Molecular Brightness Analysis Reveals Phosphatidylinositol 4-Kinase IIβ Association with Clathrin-Coated Vesicles in Living Cells

Mammalian cells express two classes of phosphatidylinositol 4-kinase (PI4K), designated as Types II and III, that phosphorylate phosphatidylinositol to generate PI4P. A number of studies have indicated that these enzymes are important for Golgi trafficking and both early and late stages of endocytosis. In this study, we focus on PI4KIIβ, a protein that is evenly distributed between membrane and soluble fractions, and is believed to participate in stimulus-dependent phosphoinositide signaling. Using molecular brightness analysis, we found that EGFP-tagged PI4KIIβ exists as two distinct species in the cytoplasm: a soluble monomer and a high-order complex enriched with multiple copies of PI4KIIβ. This observation was confirmed by an autocorrelation analysis that identified two species with distinct mobilities. We further demonstrate that the high-order complex enriched with PI4KIIβ is sensitive to inhibition of palmitoylation, indicating that it is associated with membranes, very likely vesicles. Indeed, we show that the high-order PI4KIIβ complex is sensitive to expression of dynamin 2 (K44A), a dominant-negative inhibitor of endocytosis. Using dual-color heterospecies partition analysis, we directly detected that PI4KIIβ comoves with clathrin light chain on vesicles. This analysis allows us to isolate the comobile species in the presence of strong background contribution from the monomeric pool of PI4KIIβ. Our results strongly suggest that PI4KIIβ is involved in an early stage of endocytosis and is associated with clathrin-coated vesicles. Moreover, we establish molecular brightness as a powerful tool for characterizing cellular cytosolic vesicles that are otherwise difficult to characterize by other techniques.     

A New Dimension to Ras Function: A Novel Role for Nucleotide-Free Ras in Class II Phosphatidylinositol 3-Kinase Beta (PI3KC2β) Regulation

The intersectin 1 (ITSN1) scaffold stimulates Ras activation on endocytic vesicles without activating classic Ras effectors. The identification of Class II phosphatidylinositol 3-kinase beta, PI3KC2β, as an ITSN1 target on vesicles and the presence of a Ras binding domain (RBD) in PI3KC2β suggests a role for Ras in PI3KC2β activation. Here, we demonstrate that nucleotide-free Ras negatively regulates PI3KC2β activity. PI3KC2β preferentially interacts in vivo with dominant-negative (DN) Ras, which possesses a low affinity for nucleotides. PI3KC2β interaction with DN Ras is disrupted by switch 1 domain mutations in Ras as well as RBD mutations in PI3KC2β. Using purified proteins, we demonstrate that the PI3KC2β-RBD directly binds nucleotide-free Ras in vitro and that this interaction is not disrupted by nucleotide addition. Finally, nucleotide-free Ras but not GTP-loaded Ras inhibits PI3KC2β lipid kinase activity in vitro. Our findings indicate that PI3KC2β interacts with and is regulated by nucleotide-free Ras. These data suggest a novel role for nucleotide-free Ras in cell signaling in which PI3KC2β stabilizes nucleotide-free Ras and that interaction of Ras and PI3KC2β mutually inhibit one another.     

Class IA Phosphatidylinositol 3-Kinase p110α Regulates Phagosome Maturation

Of the various phosphatidylinositol 3- kinases (PI3Ks), only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110α, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110α and contained PI3P and PI(3,4,5)P3; however, p110α and PI(3,4,5)P3 levels in phagosomes from p110α knockdown cells were decreased. Phagosomes from p110α knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, β-galactosidase. Phagosomes from p110α deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110α deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP) and homotypic vacuole fusion and protein sorting (HOPs) components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K.     

Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P₁ and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ.     

Involvement of Class II Phosphoinositide 3-Kinase α-Isoform in Antigen-Induced Degranulation in RBL-2H3 Cells

In this study, we present findings that suggest that PI3K-C2α, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of FcεRI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2α. The knockdown impaired the FcεRI-induced release of a lysosome enzyme, β-hexosaminidase, without affecting the intracellular Ca²⁺ mobilization. The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells. The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α. In wild-type cells, FcεRI stimulation induced the formation of large vesicles, which were associated with CD63, a marker protein of secretory granules. On the vesicles, the existence of PI3K-C2α and PtdIns(3,4)P₂ was observed. These results indicated that PI3K-C2α and its product PtdIns(3,4)P₂ may play roles in the secretory process.     

