Phosphoinositide 3-kinase Binding Research Articles

Is the endogenous ligand for PEAR1 a proteoglycan: clues from the sea

Platelet Endothelial Aggregation Receptor 1 (PEAR1) is an orphan receptor of unknown function which mediates powerful activation of platelets and endothelial cells in response to crosslinking by antibodies and sulfated polysaccharides belonging to the dextran and fucoidan families. PEAR1 is a single transmembrane protein composed of 15 epidermal growth factor-like repeat sequences and with a conserved binding motif, YXXM, which when phosphorylated binds to phosphoinositide 3-kinase (PI3K). The 13th of the repeats has a heparin-binding sequence that is the site of interaction with the sulfated fucoidans and the only known endogenous ligand FcεRIα. Crosslinking of PEAR1 drives Src family kinase phosphorylation of the cytosolic tail leading to binding and activation of PI3K. In this Opinion Article, we summarize the literature on PEAR1 expression, structure and signaling, and the search for further endogenous ligands. We highlight one article in which phosphorylation of a 150 kDa platelet protein by heparin-containing ligands has been reported and propose that PEAR1 is a receptor for one or more glycosaminoglycan-conjugated proteins (proteoglycans). The up-regulation of PEAR1 at sites of inflammation in the vasculature and its role in angiogenesis suggests a role in the interplay of inflammation, platelets, coagulation, and thromboinflammation. We speculate that this may explain the link between single nucleotide variants in PEAR1 and cardiovascular disease.

Pharmacological explorations of eco-friendly amide substituted (Z)-β-enaminones as anti-breast cancer drugs.

Amide substituted (Z)-β-enaminones were synthesized by green chemistry and stereo-specific synthetic pathway as novel phosphoinositide 3-kinase (PI3K) inhibitors and breast cancer drugs. PI3K inhibition was measured by competitive ELISA. A panel of cancer cell lines including MCF-7 (breast cancer), G-361 (skin cancer), and HCT 116 (colon cancer) were used to assess the anticancer potentials. In the PI3K assay, 2c and 2f were indolent for the proposed inhibitory action, which was recognized from the obtained IC50 (>1.0 μM). Excellent activity potential of 2a, 2b, and 2d was recognized from the IC50 range (<0.05 μM) whereas an intermediate action potential was observed for compounds 2e and 2i (IC50 0.1 and 0.25, respectively). The docking results exclusively proposed that the hydrophobic interactions in the PI3K binding pocket were overwhelmed by the binding affinity of the most potent ligands (2a, 2b, and 2d: inhibitory constant Ki = 18.16, 84.87, and 56.14 nM). MTT assay results revealed the antiproliferative activity domination of selected compounds (2a, 2b, and 2d) toward MCF-7. The relative activities of 2a, 2b, and 2d of 84.56, 80.87, and 90.12%, respectively, were comparable to that of the standard, doxorubicin (82.16%). SAR studies demonstrated amide substituted (Z)-β-enaminones as precise PI3K inhibitors to treat breast cancer.

Molecular explorations of substituted 2-(4-phenylquinolin-2-yl) phenols as phosphoinositide 3-kinase inhibitors and anticancer agents.

Substituted 2-(4-phenylquinolin-2-yl) phenols (PQPDs) emerged as the inhibitors of phosphoinositide 3-kinase (PI3K) and anticancer agents. PI3K inhibition was assessed by competitive ELISA. Anticancer activity was evaluated against breast cancer (MCF-7), skin cancer (G-361), and colon cancer (HCT 116) cell lines. In PI3 Kinase assay, PQPDs 4c, 4d, and 4k were inactive with IC50>5µM. IC50 for 4a, 4b, 4f-h, and 4j was ≥0.05µM. Rest PQPDs IC50 was <1.0µM. Anticancer activity found selective toward breast cancer (MCF-7); 4a, 4b, and 4j were showed excellent inhibitory (73.95, 68.36, and 70.06%) and IC50 1.16µM (4a), 2.07µM (4b), 1.021µM (4f) and 1.981µM (4j) while the standard (Doxorubicin) found with IC50 1.812µM (72% inhibition). PQPDs were docked into the active site of PI3 Kinase p110α (PDB ID: 2RD0). Docking results suggested the hydrophobic interactions in PI3K binding pocket conquered affinity of the most favorable binding ligands [4a, 4b: inhibitory constant (ki)=53.33, 41.23pM]. PI3K assay and cancer cell line experimental results ensured that the inhibitory and anticancer activity potentials of PQPDs are more selective toward breast cancer treatments. PQPDs 4a, 4b, 4f, 4g, and 4j were displayed potent PI3 Kinase and anticancer activities. SAR studies demonstrated PQPDs as the PI3K precise inhibitors with the impending to treat various cancers.

Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway

In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca2+-protein kinase C (Ca2+-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP2), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca2+-PKC and the PIP2-binding peptide of MARCKS modulate the level of free PIP2, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP2 and thereby inhibits PI3K binding to the membrane. In the on state, Ca2+-PKC phosphorylation of the MARCKS peptide reverses the PIP2 sequestration, thereby releasing multiple PIP2 molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca2+-PKC-stimulated release of free PIP2 may well regulate the membrane association of other PIP2-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.

The Early Stage Formation of PI3K-AMPAR GluR2 Subunit Complex Facilitates the Long Term Neuroprotection Induced by Propofol Post-Conditioning in Rats

Previously, we have shown that the phosphoinositide-3-kinase (PI3K) mediated acute (24 h) post-conditioning neuroprotection induced by propofol. We also found that propofol post-conditioning produced long term neuroprotection and inhibited the internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit up to 28 days post middle cerebral artery occlusion (MCAO). However, the relationship between PI3K with AMPA receptor GluR2 subunit trafficking in propofol post-conditioning has never been explored. Here we showed that propofol post-conditioning promoted the binding of PI3K to the C-terminal of AMPA receptor GluR2 subunit and formed a complex within 1 day after transient MCAO. Interestingly, the enhanced activity of PI3K was observed in the hippocampus of post-conditioning rats at day 1 post ischemia, whereas the decrease of AMPA receptor GluR2 subunit internalization was found up to 28 days in the same group. Administration of PI3K selective antagonist wortmannin inhibited the improvement of spatial learning memory and the increase of neurogenesis in the dentate gyrus up to 28 days post ischemia. It also reversed the inhibition of AMPA receptor GluR2 internalization induced by propofol post-conditioning. Together, our data indicated the critical role of PI3K in regulating the long term neuroprotection induced by propofol post-conditioning. Moreover, this role was established by first day activation of PI3K and formation of PI3K-AMPA receptor GluR2 complex, thus stabilized the structure of postsnaptic AMPA receptor and inhibited the internalization of GluR2 subunit during the early stage of propofol post-conditioning.

Specific inhibition of PI3K p110δ inhibits CSF‐1‐induced macrophage spreading and invasive capacity

Colony stimulating factor-1 (CSF-1) stimulates mononuclear phagocytic cell survival, growth and differentiation into macrophages through activation and autophosphorylation of the CSF-1 receptor (CSF-1R). We have previously demonstrated that CSF-1-induced phosphorylation of Y721 (pY721) in the receptor kinase insert triggers its association with the p85 regulatory subunit of phosphoinositide 3'-kinase (PI3K). Binding of p85 PI3K to the CSF-1R pY721 motif activates the associated p110 PI3K catalytic subunit and stimulates spreading and motility in macrophages and enhancement of tumor cell invasion. Here we show that pY721-based signaling is necessary for CSF-1-stimulated PtdIns(3,4,5)P production. While primary bone marrow-derived macrophages and the immortalized bone marrow-derived macrophage cell line M-/-.WT express all three class IA PI3K isoforms, p110δ predominates in the cell line. Treatment with p110δ-specific inhibitors demonstrates that the hematopoietically enriched isoform, p110δ, mediates CSF-1-regulated spreading and invasion in macrophages. Thus GS-1101, a potent and selective p110δ inhibitor, may have therapeutic potential by targeting the infiltrative capacity of tumor-associated macrophages that is critical for their enhancement of tumor invasion and metastasis.

