P85 Subunit Of Phosphoinositide 3-kinase Research Articles

CAR-Macrophages and CAR-T Cells Synergistically Kill Tumor Cells In Vitro.

Chimeric antigen receptor (CAR)-expressing macrophages (CAR-M) have a great potential to improve cancer therapy, as shown from several recent preclinical studies. However, unlike CAR-T cell therapy, which has been widely studied, the efficacy and limitations of CAR-M cells remain to be established. To address this issue, in the present study, we compared three intracellular signaling domains (derived from common γ subunit of Fc receptors (FcRγ), multiple EGF-like-domains protein 10 (Megf10), and the CD19 cytoplasmic domain that recruits the p85 subunit of phosphoinositide-3 kinase (PI3K), respectively) for their ability to promote primary CAR-M functions, and investigated the potential synergistic effect between CAR-M and CAR-T cells in their ability to kill tumor cells. We found that CAR-MFcRγ exerted more potent phagocytic and tumor-killing capacity than CAR-MMegf10 and CAR-MPI3K. CAR-M and CAR-T demonstrated synergistic cytotoxicity against tumor cells in vitro. Mechanistically, the inflammatory factors secreted by CAR-T increased the expression of costimulatory ligands (CD86 and CD80) on CAR-M and augmented the cytotoxicity of CAR-M by inducing macrophage M1 polarization. The upregulated costimulatory ligands may promote the fitness and activation of CAR-T cells in turn, achieving significantly enhanced cytotoxicity. Taken together, our study demonstrated for the first time that CAR-M could synergize with CAR-T cells to kill tumor cells, which provides proof-of-concept for a novel combinational immunotherapy.

Phosphorylation of the multifunctional signal transducer B-cell adaptor protein (BCAP) promotes recruitment of multiple SH2/SH3 proteins including GRB2

B-cell adaptor protein (BCAP) is a multimodular, multifunctional signal transducer that regulates signal transduction pathways in leukocytes, including macrophages, B-cells, and T-cells. In particular, BCAP suppresses inflammatory signaling by Toll-like receptors (TLRs). However, how BCAP itself is regulated and what its interaction partners are is unclear. Here, using human immune cell lines, including THP-1 cells, we characterized the complex phosphorylation patterns of BCAP and used a novel protein complex trapping strategy, called virotrap, to identify its interaction partners. This analysis identified known interactions of BCAP with phosphoinositide 3-kinase (PI3K) p85 subunit and NCK adaptor protein (NCK), together with previously unknown interactions of BCAP with Src homology 2 (SH2) and SH3 domain-containing adaptor proteins, notably growth factor receptor-bound protein 2 (GRB2) and CRK-like proto-oncogene, adaptor protein (CRKL). We show that the SH3 domain of GRB2 can bind to BCAP independently of BCAP phosphorylation status, suggesting that the SH2 domains mediate interactions with activated receptor tyrosine kinase complexes including the CD19 subunit of the B-cell receptor. Our results also suggested that the PI3K p85 subunit binds to BCAP via SH3 domains forming an inactive complex that is then activated by sequential binding with the SH2 domains. Taken together, our results indicate that BCAP is a complex hub that processes signals from multiple pathways in diverse cell types of the immune system.

Lens differentiation is controlled by the balance between PDGF and FGF signaling.