Discovery of a Small Molecule Agonist of Phosphatidylinositol 3-Kinase p110α That Reactivates Latent HIV-1

Combination antiretroviral therapy (cART) can effectively suppress HIV-1 replication, but the latent viral reservoir in resting memory CD4⁺ T cells is impervious to cART and represents a major barrier to curing HIV-1 infection. Reactivation of latent HIV-1 represents a possible strategy for elimination of this reservoir. In this study we describe the discovery of 1,2,9,10-tetramethoxy-7H-dibenzo[de,g]quinolin-7-one (57704) which reactivates latent HIV-1 in several cell-line models of latency (J89GFP, U1 and ACH-2). 57704 also increased HIV-1 expression in 3 of 4 CD8⁺-depleted blood mononuclear cell preparations isolated from HIV-1-infected individuals on suppressive cART. In contrast, vorinostat increased HIV-1 expression in only 1 of the 4 donors tested. Importantly, 57704 does not induce global T cell activation. Mechanistic studies revealed that 57704 reactivates latent HIV-1 via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. 57704 was found to be an agonist of PI3K with specificity to the p110α isoform, but not the p110β, δ or γ isoforms. Taken together, our work suggests that 57704 could serve as a scaffold for the development of more potent activators of latent HIV-1. Furthermore, it highlights the involvement of the PI3K/Akt pathway in the maintenance of HIV-1 latency.     

Phosphatidylinositol 3-Kinase γ Is Required for the Development of Experimental Cerebral Malaria

Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ^-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by Plasmodium berghei ANKA (PbA) infection. Infected PI3Kγ^-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ^-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3⁺CD8⁺ T cells were significantly reduced in the brain of infected PI3Kγ^-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ^-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.     

Structural,Biochemical, and Biophysical Characterization of Idelalisib Binding to Phosphoinositide 3-Kinase δ

Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.     

Molecular Basis for Membrane Recruitment by the PX and C2 Domains of Class II Phosphoinositide 3-Kinase-C2α

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LRP1 Regulates Architecture of the Vascular Wall by Controlling PDGFRβ-Dependent Phosphatidylinositol 3-Kinase Activation

Background

Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the activation of platelet-derived growth factor receptor β (PDGFRβ) in vascular smooth muscle cells (SMCs). Activated PDGFRβ undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphatidylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin polymerization and cell movement.

Methods and Principal Findings

In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1−/−) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor β (TGFβ) signaling are present in smooth muscle-specific LRP1 knockout (smLRP1−/−) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRβ in atherogenesis, we generated a strain of smLRP1−/− mice in which tyrosine 739/750 of the PDGFRβ had been mutated to phenylalanines (PDGFRβ F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP1−/− animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFβ signaling, as indicated by high levels of nuclear phospho-Smad2.

Conclusions and Significance

Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRβ-dependent activation of PI3K. TGFβ activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRβ is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome.     

Metabolism of Phosphatidylinositol 4-Kinase III��-Dependent PI4P Is Subverted by HCV and Is Targeted by a 4-Anilino Quinazoline with Antiviral Activity

4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIβ. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIβ isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4-anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV – by recruiting PI4KIIIα in the RNA replication complex – hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIα product.     

The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K+ Channel KCa3.1 and CD4 T-Cells

The Ca²⁺-activated K⁺ channel KCa3.1 is required for Ca²⁺ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca²⁺ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.     

Bipartite Nuclear Localization Signals in the C Terminus of Human Topoisomerase IIα

DNA topoisomerase IIα is the intracellular target for several important chemotherapeutic agents, and drug-resistant human tumor cell lines have been described in which deletions in the C-proximal region of this enzyme are associated with its cytoplasmic localization. We have identified multiple potential bipartite nuclear localization signal (NLS) sequences in this region using a modified definition of the motif, and in the present study, we have expressed five of these as fusion proteins with β-galactosidase. Only one sequence (spanning amino acids 1454 to 1497) was sufficient to cause strong nuclear localization. Subsequent mutation analyses indicated that this NLS sequence was bipartite and that both domains contain more than two basic amino acids. Substitution of the lysine residue at position 1492 in the second basic domain with glutamine resulted in a fusion protein that localized inefficiently to the nucleus, indicating that all three basic residues in this domain are necessary. Our results confirm that a broader definition is required to detect all potential bipartite NLS motifs in a polypeptide sequence, although functional tests are still essential for identification of those sequences actually capable of directing nuclear localization.     

Oncogenic Mutations of p110α Isoform of PI 3-Kinase Upregulate Its Protein Kinase Activity

In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 βc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 βc receptor (βic). We find p110α, p110β and p110γ all phosphorylate βic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards βic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn²⁺ dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110β and p110γ all display significant activity in the presence of Mg²⁺. Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.     

PI3K Class II α Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function

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