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Alfonso Bellacosa - One of the best experts on this subject based on the ideXlab platform.

Lionel Larue - One of the best experts on this subject based on the ideXlab platform.

Otilia V Vieira - One of the best experts on this subject based on the ideXlab platform.

  • distinct roles of class i and class iii Phosphatidylinositol 3 kinases in phagosome formation and maturation
    Journal of Cell Biology, 2001
    Co-Authors: Tsuyoshi Matsuo, Otilia V Vieira, Roberto J Botelho, Lucia E Rameh, Saskia M Brachmann, Howard W Davidson, Alan D Schreiber, Jonathan M Backer
    Abstract:

    Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of Phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that Phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3′-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

Harald Alfred Stenmark - One of the best experts on this subject based on the ideXlab platform.

  • Class III Phosphatidylinositol 3–kinase and its catalytic product PtdIns3P in regulation of endocytic membrane traffic
    FEBS Journal, 2013
    Co-Authors: Camilla Raiborg, Kay Oliver Schink, Harald Alfred Stenmark
    Abstract:

    Endocytosis and subsequent membrane traffic through endosomes are cellular processes that are integral to eukaryotic evolution, and numerous human diseases are associated with their dysfunction. Consequently, it is important to untangle the molecular machineries that regulate membrane dynamics and protein flow in the endocytic pathway. Central in this context is class III Phosphatidylinositol 3–kinase, an evolutionarily conserved enzyme complex that phosphorylates Phosphatidylinositol into Phosphatidylinositol 3–phosphate. Phosphatidylinositol 3–phosphate recruits specific effector proteins, most of which contain FYVE or PX domains, to promote endocytosis, endosome fusion, endosome motility and endosome maturation, as well as cargo sorting to lysosomes, the biosynthetic pathway or the plasma membrane. Here we review the functions of key Phosphatidylinositol 3–phosphate effectors in regulation of endocytic membrane dynamics and protein sorting.

  • Phosphatidylinositol 3-phosphate is found in microdomains of early endosomes
    Histochemistry and Cell Biology, 2003
    Co-Authors: David J. Gillooly, Camilla Raiborg, Harald Alfred Stenmark
    Abstract:

    Phosphatidylinositol 3-phosphate [PI(3)P] is a Phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P2, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling.

  • The Phosphatidylinositol 3-phosphate-binding FYVE finger
    FEBS Letters, 2002
    Co-Authors: Harald Alfred Stenmark, Rein Aasland, Paul C. Driscoll
    Abstract:

    The FYVE zinc finger domain is conserved from yeast (five proteins) to man (27 proteins). It functions in the membrane recruitment of cytosolic proteins by binding to Phosphatidylinositol 3-phosphate (PI3P), which is found mainly on endosomes. Here we review recent work that sheds light on the targeting of FYVE finger proteins to PI3P-containing membranes, and how these proteins serve to regulate multiple cellular functions.

  • Intracellular trafficking and turnover of Phosphatidylinositol 3-phosphate
    Seminars in Cell & Developmental Biology, 2001
    Co-Authors: Harald Alfred Stenmark, David J. Gillooly
    Abstract:

    Abstract Phosphatidylinositol 3-kinases (PI 3-kinases) regulate cellular functions through the 3′ -phosphorylation of Phosphatidylinositol (PI) and its derivatives. The PI 3-kinase product Phosphatidylinositol 3-phosphate [PI(3)P] functions to recruit and activate effector proteins containing FYVE zinc finger domains. These proteins have various functions in endocytic membrane trafficking, cytoskeletal regulation and signal transduction. In order to understand the function of FYVE proteins, it is essential to study the formation, localisation, trafficking and turnover of PI(3)P. Here we review recent evidence that PI(3)P is formed on early endosomes through the activity of a PI 3-kinase which is recruited by the GTPase Rab5, and that the PI(3)P is subsequently internalised into intralumenal vesicles of multivesicular endosomes for turnover.

  • localization of Phosphatidylinositol 3 phosphate in yeast and mammalian cells
    The EMBO Journal, 2000
    Co-Authors: David J. Gillooly, Isabel C. Morrow, Robert G. Parton, Margaret R Lindsay, R J Gould, Nia J Bryant, Jean Michel Gaullier, Harald Alfred Stenmark
    Abstract:

    Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, Phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Δ yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

Alfred J. Meijer - One of the best experts on this subject based on the ideXlab platform.