Tyr740 and Tyr751 residues of platelet-derived growth factor beta receptor are responsible for the redox regulation of phosphatase and tensin homolog in the cells stimulated with platelet-derived growth factor

Exposure of cells to hydrogen peroxide or platelet-derived growth factor (PDGF) induced Akt phosphorylation and oxidation of phosphatase and tensin homolog (PTEN). The Cys124 and Cys71 residues of PTEN were critical for the formation of a disulfide bond and the intermediate glutathionylation in the process of reduction of the disulfide bond. To determine which specific tyrosine residues of the PDGF beta receptor (PDGFβR) is involved in PDGF-induced PTEN oxidation and Akt phosphorylation, we investigated a kinase activity-deficient mutant and PDGFβR mutants where the tyrosine residues in the binding site for phosphoinositide 3-kinase (PI3K), GTPase-activating protein of Ras, Src homology 2 domain containing protein-tyrosine phosphatase-2, and phospholipase C-1 were replaced by Phe. Both PTEN oxidation and Akt phosphorylation did not occur in response to PDGF in the kinase-deficient mutant and in the PDGFβR mutant with a mutation in the PI3K binding site (Tyr740 and Tyr751). Thus, the kinase activity and the constituent Tyr740 and Tyr751 residues of PDGFβR in the cells stimulated with PDGF are responsible for the oxidation of PTEN and the Akt phosphorylation.

G protein–independent Ras/PI3K/F-actin circuit regulates basic cell motility

Phosphoinositide 3-kinase (PI3K)γ and Dictyostelium PI3K are activated via G protein–coupled receptors through binding to the Gβγ subunit and Ras. However, the mechanistic role(s) of Gβγ and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gβ null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P3. Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras–guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.

Phosphoinositide 3-kinase is activated by MUC1 but not responsible for MUC1-induced suppression of Toll-like receptor 5 signaling

MUC1 is a membrane-tethered mucin-like glycoprotein expressed on the surface of various mucosal epithelial cells as well as hematopoietic cells. Recently, we showed that MUC1 suppresses flagellin-induced Toll-like receptor (TLR) 5 signaling both in vivo and in vitro through cross talk with TLR5. In this study, we determined whether phosphoinositide 3-kinase (PI3K), a negative regulator of TLR5 signaling, is involved in the cross talk between MUC1 and TLR5 using various genetically modified epithelial cell lines. Our results showed 1) activation of MUC1 induced recruitment of the PI3K regulatory subunit p85 to the MUC1 cytoplasmic tail (CT) as well as Akt phosphorylation, 2) MUC1-induced Akt phosphorylation required the presence of Tyr(20) within the PI3K binding motif of the MUC1 CT, and 3) mutation of Tyr(20) or pharmacological inhibition of PI3K activation failed to block MUC1-induced suppression of TLR5 signaling. We conclude that whereas PI3K is downstream of MUC1 activation and negatively regulates TLR5 signaling, it is not responsible for MUC1-induced suppression of TLR5 signaling.