How multiple receptor tyrosine kinases coordinate cell fate determination is yet to be elucidated. We show here that the receptor for platelet-derived growth factor (PDGF) signaling recruits the p85 subunit of Phosphoinositide 3-kinase (PI3K) to regulate mammalian lens development. Activation of PI3K signaling not only prevents B-cell lymphoma 2 (BCL2)-Associated X (Bax)- and BCL2 Antagonist/Killer (Bak)-mediated apoptosis but also promotes Notch signaling to prevent premature cell differentiation. Reducing PI3K activity destabilizes the Notch intracellular domain, while the constitutive activation of Notch reverses the PI3K deficiency phenotype. In contrast, fibroblast growth factor receptors (FGFRs) recruit Fibroblast Growth Factor Receptor Substrate 2 (Frs2) and Rous sarcoma oncogene (Src) Homology Phosphatase 2 (Shp2) to activate Mitogen-Activated Protein Kinase (MAPK) signaling, which induces the Notch ligand Jagged 1 (Jag1) and promotes cell differentiation. Inactivation of Shp2 restored the proper timing of differentiation in the p85 mutant lens, demonstrating the antagonistic interaction between FGF-induced MAPK and PDGF-induced PI3K signaling. By selective activation of PI3K and MAPK, PDGF and FGF cooperate with and oppose each other to balance progenitor cell maintenance and differentiation.

Relationships of SLC2A4, RBP4, PCK1, and PI3K Gene Polymorphisms with Gestational Diabetes Mellitus in a Chinese Population.

Background Solute carrier family 2 member 4- (SLC2A4-) retinol binding protein-4- (RBP4-) phosphoenolpyruvate carboxykinase 1 (PCK1)/phosphoinositide 3-kinase (PI3K) is an adipocyte derived “signalling pathway” that may contribute to the pathogenesis of type 2 diabetes mellitus (T2DM). We explored whether single nucleotide polymorphisms (SNPs) of these “signalling pathway” genes are associated with gestational diabetes mellitus (GDM). Methods Case-control studies were conducted to compare GDM and control groups. A total of 334 cases and 367 controls were recruited. Seventeen candidate SNPs of the pathway were selected. Chi-square tests, logistic regression, and linear regression were used to estimate the relationships of SNPs with GDM risk and oral glucose tolerance test (OGTT), fasting insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) levels. Model-based multifactor dimensionality reduction was used to estimate the adjusted interactions between genes. Regression and interaction analyses were adjusted by maternal age, prepregnancy BMI, and weekly BMI growth. The Bonferroni correction was applied for multiple comparisons. Results RBP4 rs7091052 was significantly associated with GDM risk. SLC2A4 rs5435, RBP4 rs7091052, PCK1 rs1042531 and rs2236745, and PIK3R1 (coding gene of the PI3K P85 subunit) rs34309 were associated with OGTT, fasting insulin, and HOMA-IR levels in the linear regression analysis. The gene-gene interaction analysis showed that, compared with pregnant women with other genotype combinations, women with SLC2A4 rs5435 (CC/CT), RBP4 rs7091052 (CC), PCK1 rs1042531 (TT/TG) and rs2236745 (TT), and PIK3R1 rs34309 (AA) had lower GDM risk. Conclusion SLC2A4, RBP4, PCK1, and PIK3R1 genes may be involved in the pathogenesis of GDM.

Cell Unroofing to Study the PI3K-TRPV1 Interaction

Transient receptor potential vanilloid 1 (TRPV1) channels play an essential role in the sensitized response to painful stimuli that occurs in response to inflammatory mediators including nerve growth factor (NGF). Sensitization involves NGF activation of phosphoinositide 3-kinase (PI3K), which increases the concentration of the signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 then triggers exocytosis of TRPV1-containing vesicles, increasing the number of TRPV1 channels on the plasma membrane. We have previously shown that TRPV1 and PI3K interact directly through the channel's N-terminus and PI3K's p85 subunit. However, little is known about the dynamics behind the interaction and its consequences. Cell unroofing is an effective method for accessing biological membranes from the inside. In cell unroofing, a probe sonicator is mounted on the microscope stage and positioned within about 1 mm of adherent cells. A single pulse of the sonicator shears off the top of the cells, causing loss of organelles and cytosolic components, and leaving a plasma membrane sheet with its proteins and lipids intact. Because of the near-instantaneous disruption of cellular contents, this system is ideal for measuring dissociation rate of membrane-associated proteins. By labeling proteins of interest with fluorescent tags, we can measure the time course of the decay in fluorescence that occurs upon unroofing. We used PI3K fused to YFP and compared the dissociation rates in cells without TRPV1 to those that were transiently transfected with TRPV1. We quantified the fluorescent signals and obtained the dissociation rates (koff) by fitting the data with an exponential decay model. These studies revealed that PI3K dissociation from the plasma membrane was slower in TRPV1-expressing cells than in those not expressing TRPV1. We conclude that PI3K interacts with TRPV1 in resting cells. Whether the interaction is altered in NGF-stimulated cells is still an open question.

