Academic literature on the topic 'P84 adaptor subunit of PI3Kγ'

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein–coupled receptors. Here, we report the cryo–electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gβγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ–adaptor binding domain interfaces enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation, providing a novel tool to study and target p110γ-p101–specific signaling events in vivo.

Class IA PI3Ks (phosphoinositide 3-kinases) consist of a p110 catalytic subunit bound to one of five regulatory subunits, known as p85s. Under unstimulated conditions, p85 stabilizes the labile p110 protein, while inhibiting its catalytic activity. Recruitment of the p85–p110 complex to receptors and adaptor proteins via the p85 SH2 (Src homology 2) domains alleviates this inhibition, leading to PI3K activation and production of PIP3 (phosphatidylinositol 3,4,5-trisphosphate). Four independent p85 KO (knockout) mouse lines have been generated. Remarkably, PI3K signalling in insulin-sensitive tissues of these mice is increased. The existence of p110-free p85 in insulin-responsive cells has been invoked to explain this observation. Such a monomeric p85 would compete with heterodimeric p85–p110 for pTyr (phosphotyrosine) recruitment, and thus repress PI3K activity. Reduction in the pool of p110-free p85 in p85 KO mice was thought to allow recruitment of functional heterodimeric p85–p110, leading to increased PI3K activity. However, recent results indicate that monomeric p85, like p110, is unstable in cells. Moreover, overexpressed free p85 does not necessarily compete with heterodimeric p85–p110 for receptor binding. Using a variety of approaches, we have observed a 1:1 ratio between the p85 and p110 subunits in murine cell lines and primary tissues. Alternative models to explain the increase in PI3K signalling in insulin-responsive cells of p85 KO mice, based on possible effects of p85 deletion on phosphatases acting on PIP3, are discussed.

The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gβγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gβγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gβγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gβγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP3 production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101VVKR777AAAA neutrophils showed enhanced p84-dependent ROS responses to fMLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gβγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gβγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.

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.

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical regulator of many cellular processes including cell survival, growth, proliferation and motility. Not surprisingly therefore, the PI3K pathway is one of the most frequently mutated pathways in human cancers. In addition to their canonical role as part of the PI3K holoenzyme, the class IA PI3K regulatory subunits undertake critical functions independent of PI3K. The PI3K regulatory subunits exist in excess over the p110 catalytic subunits and therefore free in the cell. p110-independent p85 is unstable and exists in a monomer-dimer equilibrium. Two conformations of dimeric p85 have been reported that are mediated by N-terminal and C-terminal protein domain interactions, respectively. The role of p110-independent p85 is under investigation and it has been found to perform critical adaptor functions, sequestering or influencing compartmentalisation of key signalling proteins. Free p85 has roles in glucose homeostasis, cellular stress pathways, receptor trafficking and cell migration. As a regulator of fundamental pathways, the amount of p110-independent p85 in the cell is critical. Factors that influence the monomer-dimer equilibrium of p110-independent p85 offer additional control over this system, disruption to which likely results in disease. Here we review the current knowledge of the structure and functions of p110-independent class IA PI3K regulatory subunits.

Enzymes of the phosphodiesterase 3 (PDE3) and PDE4 families each regulate the activities of both protein kinases A (PKAs) and exchange proteins activated by cAMP (EPACs) in cells of the cardiovascular system. At present, the mechanisms that allow selected PDEs to individually regulate the activities of these two effectors are ill understood. The objective of this study was to determine how a specific PDE3 variant, namely PDE3B, interacts with and regulates EPAC1-based signaling in human arterial endothelial cells (HAECs). Using several biochemical approaches, we show that PDE3B and EPAC1 bind directly through protein-protein interactions. By knocking down PDE3B expression or by antagonizing EPAC1 binding with PDE3B, we show that PDE3B regulates cAMP binding by its tethered EPAC1. Interestingly, we also show that PDE3B binds directly to p84, a PI3Kγ regulatory subunit, and that this interaction allows PI3Kγ recruitment to the PDE3B-EPAC1 complex. Of potential cardiovascular importance, we demonstrate that PDE3B-tethered EPAC1 regulates HAEC PI3Kγ activity and that this allows dynamic cAMP-dependent regulation of HAEC adhesion, spreading, and tubule formation. We identify and molecularly characterize a PDE3B-based “signalosome” that integrates cAMP- and PI3Kγ-encoded signals and show how this signal integration regulates HAEC functions of importance in angiogenesis.

Grb2-associated binder 2 (Gab2), a member of the Dos/Gab subfamily scaffolding molecules, plays important roles in regulating the growth, differentiation, and function of many hematopoietic cell types. In this paper, we reveal a novel function of Gab2 in Fcγ receptor (FcγR)–initiated phagocytosis in macrophages. Upon FcγR activation, Gab2 becomes tyrosyl phosphorylated and associated with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and the protein–tyrosine phosphatidylinositol Shp-2. FcγR-mediated phagocytosis is severely impaired in bone marrow–derived macrophages from Gab2−/− mice. The defect in phagocytosis correlates with decreased FcγR-evoked activation of Akt, a downstream target of PI3K. Using confocal fluorescence microscopy, we find that Gab2 is recruited to the nascent phagosome, where de novo PI3K lipid production occurs. Gab2 recruitment requires the pleckstrin homology domain of Gab2 and is sensitive to treatment with the PI3K inhibitor wortmannin. The Grb2 binding site on Gab2 also plays an auxiliary role in recruitment to the phagosome. Because PI3K activity is required for FcγR-mediated phagocytosis, our results indicate that Gab2 acts as a key component of FcγR-mediated phagocytosis, most likely by amplifying PI3K signaling in the nascent phagosome.

