table_1.PDF

<p>The signaling lipid phosphatidylinositol 3,4,5, trisphosphate (PIP₃) is an essential mediator of many vital cellular processes, including growth, survival, and metabolism. PIP₃ is generated through the action of the class I phosphoinositide 3-kinases (PI3K), and their activity is tightly controlled through interactions with regulatory proteins and activating stimuli. The class IA PI3Ks are composed of three distinct p110 catalytic subunits (p110α, p110β, and p110δ), and they play different roles in specific tissues due to disparities in both expression and engagement downstream of cell-surface receptors. Disruption of PI3K regulation is a frequent driver of numerous human diseases. Activating mutations in the PIK3CA gene encoding the p110α catalytic subunit of class IA PI3K are frequently mutated in several cancer types, and mutations in the PIK3CD gene encoding the p110δ catalytic subunit have been identified in primary immunodeficiency patients. All class IA p110 subunits interact with p85 regulatory subunits, and mutations/deletions in different p85 regulatory subunits have been identified in both cancer and primary immunodeficiencies. In this review, we will summarize our current understanding for the molecular basis of how class IA PI3K catalytic activity is regulated by p85 regulatory subunits, and how activating mutations in the PI3K catalytic subunits PIK3CA and PIK3CD (p110α, p110δ) and regulatory subunits PIK3R1 (p85α) mediate PI3K activation and human disease.</p

Expanding the Scope of Electrophiles Capable of Targeting K‑Ras Oncogenes

There is growing interest in reversible and irreversible covalent inhibitors that target noncatalytic amino acids in target proteins. With a goal of targeting oncogenic K-Ras variants (e.g., G12D) by expanding the types of amino acids that can be targeted by covalent inhibitors, we survey a set of electrophiles for their ability to label carboxylates. We functionalized an optimized ligand for the K-Ras switch II pocket with a set of electrophiles previously reported to react with carboxylates and characterized the ability of these compounds to react with model nucleophiles and oncogenic K-Ras proteins. Here, we report that aziridines and stabilized diazo groups preferentially react with free carboxylates over thiols. Although we did not identify a warhead that potently labels K-Ras G12D, we were able to study the interactions of many electrophiles with K-Ras, as most of the electrophiles rapidly label K-Ras G12C. We characterized the resulting complexes by crystallography, hydrogen/deuterium exchange, and differential scanning fluorimetry. Our results both demonstrate the ability of a noncatalytic cysteine to react with a diverse set of electrophiles and emphasize the importance of proper spatial arrangements between a covalent inhibitor and its intended nucleophile. We hope that these results can expand the range of electrophiles and nucleophiles of use in covalent protein modulation

Regulation of a Coupled MARCKS–PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module

Amoeboid cells that employ chemotaxis to travel up an attractant gradient possess a signaling network assembled on the leading edge of the plasma membrane that senses the gradient and remodels the actin mesh and cell membrane to drive movement in the appropriate direction. In leukocytes such as macrophages and neutrophils, and perhaps in other amoeboid cells as well, the leading edge network includes a positive feedback loop in which the signaling of multiple pathway components is cooperatively coupled. Cytoplasmic Ca²⁺ is a recently recognized component of the feedback loop at the leading edge where it stimulates phosphoinositide-3-kinase (PI3K) and the production of its product signaling lipid phosphatidylinositol 3,4,5-trisphosphate (PIP₃). A previous study implicated Ca²⁺-activated protein kinase C (PKC) and the phosphatidylinositol 4,5-bisphosphate (PIP₂) binding protein MARCKS as two important players in this signaling, because PKC phosphorylation of MARCKS releases free PIP₂ that serves as the membrane binding target and substrate for PI3K. This study asks whether calmodulin (CaM), which is known to directly bind MARCKS, also stimulates PIP₃ production by releasing free PIP₂. Single-molecule fluorescence microscopy is used to quantify the surface density and enzyme activity of key protein components of the hypothesized Ca²⁺–CaM–MARCKS–PIP₂–PI3K–PIP₃ circuit. The findings show that CaM does stimulate PI3K lipid kinase activity by binding MARCKS and displacing it from PIP₂ headgroups, thereby releasing free PIP₂ that recruits active PI3K to the membrane and serves as the substrate for the generation of PIP₃. The resulting CaM-triggered activation of PI3K is complete in seconds and is much faster than PKC-triggered activation, which takes minutes. Overall, the available evidence implicates both PKC and CaM in the coupling of Ca²⁺ and PIP₃ signals and suggests these two different pathways have slow and fast activation kinetics, respectively

Novel K‑Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange

The success of targeted covalent inhibitors in the global pharmaceutical industry has led to a resurgence of covalent drug discovery. However, covalent inhibitor design for flexible binding sites remains a difficult task due to a lack of methodological development. Here, we compared covalent docking to empirical electrophile screening against the highly dynamic target K-Ras^G12C. While the overall hit rate of both methods was comparable, we were able to rapidly progress a docking hit to a potent irreversible covalent binder that modifies the inactive, GDP-bound state of K-Ras^G12C. Hydrogen–deuterium exchange mass spectrometry was used to probe the protein dynamics of compound binding to the switch-II pocket and subsequent destabilization of the nucleotide-binding region. SOS-mediated nucleotide exchange assays showed that, contrary to prior switch-II pocket inhibitors, these new compounds appear to accelerate nucleotide exchange. This study highlights the efficiency of covalent docking as a tool for the discovery of chemically novel hits against challenging targets

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

Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIβ

Type III phosphatidylinositol 4-kinase (PI4KIIIβ) is an essential enzyme in mediating membrane trafficking and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles and may lead to novel antiviral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIβ. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design, we have designed novel potent and selective (>1000-fold over class I and class III PI3Ks) PI4KIIIβ inhibitors. These compounds showed antiviral activity against hepatitis C virus. The co-crystal structure of PI4KIIIβ bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIβ inhibitors, which may play significant roles as antiviral therapeutics

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

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

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

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