ErbB3 interacts with Hrs and is sorted to lysosomes for degradation

The ErbB family of receptor tyrosine kinases mediates activation of a wide network of signaling pathways. ErbB3 has weak kinase activity, but its six docking sites for the p85 subunit of phosphoinositide 3-kinase make it an important contributor to proliferative signaling. ErbB3 has a relatively short half-life but the exact mechanisms controlling its turnover are unclear as contradictory reports exist. ErbB-mediated signaling is, however, negatively regulated by endocytosis of the receptors, followed by either recycling or degradation. Our previous studies showed that ErbB3 can be endocytosed and degraded in the absence of its ligand heregulin. However, binding of heregulin increased the degradation rate. In the current study we have investigated in more detail the trafficking and degradation of ErbB3 in the presence or absence of heregulin. We report that ErbB3 is internalized by clathrin-mediated endocytosis both in the presence and absence of heregulin. Moreover, we show that both proteasomal and lysosomal activity regulate ErbB3 degradation. Although steady-state expression of ErbB3 is regulated by proteasomal activity to a large extent, probably linked to a previously identified ER-localized quantity control, the results indicate that internalization, both constitutive and ligand-induced, causes lysosomal degradation of ErbB3. Furthermore, we show that ErbB3 interacts with the ESCRT-0 subunit Hrs both in the presence and absence of heregulin. This indicates an ESCRT-mediated sorting of ErbB3 to late endosomes and lysosomes, and in line with this we show that impaired ESCRT function leads to an endosomal accumulation of ErbB3.

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.

C-terminal domain of p42 Ebp1 is essential for down regulation of p85 subunit of PI3K, inhibiting tumor growth.

Potential tumor suppressor p42, ErbB3-binding protein 1 (EBP1) inhibits phosphoinositide 3-kinase (PI3K) activity reducing the p85 regulatory subunit. In this study, we demonstrated that overexpression of p42 promoted not only a reduction of wild type of p85 subunit but also oncogenic mutant forms of p85 which were identified in human cancers. Moreover, we identified the small fragment of C-terminal domain of p42 is sufficient to exhibit tumor suppressing activity of p42-WT, revealing that this small fragment (280–394) of p42 is required for the binding of both HSP70 and CHIP for a degradation of p85. Furthermore, we showed the small fragment of p42 markedly inhibited the tumor growth in mouse xenograft models of brain and breast cancer, resembling tumor suppressing activity of p42. Through identification of the smallest fragment of p42 that is responsible for its tumor suppressor activity, our findings represent a novel approach for targeted therapy of cancers that overexpress PI3K.

Metformin treatment in the period after stroke prevents nitrative stress and restores angiogenic signaling in the brain in diabetes.

Diabetes impedes vascular repair and causes vasoregression in the brain after stroke, but mechanisms underlying this response are still unclear. We hypothesized that excess peroxynitrite formation in diabetic ischemia/reperfusion (I/R) injury inactivates the p85 subunit of phosphoinositide 3-kinase (PI3K) by nitration and diverts the PI3K–Akt survival signal to the p38–mitogen-activated protein kinase apoptosis pathway. Nitrotyrosine (NY), Akt and p38 activity, p85 nitration, and caspase-3 cleavage were measured in brains from control, diabetic (GK), or metformin-treated GK rats subjected to sham or stroke surgery and in brain microvascular endothelial cells (BMVECs) from Wistar and GK rats subjected to hypoxia/reoxygenation injury. GK rat brains showed increased NY, caspase-3 cleavage, and p38 activation and decreased Akt activation. Metformin attenuated stroke-induced nitrative signaling in GK rats. GK rat BMVECs showed increased basal nitrative stress compared with controls. A second hit by hypoxia/reoxygenation injury dramatically increased the nitration of p85 and activation of p38 but decreased Akt. These effects were associated with impairment of angiogenic response and were restored by treatment with the peroxynitrite scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride or the nitration inhibitor epicatechin. Our results provide evidence that I/R-induced peroxynitrite inhibits survival, induces apoptosis, and promotes peroxynitrite as a novel therapeutic target for the improvement of reparative angiogenesis after stroke in diabetes.

