The p101 subunit of PI3Kγ restores activation by Gβ mutants deficient in stimulating p110γ

G-protein-regulated PI3Kγ (phosphoinositide 3-kinase γ) plays a crucial role in inflammatory and allergic processes. PI3Kγ, a dimeric protein formed by the non-catalytic p101 and catalytic p110γ subunits, is stimulated by receptor-released Gβγ complexes. We have demonstrated previously that Gβγ stimulates both monomeric p110γ and dimeric p110γ/p101 lipid kinase activity in vitro. In order to identify the Gβ residues responsible for the Gβγ-PI3Kγ interaction, we examined Gβ1 mutants for their ability to stimulate lipid and protein kinase activities and to recruit PI3Kγ to lipid vesicles. Our findings revealed different interaction profiles of Gβ residues interacting with p110γ or p110γ/p101. Moreover, p101 was able to rescue the stimulatory activity of Gβ1 mutants incapable of modulating monomeric p110γ. In addition to the known adaptor function of p101, in the present paper we show a novel regulatory role of p101 in the activation of PI3Kγ.     

Lysophosphatidic acid-stimulated phosphorylation of PKD2 is mediated by PI3K p110β and PKCδ in myoblasts

Abstract

Context: G-protein coupled receptor (GPCR) signaling in skeletal muscle is incompletely understood; in particular, the signaling pathways that regulate GPCR-mediated signaling in skeletal muscle are only beginning to be established. Lysophosphatidic acid (LPA) is a GPCR agonist that has previously been shown to activate protein kinase D (PKD) in non-muscle cells; however, whether PKD is activated in response to LPA in skeletal muscle myoblasts, and the identities of signaling intermediates that regulate this activation, have not been defined. Objective: To determine whether PKD is activated in response to LPA administration in myoblasts, and to define the signaling pathways that mediate LPA-stimulated PKD phosphorylation. Methods: C2C12 myoblasts were treated with LPA and signaling pathways examined by means of Western immunoblotting and real-time PCR (RT-PCR). Pharmacological inhibition and RNA-interference were used to target specific molecules to determine their involvement in LPA-induced PKD phosphorylation. Results: Treatment of myoblasts with exogenous LPA revealed that PI3K p110β mediated PKD phosphorylation at Ser 748 and at Ser 916 through kinase-dependent and kinase-independent mechanisms. Loss of PKCδ, but not the loss of PKCα, prevented LPA-induced PKD phosphorylation. The PKD isoform responsive to LPA treatment was identified as PKD2. Conclusion: These results indicate that LPA-stimulated PKD2 phosphorylation requires PKCδ and non-catalytic actions of PI3K p110β, and provide new information with respect to GPCR-mediated signal transduction in myoblasts.     

The phosphoinositide 3'-kinase p110δ modulates contractile protein production and IL-6 release in human airway smooth muscle

Transforming growth factor (TGF) β1 increases pro‐inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3′ kinase (PI3K) is one of the signaling pathways implicated in TGFβ1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFβ1 induced pro‐inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFβ1 increased 110δ mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 µM), the p110β inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10 µM) had any effect on basal IL‐6, calponin or smooth muscle α‐actin (α‐SMA) expression. However, TGFβ1 increased calponin and α‐SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFβ1 induced IL‐6 release in a dose related manner in all groups of ASM cells. PI3K p110δ is important for TGFβ1 induced production of the contractile proteins calponin and α‐SMA and the proinflammatory cytokine IL‐6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling. J. Cell. Physiol. 227: 3044–3052, 2012. © 2011 Wiley Periodicals, Inc.     

Hot-spot mutations in p110α of phosphatidylinositol 3-kinase (PI3K): Differential interactions with the regulatory subunit p85 and with RAS

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently upregulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, is mutated in about 12% of all human cancers. Most of these mutants are single amino acid substitutions that map to three positions (hot spots) in the helical or kinase domains of the enzyme. The mutant proteins show gain of enzymatic function, constitutively activate AKT signaling and induce oncogenic transformation in vitro and in animal model systems. We have shown previously that hot-spot mutations in the helical domain and kinase domain of the avian p110α have different requirements for interaction with the regulatory subunit p85 and with RAS-GTP. Here, we have carried out a genetic and biochemical analysis of these "hot-spot" mutations in human p110α. The present studies add support to the proposal that helical and kinase domain mutations in p110α trigger a gain of function by different molecular mechanisms. The gain of function induced by helical domain mutations requires interaction with RAS-GTP. In contrast, the kinase domain mutation is active in the absence of RAS-GTP binding, but depends on the interaction with p85.     