  • Vps34 is a Phosphatidylinositol 3-kinase, not a phosphoinositide 3-kinase.
    Autophagy, 2011
    Co-Authors: Alfred J. Meijer, Daniel J. Klionsky
    Abstract:

    There seems to be some confusion regarding the naming of enzymes that phosphorylate certain lipids. In particular, we are referring to the lipid Phosphatidylinositol and to phosphoinositides. Inositides are inositol-containing derivatives of phosphatidic acid (Fig. 1). The term phosphoinositide indicates that one or more of the hydroxyl groups of Phosphatidylinositol is esterified with inorganic phosphate. Phosphatidylinositol is an inositide, but it is not a phosphoinositide, whereas Phosphatidylinositol 3-phosphate (Phosphatidylinositol that is phosphorylated on the three hydroxyl group of inositol) is a phosphoinositide (Fig. 1). In addition to phophatidylinositol 3-phosphate, the lipids Phosphatidylinositol 3,4-bisphosphate, Phosphatidylinositol 3,5-bisphosphate and Phosphatidylinositol 3,4,5-trisphosphate are all phosphoinositides. Figure 1. Chemical structures of inositol, phosphatidic acid, Phosphatidylinositol (an inositide, but not a phosphoinositide) and Phosphatidylinositol 3-phosphate (a phosphoinositide). There is a single enzyme that converts Phosphatidylinositol to Phosphatidylinositol 3-phosphate, and that is Vps34. Furthermore, Vps34 only phosphorylates Phosphatidylinositol, and does not act on other substrates. Thus, Vps34 is not a phosphoinositide kinase, but rather is a Phosphatidylinositol kinase that phosphorylates the three hydroxyl group, making it in particular a Phosphatidylinositol 3-kinase. In contrast, Vps34 is not a phosphoinositide 3-kinase, because its substrate, Phosphatidylinositol, is not a phosphoinositide. At this point it might be worth considering, albeit briefly, the origin of these terms. That is, the ending “-ide” refers to a chemical compound derived from or related to another such compound, or indicating one of a class of compounds. Thus, a phosphoinositide is related to Phosphatidylinositol, and Phosphatidylinositol 3 phosphate is one of a class of phosphoinositides. The suffix “-ol” is used in the names of chemical derivatives representing alcohol (inositol is a sugar alcohol; Fig. 1). When you see the abbreviation “PI3K” it is not clear whether the author is referring to a phosphoinositide 3-kinase or a Phosphatidylinositol 3-kinase. In contrast, “PtdIns3K” makes it quite clear that the enzyme is acting upon Phosphatidylinositol. Perhaps we are being obsessive, but when it comes to Vps34 we think people should refer to it as a Phosphatidylinositol 3-kinase because they are discussing the generation of Phosphatidylinositol 3-phosphate or PtdIns(3)P, which is one of the lipids that we tend to be most concerned about with regard to macroautophagy. This is the reason that Autophagy uses the abbreviation “PtdIns3K” as the standard for the enzyme complex containing Vps34. Furthermore, you will not go wrong if you describe a phosphoinositide kinase such as Fab1 as a Phosphatidylinositol 3-phosphate 5-kinase (PtdIns(3)P 5-kinase), for example, whereas you run the risk of incorrectly referring to an enzyme such as Vps34 as a phosphoinositide kinase. There is one additional issue, and that concerns the distinction between the different classes of Phosphatidylinositol kinases, and in particular the class I and class III enzymes. In this case, many people refer to the PI3KC3, which is a short and handy name, except when you consider that the official gene name in humans is “PIK3C3,” with the “3” and the “K” transposed, and both of these suffer from the problem of not making it clear whether we are really referring to a Phosphatidylinositol 3-kinase or a phosphoinositide 3-kinase. Again, the class III enzyme that generates Phosphatidylinositol 3-phosphate is not a phosphoinositide 3-kinase. In contrast, the enzyme that phosphorylates PtdIns(4,5)P2 to PtdIns(3,4,5)P3 can be correctly referred to as a phosphoinositide-3-kinase, but the substrate specificity is still not indicated. Therefore, Autophagy will use “PtdIns3KC3” to refer to the class III enzyme Vps34 when necessary, as this makes it clear that the substrate is Phosphatidylinositol 3-phosphate. We certainly hope this clears up any confusion.

  • the Phosphatidylinositol 3 kinase inhibitors wortmannin and ly294002 inhibit autophagy in isolated rat hepatocytes
    FEBS Journal, 1997
    Co-Authors: E F C Blommaart, Ulrike Krause, Jacques P M Schellens, Heleen Vreelingsindelarova, Alfred J. Meijer
    Abstract:

    Recent studies indicate that Phosphatidylinositol 3-kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether Phosphatidylinositol 3-kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated Phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenylchromone (LY294002). The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC50 30 nM) and LY294002 (IC50 10 mu M) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids. 3-Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of Phosphatidylinositol 3-kinase, thus providing a target for its action. It is proposed that Phosphatidylinositol 3-kinase activity is required for autophagy. 3-Methyladenine inhibits autophagy by inhibition of this enzyme.