Bernard-Soulier Syndrome: An Update

Bernard-Soulier syndrome (BSS) is a rare inherited platelet bleeding disorder characterized by low platelet count and abnormally large platelets (macrothrombocytopenia). Platelets from BSS patients are typically defective in surface expression of glycoprotein (GP)Ib-IX-V, a platelet-specific adhesion-signaling complex, composed of GPIbα disulfide linked to GPIbβ, and noncovalently associated with GPIX and GPV. The major ligand-binding subunit, GPIbα, binds the adhesive ligands von Willebrand factor (VWF) or thrombospondin, counterreceptors on activated endothelial cells (P-selectin) or activated leukocytes (integrin αMβ2), and coagulation factors (thrombin, factors XI and XII, high-molecular-weight kininogen). The cytoplasmic domain of GPIb-IX-V interacts with the cytoskeletal protein, filamin-A via a binding site within the GPIbα cytoplasmic tail, and with structural-signaling proteins including calmodulin, 14-3-3ζ and the p85 subunit of phosphoinositide 3-kinase. GPIbα is physically/functionally co-associated on the platelet surface with the major platelet collagen receptor, GPVI. As such, it is easy to see how genetic defects impacting GPIb-IX-V expression or function can have significant consequences on normal platelet size, adhesion to VWF/collagen and/or stable thrombus formation, and why BSS is often associated with clinical bleeding. Furthermore, the rarity, multiple genetic causes, and variable clinical phenotype of BSS can complicate routine diagnosis. Here, we discuss how studies of BSS have contributed to platelet biology and recent studies to improve diagnosis and treatment.

A novel TIR domain containing adapter, BCAP, links Toll-like receptors to PI3K/AKT activation and regulates inflammation (P1203)

Abstract Stimulation of TLRs leads to recruitment of several adaptor proteins leading to activation of NF-κB, MAP kinases, and IRFs, culminating in an inflammatory response. Interaction between TLRs and all known adaptor proteins depends on heterotypic interactions between respective Toll-Interleukin-1 receptor (TIR) homology domains. In addition to activation of NF-κB, MAP kinases, and IRFs, TLR signaling also leads to activation of phosphoinositide 3-kinase (PI3K). Activation of PI3K regulates the TLR induced inflammatory response, however, the mechanism of TLR mediated PI3K activation is unclear. Using in silico analysis, we identified an uncharacterized cryptic TIR domain at the amino-terminus of B cell adaptor for PI3K (BCAP, also known as Pik3ap1). We found that BCAP associated with MyD88 and TIRAP and attenuated TLR mediated activation of NF-κB. Both of these functions were dependent on BCAP’s TIR domain. Mechanistically, BCAP was phosphorylated upon TLR activation and the PI3K p85 subunit was recruited to BCAP upon TLR stimulation. BCAP deficient macrophages were severely limited in activation of PI3K/AKT and secreted increased pro-inflammatory cytokines upon TLR stimulation. In vivo studies revealed BCAP deficient mice to be highly susceptible to inflammatory colitis, as evidenced by increased weight loss, as well as enhanced tissue destruction. Additionally BCAP has a major role in wound healing and tissue repair.Together, our results demonstrate that BCAP is a TIR domain containing TLR signaling adaptor crucial for connecting TLRs to PI3K/AKT activation and regulation of inflammatory responses both in vitro and in vivo.