Identification of highly potent and selective PI3Kδ inhibitors

Selective PI3Kδ inhibitors have recently been hypothesized to be appropriate immunosuppressive agents for the treatment of immunological disorders such as rheumatoid arthritis. However, few reports have highlighted molecules that are highly selective for PI3Kδ over the other PI3K isoforms. In this letter, isoform and kinome selective PI3Kδ inhibitors are presented. The Structural Activity Relationship leading to such molecules is outlined.     

Multiple roles for the p85α isoform in the regulation and function of PI3K signalling and receptor trafficking

The p85α protein is best known as the regulatory subunit of class 1A PI3Ks (phosphoinositide 3-kinases) through its interaction, stabilization and repression of p110-PI3K catalytic subunits. PI3Ks play multiple roles in the regulation of cell survival, signalling, proliferation, migration and vesicle trafficking. The present review will focus on p85α, with special emphasis on its important roles in the regulation of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and Rab5 functions. The phosphatidylinositol-3-phosphatase PTEN directly counteracts PI3K signalling through dephosphorylation of PI3K lipid products. Thus the balance of p85α-p110 and p85α-PTEN complexes determines the signalling output of the PI3K/PTEN pathway, and under conditions of reduced p85α levels, the p85α-PTEN complex is selectively reduced, promoting PI3K signalling. Rab5 GTPases are important during the endocytosis, intracellular trafficking and degradation of activated receptor complexes. The p85α protein helps switch off Rab5, and if defective in this p85α function, results in sustained activated receptor tyrosine kinase signalling and cell transformation through disrupted receptor trafficking. The central role for p85α in the regulation of PTEN and Rab5 has widened the scope of p85α functions to include integration of PI3K activation (p110-mediated), deactivation (PTEN-mediated) and receptor trafficking/signalling (Rab5-mediated) functions, all with key roles in maintaining cellular homoeostasis.     

Engineering of an isolated p110α subunit of PI3Kα permits crystallization and provides a platform for structure-based drug design

PI3Kα remains an attractive target for the development of anticancer targeted therapy. A number of p110α crystal structures in complex with the nSH2-iSH2 fragment of p85 regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the ATP binding site. Taking advantage of recent data on the functional characterization of the lipid binding properties of p110α, we designed a set of novel constructs allowing production of isolated stable p110α subunit missing the Adapter Binding Domain and lacking or featuring a modified C-terminal lipid binding motif. While this protein is not catalytically competent to phosphorylate its substrate PIP2, it retains ligand binding properties as indicated by direct binding studies with a pan-PI3Kα inhibitor. Additionally, we determined apo and PF-04691502 bound crystal structures of the p110α (105-1048) subunit at 2.65 and 2.85 Å, respectively. Comparison of isolated p110α(105-1048) with the p110α/p85 complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p110α for iterative SBDD. Importantly, this crystal form of p110α readily accommodates the binding of noncovalent inhibitor by means of a fully accessible ATP site. The strategy presented here can be also applied to structural studies of other members of PI3KIA family.     

MicroRNA-384-5p regulates ischemia-induced cardioprotection by targeting phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta (PI3K p110δ)

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. This study identified 16 differentially expressed miRNAs in ischemic myocardium of rats using TaqMan Low Density Array. In addition, bioinformatics analyses, such as Gene ontology and Pathway assays, were applied to determine the apoptosis pathway, only regulated by miR-384-5p, and all the associated target genes (PIK3CD, PPP3CA, PPP3CB, PPP3R1, CASP3 and IL1A). These target genes, besides PIK3CB, were shown to be significantly up-regulated by qRT-PCR assay, which further suggested that PIK3CD, PPP3CA, PPP3R1, CASP3, IL1A could be regulated by miR-384-5p. MTT, Western blot, qRT-PCR and luciferase assays were used to investigate the role of miR-384-5p in myocardial ischemia. We found that cleaved caspase3 expression was up-regulated by miR-384-5p and down-regulated by miR-384-5p inhibitor suggesting that apoptosis pathway was regulated by miR-384-5p. We also found that miR-384-5p suppressed cell viability while miR-384-5p inhibitor improved it, confirming H9c2 cell survival was affected by miR-384-5p. In addition, the PIK3CD protein level in H9c2 cells was up-regulated by miR-384-5p inhibitor. We found that miR-384-5p expression level decreased and PIK3CD protein level increased in both ischemic myocardium of rats and hypoxic H9c2 cells, and that miR-384-5p suppress PIK3CD expression through a miR-384-5p binding site within the 3′ untranslational region of PIK3CD. These results show that miR-384-5p, an important protecting factor, plays a significant role in cardioprotection by regulating PIK3CD in myocardial ischemia.     

