n</i> = 3; * depict analysis using unstimulated control. & reference point is IgE only). (B) IgE/antigen-induced Ser582 phosphorylation of p110γ requires Ca²⁺ influx. Cells were stimulated as in (A), but exposed to EDTA, EGTA, or loaded with BAPTA/AM where indicated (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio-1001587-g002" target="_blank">Figure 2</a>). Phosphorylated p110γ was detected as in (A); mean ± SEM, <i>n</i> = 3; * comparison with unstimulated control; ^&analysis of stimulated versus chelator treated). (C) Phosphorylation of p110γ in wild type and PKCβ^−/− BMMCs. Experimental settings were as in (A), and (D) depicts quantification of pp110γ in relation to total p110γ protein (mean ± SEM; PMA <i>n</i> = 4, antigen <i>n</i> = 3). Cells devoid of p110γ were included as negative control.</p

p84 Interacts with the helical domain of p110γ.

<p>(A) Changes in deuteration levels between free and p84-bound PI3Kγ are mapped onto the crystal structure of PI3Kγ (PDB ID: 2CHX). Regions that are covered by peptides of PI3Kγ (labeled A–R) that showed greater than 0.5 or 1.0 Da changes in deuteration are colored light or dark blue, respectively. The greatest difference in exchange observed at any time was used for the mapping. S582 is labeled red. The ATP competitive inhibitor PIK-90 in the crystal structure is shown in green as a reference point for the kinase domain. The linker regions between the RBD and the C2 domain and the C2 and the helical domain are shown as dotted lines (right part). (B) The percent deuterium exchange differences between free and p84-bound PI3Kγ were summed up over all seven time points for every identified peptide (<i>y</i>-axis), which were graphed according to their central residue number (<i>x</i>-axis). (C) A selected peptide (623–630) from the helical domain is shown at four time points of H/D on-exchange +/− the p84 subunit. In the absence of the p84 adaptor the majority of peptides in the helical domain showed broadening of the isotopic profiles indicative of EX1 kinetics (see 30, or 300 s in free p110γ). The helix A3 (624–631) selected is located at the interface of the helical domain with the C-lobe. Ser582 and Thr1024 have been highlighted as a reference. (D) p84 was coexpressed with GST-tagged or untagged PI3Kγ constructs in HEK293 cells. N-terminal deletions of 37 or 130 amino acids are denoted Δ37 or Δ130, respectively. HA-p84 (left) or PI3Kγ (right) was immunoprecipitated from cell lysates with anti-HA or anti-PI3Kγ antibodies and protein G beads. PI3Kγ-p84 interactions were analyzed by Western blotting, quantified with Odyssey Imager software and expressed as fold of untagged, full-length p110γ-p84 association (mean ± standard error of the mean [SEM], left: <i>n</i> = 4, 6, 6, 6; right: <i>n</i> = 2, 4, 4, 4).</p