18F]-FLT Positron Emission Tomography Can Be Used to Image the Response of Sensitive Tumors to PI3-Kinase Inhibition with the Novel Agent GDC-0941

The phosphoinositide 3-kinase (PI3K) pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the positron emission tomography tracer 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]-FLT) is suitable to mark the effect of the novel PI3K inhibitor GDC-0941, which has entered phase II clinical trial. CBA nude mice bearing U87 glioma and HCT116 colorectal xenografts were imaged at baseline with [(18)F]-FLT and at acute (18 hours) and chronic (186 hours) time points after twice-daily administration of GDC-0941 (50 mg/kg) or vehicle. Tumor uptake normalized to blood pool was calculated, and tissue was analyzed at sacrifice for PI3K pathway inhibition and thymidine kinase (TK1) expression. Uptake of [(18)F]-FLT was also assessed in tumors inducibly overexpressing a dominant-negative form of the PI3K p85 subunit p85α, as well as HCT116 liver metastases after GDC-0941 therapy. GDC-0941 treatment induced tumor stasis in U87 xenografts, whereas inhibition of HCT116 tumors was more variable. Tumor uptake of [(18)F]-FLT was significantly reduced following GDC-0941 dosing in responsive tumors at the acute time point and correlated with pharmacodynamic markers of PI3K signaling inhibition and significant reduction in TK1 expression in U87, but not HCT116, tumors. Reduction of PI3K signaling via expression of Δp85α significantly reduced tumor growth and [(18)F]-FLT uptake, as did treatment of HCT116 liver metastases with GDC-0941. These results indicate that [(18)F]-FLT is a strong candidate for the noninvasive measurement of GDC-0941 action.

Abstract 2738: PIK3CA and APC mutations are synergistic in the development of intestinal cancers.

Abstract Background: Human colorectal cancers are known to possess multiple mutations, though how these mutations interact in tumor development and progression has not been thoroughly investigated. We have previously described the FC PIK3ca* murine colon cancer model which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K results in hyperplasia and invasive mucinous adenocarcinomas in the proximal colon. These cancers form via a non-canonical mechanism without aberrations in Wnt signaling. Since the Adenomatous Polyposis Coli (APC) gene is mutated in the vast majority of human colon cancers and often occurs concurrently with PIK3CA mutations, we sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine using novel murine models. Methods: B6 females carrying a transgene encoding for a chimeric protein with the iSH2 domain of the p85 subunit of PI3K fused to the N-terminus of p110 (PIK3ca*) were crossed to B6 males carrying the Min allele of Apc (ApcMin/+). The resulting B6 PIK3ca* ApcMin/+ mice were then crossed to mice carrying a transgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC). (FVB x B6)F1 FC PIK3ca* ApcMin/+ progeny express this constitutively active form of PI3K in epithelial cells of the distal small bowel and colon, as well as having lost one allele of Apc. An additional novel model system was developed to induce mutations in the cells of the mid to distal colon using a minimally invasive technique to instil adenovirus expressing the Cre recombinase. These tumors could then be monitored over time with colonoscopy with biopsies taken for histological and molecular analyses. Results: FC PIK3ca* ApcMin/+ mice develop multiple small intestinal and colon tumors, including invasive adenocarcinomas with lymphatic and mesenteric metastases. Tumor multiplicity and size were increased. Moreover, the tumors appeared less differentiated and were more invasive as compared to those in controls. Advanced adenomas and adenocarcinomas were easily detectible with a novel near-infrared phospholipid ether analog, CLR1502, that selectively accumulates in tumors. In the proximal colon, mucinous adenocarcinomas that were initiated by the dominant active PI3K rarely exhibited nuclear β-catenin and when observed, this localization was limited to select areas. This indicates that in these tumors loss of APC in these cells was a late event and can be a source of clonal outgrowth. The tumors that form in this model are microsatellite stable. Interestingly this synergy appears to be independent of PI3K mediated regulation of GSK-3β and β-catenin signaling. Conclusion: Expression of a dominant active PI3K synergizes with loss of APC activity resulting in dramatic changes in tumor multiplicity, size, morphology, and invasiveness. Citation Format: Dustin Deming, Alyssa Leystra, Laura Nettekoven, Chelsie Kohns, Linda Clipson, Dawn Albrecht, Jeff Bacher, Mary Kay Washington, Christopher Pazoles, Jamey Weichert, Richard Halberg. PIK3CA and APC mutations are synergistic in the development of intestinal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2738. doi:10.1158/1538-7445.AM2013-2738