Optimization and in vivo evaluation of pyrazolopyridines as a potent and selective PI3Kδ inhibitor

Chemical optimization of pyrazolopyridine 1, focused on cellular potency, isoform selectivity and microsomal stability, led to the discovery of the potent, selective and orally available PI3Kδ inhibitor 5d. On the basis of its desirable potency, selectivity and pharmacokinetic profiles, 5d was tested in the trinitrophenylated aminoethylcarboxymethyl-Ficoll (TNP-Ficoll)-induced antibody production model, and showed higher antibody inhibition than a 4-fold oral dose of the starting compound 1. These excellent results suggest that 5d is a potential candidate for further studies in the treatment of autoimmune diseases and leukocyte malignancies.     

Both p110α and p110β isoforms of PI3K can modulate the impact of loss-of-function of the PTEN tumour suppressor

The PI3K (phosphoinositide 3-kinase) pathway is commonly activated in cancer as a consequence of inactivation of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), a major negative regulator of PI3K signalling. In line with this important role of PTEN, mice that are heterozygous for a PTEN-null allele (PTEN+/? mice) spontaneously develop a variety of tumours in multiple organs. PTEN is a phosphatase with selectivity for PtdIns(3,4,5)P3, which is produced by the class I isoforms of PI3K (p110α, p110β, p110γ and p110δ). Previous studies indicated that PTEN-deficient cancer cell lines mainly depend on p110β, and that p110β, but not p110α, controls mouse prostate cancer development driven by PTEN loss. In the present study, we investigated whether the ubiquitously expressed p110α can also functionally interact with PTEN in cancer. Using genetic mouse models that mimic systemic administration of p110α- or p110β-selective inhibitors, we confirm that inactivation of p110β, but not p110α, inhibits prostate cancer development in PTEN+/? mice, but also find that p110α inactivation protects from glomerulonephritis, pheochromocytoma and thyroid cancer induced by PTEN loss. This indicates that p110α can modulate the impact of PTEN loss in disease and tumourigenesis. In primary and immortalized mouse fibroblast cell lines, both p110α and p110β controlled steady-state PtdIns(3,4,5)P3 levels and Akt signalling induced by heterozygous PTEN loss. In contrast, no correlation was found in primary mouse tissues between PtdIns(3,4,5)P3 levels, PI3K/PTEN genotype and cancer development. Taken together, our results from the present study show that inactivation of either p110α or p110β can counteract the impact of PTEN inactivation. The potential implications of these findings for PI3K-targeted therapy of cancer are discussed.     

Suppression of the PI3K subunit p85α delays embryoid body development and inhibits cell adhesion

Phosphatidylinositol-3-kinases (PI3Ks) exert a variety of signaling functions in eukaryotes. We suppressed the PI3K regulatory subunit p85α using a small interfering RNA (Pik3r1 siRNA) and examined the effects on embryoid body (EB) development in hanging drop culture. We observed a 150% increase in the volume of the treated EBs within 24 h, compared to the negative controls. Fluorescence Activated Cell Sorting (FACS) assays showed that this increase in volume is not due to increased cellular proliferation. Instead, the increase in volume appears to be due to reduced cellular aggregation and adherence. This is further shown by our observation that 40% of treated EBs form twin instead of single EBs, and that they have a significantly reduced ability to adhere to culture dishes when plated. A time course over the first 96 h reveals that the impaired adherence is transient and explained by an initial 12-hour delay in EB development. Quantitative PCR expression analysis suggests that the adhesion molecule integrin-β1 (ITGB1) is transiently downregulated by the p85α suppression. In conclusion we found that suppressing p85α leads to a delay in forming compact EBs, accompanied by a transient inability of the EBs to undergo normal cell-cell and cell-substrate adhesion.     