Phase Transition as a Gradient Amplifier

Chemotactic cells can detect and move toward a shallow gradient of chemoattractant. This directional movement involves the localized synthesis of membrane lipids, specifically phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), which is formed in response to recruitment of phosphoinositide 3-kinase (PI3K) at the site of receptor activation. PIP 3 is in turn metabolized to PIP 2 by the phosphatase PTEN. Gamba et al . performed a stochastic simulation with four components: (i) binding of PI3K to activated receptors, (ii) binding of PTEN to PIP 2 , (iii) catalytic activity of PI3K and PTEN, and (iv) diffusion of phosphoinositides in the membrane. The model detected phase separation of the phosphoinositides PIP 2 and PIP 3 within the membrane. When a uniform stimulus was applied, the phosphoinositides slowly separated into PIP 2 -rich and PIP 3 -rich phases, and the rate of separation was dependent on the concentration of activated receptors and the diffusion of the lipids. When a shallow gradient (5% gradient of activated receptors over the length of the simulated membrane) of stimulus was applied, a much more rapid separation of the lipids was observed, and the PIP 3 -rich phase accumulated on the side with the higher concentration of activated receptors. The authors state that the results can be explained by the selective recruitment of PTEN to its product PIP 2 , which serves as a positive feedback mechanism that drives the system toward phase separation. The net effect is amplification of a shallow signal. A. Gamba, A. de Candia, S. Di Talila, A. Coniglio, F. Bussolino, G. Serini, Diffusion-limited phase separation in eukaryotic chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 102 , 16927-16932 (2005). [Abstract] [Full Text]

The 14-3-3 Protein Translates the NA+,K+-ATPase α1-Subunit Phosphorylation Signal into Binding and Activation of Phosphoinositide 3-Kinase during Endocytosis

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na(+),K(+)-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the alpha-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na(+),K(+)-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the alpha-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na(+),K(+)-ATPase endocytosis.

Two Complementary, Local Excitation, Global Inhibition Mechanisms Acting in Parallel Can Explain the Chemoattractant-Induced Regulation of PI(3,4,5)P 3 Response in Dictyostelium Cells

Chemotaxing cells, such as Dictyostelium and mammalian neutrophils, sense shallow chemoattractant gradients and respond with highly polarized changes in cell morphology and motility. Uniform chemoattractant stimulation induces the transient translocations of several downstream signaling components, including phosphoinositide 3-kinase (PI3K), tensin homology protein (PTEN), and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3). In contrast, static spatial chemoattractant gradients elicit the persistent, amplified localization of these molecules. We have proposed a model in which the response to chemoattractant is regulated by a balance of a local excitation and a global inhibition, both of which are controlled by receptor occupancy. This model can account for both the transient and spatial responses to chemoattractants, but alone does not amplify the external gradient. In this article, we develop a model in which parallel local excitation, global inhibition mechanisms control the membrane binding of PI3K and PTEN. Together, the action of these enzymes induces an amplified PI(3,4,5)P 3 response that agrees quantitatively with experimentally obtained plekstrin homology-green fluorescent protein distributions in latrunculin-treated cells. We compare the model’s performance with that of several mutants in which one or both of the enzymes are disrupted. The model accounts for the observed response to multiple, simultaneous chemoattractant cues and can recreate the cellular response to combinations of temporal and spatial stimuli. Finally, we use the model to predict the response of a cell where only a fraction is stimulated by a saturating dose of chemoattractant.

Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor.

The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.

The SH2 Domain-containing Inositol 5′-Phosphatase (SHIP) Recruits the p85 Subunit of Phosphoinositide 3-Kinase during FcγRIIb1-mediated Inhibition of B Cell Receptor Signaling

Coligation of FcgammaRIIb1 with the B cell receptor (BCR) or FcepsilonRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4, 5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcgammaRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcgammaRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcgammaRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcgammaRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis.

Mechanism of insulin action--signal transductions after binding to insulin receptor

Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit and tyrosine-phosphorylates the insulin receptor substrates-1 (IRS-1). Insulin promotes the formation of a complex between tyrosine-phosphorylated IRS-1 and several proteins including phosphoinositide(PI) 3-kinase, a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits, GRB2 and Syp via the Src homology region 2 (SH2) domains. Recently, it was suggested that GRB2-Sos complex binding to IRS-1 was linked to Ras activation and that PI 3-kinase binding to IRS-1 was linked to activation of glucose transport. Since the mechanism of insulin-stimulated glucose uptake is mainly due to translocation of glucose transporters from an intracellular vesicle pool to the plasma membrane, PI 3-kinase activity may be involved in vesicle transport in mammalian cells.