GAB2—a Scaffolding Protein in Cancer

Adaptor or scaffolding proteins mediate protein-protein interactions that drive the formation of protein complexes. Grb2-associated binding protein 2 (GAB2) scaffolding protein is an intermediary molecule that links plasma membrane receptor signaling including receptor tyrosine kinases with the downstream effectors, such as protein tyrosine phosphatase, nonreceptor type 11 (SHP2), p85 subunit of phosphoinositide-3 kinase (PI3-K), phospholipase C-gamma 1 (PLC-γ), v-crk sarcoma virus CT10 (CRK), Src homology 2 domain containing transforming protein 1 (SHC), and SH2 containing inositol phosphatase (SHIP). Although, well described in signal transduction, its role in cancer has recently been emerging especially in leukemia, breast and ovarian cancer, and melanoma. GAB2 is essential for two major signal transduction pathways in cancer, the PI3-K-AKT and extracellular signal-regulated kinase (ERK) signaling pathways, and thus regulates a number of key cellular processes. This review focuses on structure and function of GAB2, its regulatory proteins, emerging role in cancer, and potential as a therapeutic target.

Helicobacter pylori‐Associated Regulation of Forkhead Transcription Factors FoxO1/3a in Human Gastric Cells

Interaction of Helicobacter pylori with gastric mucosa leads to marked cellular and humoral host immunologic responses. The signaling pathways initiated by bacteria-host interaction that result in perturbations in cell structure and function remain unclear. Forkhead transcription factors of class O (FoxO) are implicated in the regulation of apoptosis, cell survival, and pathogenesis. H. pylori infection of gastric epithelial cells induces phosphoinositide-3 kinase (PI3K)-dependent Akt activation and cell survival signaling. We investigated the role of H. pylori-activated PI3K/Akt in the regulation of FoxO1/3a in gastric cells. Immunoblot, immunoprecipitation, and fluorescence microscopy were used to assess the effect of infection of gastric epithelial cells with wild-type H. pylori and their isogenic cag pathogenicity island (PAI) or oipA mutants on the FoxO1/3a signaling pathways. Interleukin-8 release was determined by enzyme-linked immunosorbent assays. H. pylori infection resulted in activation of the PI3K p85 subunit and inactivation of FoxO1 and FoxO3a by their phosphorylation and translocation of from the nucleus to the cytoplasm. Inhibition of PI3K or Akt kinase activity reduced FoxO1/3a phosphorylation. Akt, FoxO1, or FoxO3a siRNA reduced H. pylori-induced interleukin-8 production. Infection with oipA mutants reduced PI3K/Akt activation and inhibited FoxO1/3a phosphorylation, whereas infection with cag PAI mutants reduced PI3K/Akt activity but did not inhibit FoxO1/3a activation. FoxO1 and FoxO3a are novel nuclear substrates of H. pylori-induced PI3K/Akt cell survival signaling pathways that partially control interleukin-8 production. OipA-regulated interleukin-8 release through PI3K/Akt is dependent on FoxO1/3a inactivation, whereas cag PAI-mediated interleukin-8 production employs FoxO1/3-independent signaling.