Abstract The interaction of endothelial P-selectin (CD62P) with neutrophil PSGL-1 (P-selectin glycoprotein ligand-1, CD162) mediates neutrophil rolling, which acts in concert with cytokines and chemoattractants for integrin-mediated firm adhesion of neutrophils to vascular endothelial cells. Numerous studies have established that this sequence of events is critical to the extravasation of neutrophils during an inflammatory response. We have recently shown that cross-linking of PSGL-1 with P-selectin acts cooperatively with extracellular stimuli, such as cytokines and chemoattractants released from and displayed on the activated endothelial lining of the vessel walls, for optimal activation of beta2-integrins, an increase in their apparent affinity/avidity for cognate ligands, which in turn supports firm adhesion and transendothelial migration. To identify cellular proteins which interact with human PSGL-1 and trigger this cascade of events, we used the cytoplasmic domain of human PSGL-1 as the bait for screening of human leukocyte yeast two-hybrid library. During this genetic screening, we identified Naf-1 (Nef-associated factor 1; also called ABIN, A20 binding inhibitor of NF-kB, or VAN, virion-associated nuclear shuttle protein) as a binding partner to the cytoplasmic domain of PSGL-1. The specific interaction of Naf1 with PSGL-1 was confirmed by GST-fusion protein pull-down and co-immunoprecipitation experiments. As YPPM at 552–555 of Naf1a amino acid sequence is a known binding motif for p85 subunit of phosphatidylinositol-3 kinase (PI3K; YXXM in which phosphorylated Y is required for p85 binding), we performed co-immunoprecipitation experiments to show that Naf1a actually acted as an adaptor protein for PSGL-1 and p85 subunit of PI3K. In addition, we found that P-selectin activated the enzyme activity of PI3K in human neutrophils and PI3K specific inhibitors, wortmannin and LY294002, inhibited alphaMbeta2 activation and clustering induced by P-selectin. Our data thus delineate a novel PSGL-1 signal transduction pathway essential for transactivation of beta2-integrins.

B cell adaptor for phosphoinositide 3-kinase (PI3K) (BCAP) is a signaling adaptor that activates the PI3K pathway downstream of B cell receptor signaling in B cells and Toll-like receptor (TLR) signaling in macrophages. BCAP binds to the regulatory p85 subunit of class I PI3K and is a large, multidomain protein. We used proteomic analysis to identify other BCAP-interacting proteins in macrophages and found that BCAP specifically associated with the caspase-1 pseudosubstrate inhibitor Flightless-1 and its binding partner leucine-rich repeat flightless-interacting protein 2. Because these proteins inhibit the NLRP3 inflammasome, we investigated the role of BCAP in inflammasome function. Independent of its effects on TLR priming, BCAP inhibited NLRP3- and NLRC4-induced caspase-1 activation, cell death, and IL-1β release from macrophages. Accordingly, caspase-1–dependent clearance of a Yersinia pseudotuberculosis mutant was enhanced in BCAP-deficient mice. Mechanistically, BCAP delayed the recruitment and activation of pro–caspase-1 within the NLRP3/ASC preinflammasome through its association with Flightless-1. Thus, BCAP is a multifunctional signaling adaptor that inhibits key pathogen-sensing pathways in macrophages.

Abstract B-cell adaptor for phosphoinositide 3-kinase (BCAP) connects B cell receptor (BCR) signaling to the phosphoinositide 3-kinase (PI3K)-Akt pathway. Upon BCR engagement, tyrosine kinases Syk and Btk phosphorylate BCAP and phosphorylated BCAP provides the binding site for the regulatory subunit (p85) of PI3K, leading to relocation of PI3K to cytoplasmic membrane and PI3K activation. In response to CD19 engagement, BCAP phosphorylation is also required for p85 binding of BCAP prior to PI3K and Akt activation. It has been shown that disruption of the BCAP gene in the chicken DT40 B cell line inhibits BCR-mediated phosphatidylinositol 3,4,5-trisphosphate generation, leading to an impaired Akt response through a significant reduction in the recruitment of PI3K to glycolipid-enriched microdomains (lipid rafts). Furthermore, using BCAP knock out (KO) mice, BCAP has been shown to be essential for normal Follicular B and B1 B cell development and function. Here we report that the development of another peripheral B cell subpopulation, Marginal Zone B (MZB) cells, is impaired in BCAP KO mice as well. The numbers of MZB (B220+CD23−CD24midCD21high) cells and their surface expression level of CD80 are significantly reduced, compared to the littermate or age-matched B6 controls. The serum IgG3 level of the BCAP KO mice is also significantly lower than that of control mice. This result is further supported by an in vitro co-culture system, using bone marrow (BM) cells with the OP9-DL1 cells, in which the development of MZB cells from BCAP KO BM is also reduced, compared to B6 BM. Moreover, addition of exogenous BAFF could not fully restore the development of MZB cells from BCAP KO BM. Our data ascribes a role of BCAP to MZB cell development in mice.