PI3K p110α/Akt signaling negatively regulates secretion of the intestinal peptide neurotensin through interference of granule transport

Neurotensin (NT), an intestinal peptide secreted from N cells in the small bowel, regulates a variety of physiological functions of the gastrointestinal tract, including secretion, gut motility, and intestinal growth. The class IA phosphatidylinositol 3-kinase (PI3K) family, which comprised of p110 catalytic (α, β and δ) and p85 regulatory subunits, has been implicated in the regulation of hormone secretion from endocrine cells. However, the underlying mechanisms remain poorly understood. In particular, the role of PI3K in intestinal peptide secretion is not known. Here, we show that PI3K catalytic subunit, p110α, negatively regulates NT secretion in vitro and in vivo. We demonstrate that inhibition of p110α, but not p110β, induces NT release in BON, a human endocrine cell line, which expresses NT mRNA and produces NT peptide in a manner analogous to N cells, and QGP-1, a pancreatic endocrine cell line that produces NT peptide. In contrast, overexpression of p110α decreases NT secretion. Consistently, p110α-inhibition increases plasma NT levels in mice. To further delineate the mechanisms contributing to this effect, we demonstrate that inhibition of p110α increases NT granule trafficking by up-regulating α-tubulin acetylation; NT secretion is prevented by overexpression of HDAC6, an α-tubulin deacetylase. Moreover, ras-related protein Rab27A (a small G protein) and kinase D-interacting substrate of 220 kDa (Kidins220), which are associated with NT granules, play a negative and positive role, respectively, in p110α-inhibition-induced NT secretion. Our findings identify the critical role and novel mechanisms for the PI3K signaling pathway in the control of intestinal hormone granule transport and release.     

Discovery and optimization of heteroaryl piperazines as potent and selective PI3Kδ inhibitors

A high-throughput screening (HTS) campaign identified a class of heteroaryl piperazines with excellent baseline affinity and selectivity for phosphoinositide 3-kinase δ (PI3Kδ) over closely related isoforms. Rapid evaluation and optimization of structure-activity relationships (SAR) for this class, leveraging the modular nature of this scaffold, facilitated development of this hit class into a series of potent and selective inhibitors of PI3Kδ. This effort culminated in the identification of 29, which displayed excellent potency in enzyme and cell-based assays, as well as favorable pharmacokinetic and off-target profiles.     

CoA Synthase is in complex with p85αPI3K and affects PI3K signaling pathway

The complex interplay between cellular signaling and metabolism in eukaryotic cells just start to emerge. Coenzyme A (CoA) and its derivatives play a key role in cell metabolism and also participate in regulatory processes. CoA Synthase (CoASy) is a mitochondria-associated enzyme which mediates two final stages of de novo CoA biosynthesis. Here, we report that CoASy is involved in signaling events in the cell and forms a functional complex with p85αPI3K in vivo. Importantly, observed interaction of endogenous CoASy and p85αPI3K is regulated in a growth factor dependent manner. Surprisingly, both catalytic p110α and regulatory p85α subunits of PI3K were detected in mitochondrial fraction where mitochondria-localized p85αPI3K was found in complex with CoASy. Unexpectedly, significant changes of PI3K signaling pathway activity were observed in experiments with siRNA-mediated CoASy knockdown pointing on the role of CoA biosynthetic pathway in signal transduction.     

Evidence for a role for the p110-α isoform of PI3K in skeletal function

Signaling through phosphatidylinositol-3 kinases (PI3K) regulates fundamental cellular processes such as survival and growth, and these lipid kinases are currently being investigated as therapeutic targets in several contexts. In skeletal tissue, experiments using pan-specific PI3K inhibitors have suggested that PI3K signaling influences both osteoclast and osteoblast function, but the contributions of specific PI3K isoforms to these effects have not been examined. In the current work, we assessed the effects of pharmacological inhibitors of the class Ia PI3Ks, α, β, and δ, on bone cell growth, differentiation and function in vitro. Each of the class Ia PI3K isoforms is expressed and functionally active in bone cells. No consistent effects of inhibitors of p110-β or p110-δ on bone cells were observed. Inhibitors of p110-α decreased osteoclastogenesis by 60-80% (p < 0.001 vs control) by direct actions on osteoclast precursors, and decreased the resorptive activity of mature osteoclasts by 60% (p < 0.01 vs control). The p110-α inhibitors also decreased the growth of osteoblastic and stromal cells (p < 0.001 vs control), and decreased differentiated osteoblast function by 30% (p < 0.05 vs control). These data suggest that signaling through the p110-α isoform of class Ia PI3Ks positively regulates the development and function of both osteoblasts and osteoclasts. Therapeutic agents that target this enzyme have the potential to significantly affect bone homeostasis, and evaluation of skeletal endpoints in clinical trials of such agents is warranted.