Overexpression of 14-3-3ζ in cancer cells activates PI3K via binding the p85 regulatory subunit

The ubiquitously expressed 14-3-3 proteins regulate many pathways involved in transformation. Previously, we found that 14-3-3ζ overexpression increased Akt phosphorylation in human mammary epithelial cells. Here, we investigated the clinical relevance and molecular mechanism of 14-3-3ζ overexpression-mediated Akt phosphorylation and the potential impact on breast cancer progression. We found that 14-3-3ζ overexpression was significantly (P = 0.005) associated with increased Akt phosphorylation in human breast tumors. Additionally, 14-3-3ζ overexpression combined with strong Akt phosphorylation was significantly (P=0.01) associated with increased cancer recurrence in patients. In contrast, knockdown of 14-3-3ζ expression by siRNA in cancer cell lines and tumor xenografts reduced Akt phosphorylation. Furthermore, 14-3-3ζ enhanced Akt phosphorylation through activation of PI3K. Mechanistically, 14-3-3ζ bound to the p85 regulatory subunit of PI3K and increased PI3K translocation to the cell membrane. A single 14-3-3 binding motif encompassing serine 83 on p85 is largely responsible for 14-3-3ζ-mediated p85 binding and PI3K/Akt activation. Mutation of serine 83 to alanine on p85 inhibited 14-3-3ζ binding to the p85 subunit of PI3K, reduced PI3K membrane localization and activation, impeded anchorage independent growth and enhanced stress induced apoptosis. These findings revealed a novel mechanism by which 14-3-3ζ overexpression activates PI3K, a key node in the mitogenic signaling network known to promote malignancies in many cell types.

Phosphorylation of CSF-1R Y721 mediates its association with PI3K to regulate macrophage motility and enhancement of tumor cell invasion

Colony stimulating factor-1 (CSF-1) regulates macrophage morphology and motility, as well as mononuclear phagocytic cell proliferation and differentiation. The CSF-1 receptor (CSF-1R) transduces these pleiotropic signals through autophosphorylation of eight intracellular tyrosine residues. We have used a novel bone-marrow-derived macrophage cell line system to examine specific signaling pathways activated by tyrosine-phosphorylated CSF-1R in macrophages. Screening of macrophages expressing a single species of CSF-1R with individual tyrosine-to-phenylalanine residue mutations revealed striking morphological alterations upon mutation of Y721. M⁻/⁻.Y721F cells were apolar and ruffled poorly in response to CSF-1. Y721-P-mediated CSF-1R signaling regulated adhesion and actin polymerization to control macrophage spreading and motility. Moreover, the reduced motility of M⁻/⁻.Y721F macrophages was associated with their reduced capacity to enhance carcinoma cell invasion. Y721 phosphorylation mediated the direct association of the p85 subunit of phosphoinositide 3-kinase (PI3K) with the CSF-1R, but not that of phospholipase C (PLC) γ2, and induced polarized PtdIns(3,4,5)P₃ production at the putative leading edge, implicating PI3K as a major regulator of CSF-1-induced macrophage motility. The Y721-P-motif-based motility signaling was at least partially independent of both Akt and increased Rac and Cdc42 activation but mediated the rapid and transient association of an unidentified ~170 kDa phosphorylated protein with either Rac-GTP or Cdc42-GTP. These studies identify CSF-1R-Y721-P-PI3K signaling as a major pathway in CSF-1-regulated macrophage motility and provide a starting point for the discovery of the immediate downstream signaling events.

MG53 participates in ischaemic postconditioning through the RISK signalling pathway