The Class IB phosphatidylinositol 3-kinase (PI3K) enzyme, PI3Kγ, is activated and recruited to the plasma membrane in response to G protein-coupled receptor stimulation. Upon activation, the lipid-kinase activity and downstream signalling cascades initiated by PI3Kγ lead to cytoskeletal rearrangements and the formation of a leading edge for the induction of directed cell migration. PI3Kγ consists of the catalytic subunit p110γ, which forms a mutually exclusive heterodimer with one of two regulatory adaptor subunits, p84 or p101. Although expressed by most cells in the organism, PI3Kγ subunits are expressed at highest levels in motile haematopoietic cells, where the regulation of PI3Kγ signalling is critical to controlling and maintaining coordinated cell migration during immune responses. Consistent with a central role in leukocyte chemotaxis, innate and adaptive immune cell subsets from p110γ-deficient mice have been shown to exhibit migration defects in vitro and in vivo. Furthermore, the aberrant expression of PI3Kγ subunits and dysregulation of PI3Kγ signalling pathways has been shown to contribute to pathologies such as cancer and autoimmunity where enhanced cell migration promotes disease progression. Despite this, the mechanistic basis for PI3Kγ signal regulation is not well understood, particularly with respect to the distinct contributions of the individual regulatory adaptor subunits, p84 and p101. Many PI3Kγ-dependent cell functions have been elucidated experimentally using p110γ- and p101-deficient genetically-modified mouse strains and the PI3Kγ-selective inhibitor, AS605240. However, detailed functional data regarding p84 is lacking due to the absence of a p84-deficient mouse strain and limited availability of high quality p84-specific reagents. Three major research goals were addressed in the present study to improve our understanding of the role of p84 in PI3Kγ lipid-kinase signalling and its implication in PI3Kγ-dependent cell migration. The first goal was to examine the phosphorylation status of p84 during PI3Kγ signalling and assess the role of identified regulatory phosphorylation sites for p84 function using the mammary epithelial carcinoma model cell line, MDA.MB.231. Data presented in this thesis demonstrate that in contrast to the p110γ and p101 subunits that promote the migration and metastasis of carcinoma cells, the p84 adaptor protein has tumour suppressor function in vitro and in vivo, which was determined to be dependent on a potential phosphorylation site within p84, Thr607. It was found that Thr607 was required for p84 to form an inducible heterodimer with p110γ (after initial PI3Kγ signal activation) in a complex sequestered from active signalling at the membrane. This Thr607-dependent p84/p110γ dimerisation may therefore represent a novel mechanism of negative PI3Kγ signal regulation that limits the migration and metastasis of cancer cells. Next, the contribution of p84 to PI3Kγ-dependent immune cell function was determined through the generation and characterisation of a novel p84-deficient mouse (Pik3r6⁻ʹ⁻) using CRISPR gene-editing technology. Pik3r6⁻ʹ⁻ mice were characterised in the context of immune cell development, activation and migration in a variety of haematopoietic cell subsets. It was shown that Pik3r6⁻ʹ⁻ mice develop normally with respect to lymphoid organ and circulating leukocyte populations at homeostasis. However upon stimulation, neutrophils from Pik3r6⁻ʹ⁻ mice display reduced migration in response to GPCR agonists in vitro and in a murine model of inflammatory autoimmunity (experimental autoimmune encephalomyelitis; EAE), it was found that activated Th lymphocytes display impaired trafficking and reduced infiltration to inflammatory sites. The final goal was to develop and optimise a proteomic platform to investigate and compare the proteomes of migratory CD4⁺ lymphocytes isolated from tissues at different stages of inflammatory disease progression using experimental autoimmune encephalomyelitis as a model. An isotope-coded protein-labelling (ICPL) approach was developed and optimised to assess the proteomes of CNS-infiltrating CD4⁺ lymphocytes during disease progression in two models of EAE; chronic MOG₃₅₋₅₅-induced EAE and relapsing-remitting PLP₁₃₉₋₁₅₁-induced EAE. This study identified differentially regulated proteins related to immune cell function and represented a initial feasibility study to verify the validity of ICPL as an approach to examine the differential proteomes of wildtype and p84-deficient migratory CD4⁺ lymphocytes during inflammatory disease. Collectively, the data presented in this thesis represent the identification and characterisation of novel roles for p84 within PI3Kγ lipid-kinase signalling during both the regulation of cell migration in carcinoma cells and in haematopoietic cells during immune responses. In addition to furthering the understanding of the unique roles for p84 within PI3Kγ signal regulation, the generation of a p84-deficient mouse strain constitutes an important tool to further experimental research in this area.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2015.