Recent studies show that ischaemic postconditioning (PostC), similar to the well-established ischaemic preconditioning (IPC), confers cardioprotection against ischaemia/reperfusion (IR) injury, and both IPC and PostC can activate the reperfusion injury salvage kinase (RISK) pathway and the survivor activating factor enhancement (SAFE) pathway. PostC is clinically more attractive because of its therapeutic application at the predictable onset of reperfusion. Our previous studies have demonstrated that MG53 is a primary component of the IPC machinery. Here, we investigated the potential role of MG53 in PostC-mediated myocardial protection and explored the underlying mechanism. Using Langendorff perfusion, we investigated IR injury in wild-type (wt) and MG53-deficient (mg53(-/-)) mouse hearts with or without PostC. IR-induced myocardial damage was markedly exacerbated in mg53(-/-) hearts compared with wt controls. PostC protected wt hearts against IR-induced myocardial infarction, myocyte necrosis, and apoptosis, but failed to protect mg53(-/-) hearts. The loss of PostC protection in mg53(-/-) hearts was attributed to selectively impaired PostC-activated RISK signalling. Mechanistically, MG53 is required for the interaction between caveolin 3 (CaV3) and the p85 subunit of phosphoinositide 3-kinase (p85-PI3K) and PostC-mediated activation of the RISK pathway. Importantly, a structure-function study revealed that the MG53 tripartite motif (TRIM) domain (aa1-284) physically interacted with CaV3 but not p85-PI3K, whereas the MG53 SPRY domain (aa285-477) interacted with p85-PI3K but not CaV3, indicating that MG53 binds to CaV3 and p85 at its N- and C-terminus, respectively. We conclude that MG53 participates in PostC-mediated cardioprotection largely through tethering CaV3 and PI3K and subsequent activation of the RISK pathway.

The phosphoinositide 3-kinase regulatory subunit p85alpha can exert tumor suppressor properties through negative regulation of growth factor signaling.

Phosphoinositide 3-kinase (PI3K) plays a critical role in tumorigenesis, and the PI3K p85 regulatory subunit exerts both positive and negative effects on signaling. Expression of Pik3r1, the gene encoding p85, is decreased in human prostate, lung, ovarian, bladder, and liver cancers, consistent with the possibility that p85 has tumor suppressor properties. We tested this hypothesis by studying mice with a liver-specific deletion of the Pik3r1 gene. These mice exhibited enhanced insulin and growth factor signaling and progressive changes in hepatic pathology, leading to the development of aggressive hepatocellular carcinomas with pulmonary metastases. Liver tumors that arose exhibited markedly elevated levels of phosphatidylinositol (3,4,5)-trisphosphate, along with Akt activation and decreased PTEN expression, at both the mRNA and protein levels. Together, these results substantiate the concept that the p85 subunit of PI3K has a tumor-suppressive role in the liver and possibly other tissues.

Dietary fish oil alleviates soleus atrophy during immobilization in association with Akt signaling to p70s6k and E3 ubiquitin ligases in rats

Reduced muscle activity leads to impaired insulin signaling, which leads to loss of contractile proteins and muscle mass via the Akt pathway. Dietary fish oil rich in long chain n-3 polyunsaturated fatty acids has been shown to prevent insulin signaling resistance in skeletal muscle. This study was conducted to elucidate the protective effect of dietary fish oil on disuse-induced perturbations in insulin signaling and soleus muscle atrophy. To accomplish this, rats were fed a corn-oil- (control) or fish-oil-based diet for 2 weeks, and then subjected to hindlimb immobilization while still receiving the same diets. After 10 days of immobilization, the soleus muscle mass and myosin heavy chain level had markedly decreased; however, these losses were significantly suppressed in rats fed dietary fish oil, compared with the control group. Dietary fish oil nearly completely attenuated the disturbances in activation of the Akt and p70 S6 kinase proteins, as well as the gene expression of muscle-specific E3 ubiquitin ligases (muscle atrophy F-box and muscle RING finger 1). However, insulin receptor substrate 1 associated with the p85 subunit of phosphoinositide 3-kinase was not altered during immobilization. Dietary fish oil also inhibited alterations in the gene expression of cyclooxygenase-2 and inducible nitric oxide synthase, with no additional observation of oxidative stress. Collectively, these findings indicate that dietary fish oil prior to and during immobilization may alleviate the immobilization-induced soleus muscle atrophy, at least in part, via the Akt pathway through E3 ubiquitin ligases and p70s6k.