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Richard A. Anderson - One of the best experts on this subject based on the ideXlab platform.

  • phosphoinositide 3 kinase pathways and autophagy require Phosphatidylinositol Phosphate kinases
    Advances in biological regulation, 2018
    Co-Authors: Suyong Choi, Xander Houdek, Richard A. Anderson
    Abstract:

    Abstract Phosphatidylinositol Phosphate kinases (PIPKs) generate a lipid messenger Phosphatidylinositol 4,5-bisPhosphate (PI4,5P2) that controls essentially all aspects of cellular functions. PI4,5P2 rapidly diffuses in the membrane of the lipid bilayer and does not greatly change in membrane or cellular content, and thus PI4,5P2 generation by PIPKs is tightly linked to its usage in subcellular compartments. Based on this verity, recent study of PI4,5P2 signal transduction has been focused on investigations of individual PIPKs and their underlying molecular regulation of cellular processes. Here, we will discuss recent advances in the study of how PIPKs control specific cellular events through assembly and regulation of PI4,5P2 effectors that mediate specific cellular processes. A focus will be on the roles of PIPKs in control of the phosphoinositide 3-kinase pathway and autophagy.

  • Phosphatidylinositol Phosphate 5 kinase iγ and phosphoinositide 3 kinase akt signaling couple to promote oncogenic growth
    Journal of Biological Chemistry, 2015
    Co-Authors: Narendra Thapa, Suyong Choi, Xiaojun Tan, T Wise, Richard A. Anderson
    Abstract:

    The assembly of signaling complexes at the plasma membrane is required for the initiation and propagation of cellular signaling upon cell activation. The class I PI3K and the serine/threonine-specific protein kinase Akt signaling pathways (PI3K/Akt) are often activated in tumors. These pathways are initiated by the generation of Phosphatidylinositol 3,4,5-triPhosphate (PIP3) by PI3K-mediated phosphorylation of Phosphatidylinositol 4,5-biPhosphate (PIP2), synthesized by Phosphatidylinositol 4-Phosphate 5-kinase (PIPKI) enzymes. The mechanism of how tumor cells recruit and organize the PIP2-synthesizing enzymes with PI3K in the plasma membrane for activation of PI3K/Akt signaling is not defined. Here, we demonstrated a role for the Phosphatidylinositol 4-Phosphate 5-kinase Iγ (PIPKIγ) in PI3K/Akt signaling. PIPKIγ is overexpressed in triple-negative breast cancers. Loss of PIPKIγ or its focal adhesion-targeting variant, PIPKIγi2, impaired PI3K/Akt activation upon stimulation with growth factors or extracellular matrix proteins in different tumor cells. PIPKIγi2 assembles into a complex containing Src and PI3K; Src was required for the recruitment of PI3K enzyme into the complex. PIPKIγi2 interaction with Src and its lipid kinase activity were required for promoting PI3K/Akt signaling. These results define a mechanism by which PIPKIγi2 and PI3K are integrated into a complex regulated by Src, resulting in the spatial generation of PIP2, which is the substrate PI3K required for PIP3 generation and subsequent Akt activation. This study elucidates the mechanism by which PIP2-generating enzyme controls Akt activation upstream of a PI3K enzyme. This pathway may represent a signaling nexus required for the survival and growth of metastasizing and circulating tumor cells in vivo.

  • Phosphatidylinositol Phosphate 5-kinase Iγi2 in association with Src controls anchorage-independent growth of tumor cells.
    Journal of Biological Chemistry, 2013
    Co-Authors: Narendra Thapa, Suyong Choi, Andrew C. Hedman, Xiaojun Tan, Richard A. Anderson
    Abstract:

    A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for Phosphatidylinositol 4-Phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.

  • pip kinases from the cell membrane to the nucleus
    Sub-cellular biochemistry, 2012
    Co-Authors: Mark Schramp, Andrew C. Hedman, Xiaojun Tan, Richard A. Anderson
    Abstract:

    Phosphatidylinositol 4,5-bisPhosphate (PIP2) is a membrane bound lipid molecule with capabilities to affect a wide array of signaling pathways to regulate very different cellular processes. PIP2 is used as a precursor to generate the second messengers PIP3, DAG and IP3, indispensable molecules for signaling events generated by membrane receptors. However, PIP2 can also directly regulate a vast array of proteins and is emerging as a crucial messenger with the potential to distinctly modulate biological processes critical for both normal and pathogenic cell physiology. PIP2 directly associates with effector proteins via unique phosphoinositide binding domains, altering their localization and/or enzymatic activity. The spatial and temporal generation of PIP2 synthesized by the Phosphatidylinositol Phosphate kinases (PIPKs) tightly regulates the activation of receptor signaling pathways, endocytosis and vesicle trafficking, cell polarity, focal adhesion dynamics, actin assembly and 3’ mRNA processing. Here we discuss our current understanding of PIPKs in the regulation of cellular processes from the plasma membrane to the nucleus.

  • an electrostatic switch displaces Phosphatidylinositol Phosphate kinases from the membrane during phagocytosis
    Journal of Cell Biology, 2009
    Co-Authors: Gregory D Fairn, Richard A. Anderson, Koji Ogata, Roberto J Botelho, Philip D Stahl, Pietro De Camilli, Tobias Meyer, Shoshana J Wodak, Sergio Grinstein
    Abstract:

    Plasmalemmal Phosphatidylinositol (PI) 4,5-bisPhosphate (PI4,5P2) synthesized by PI 4-Phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-γ prevented the disappearance of PI4,5P2 and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.

Mark S P Sansom - One of the best experts on this subject based on the ideXlab platform.

  • Coarse-Grained Simulations Suggest the Epsin N-Terminal Homology Domain Can Sense Membrane Curvature without Its Terminal Amphipathic Helix.
    ACS nano, 2020
    Co-Authors: Mark S P Sansom, Alexis Belessiotis-richards, Stuart G. Higgins, Molly M. Stevens, Alfredo Alexander-katz
    Abstract:

    Nanoscale membrane curvature is a common feature in cell biology required for functions such as endocytosis, exocytosis and cell migration. These processes require the cytoskeleton to exert forces on the membrane to deform it. Cytosolic proteins contain specific motifs which bind to the membrane, connecting it to the internal cytoskeletal machinery. These motifs often bind charged Phosphatidylinositol Phosphate lipids present in the cell membrane which play significant roles in signaling. These lipids are important for membrane deforming processes, such as endocytosis, but much remains unknown about their role in the sensing of membrane nanocurvature by protein domains. Using coarse-grained molecular dynamics simulations, we investigated the interaction of a model curvature active protein domain, the epsin N-terminal homology domain (ENTH), with curved lipid membranes. The combination of anionic lipids (Phosphatidylinositol 4,5-bisPhosphate and phosphatidylserine) within the membrane, protein backbone flexibility, and structural changes within the domain were found to affect the domain's ability to sense, bind, and localize with nanoscale precision at curved membrane regions. The findings suggest that the ENTH domain can sense membrane curvature without the presence of its terminal amphipathic α helix via another structural region we have denoted as H3, re-emphasizing the critical relationship between nanoscale membrane curvature and protein function.

  • modes of interaction of pleckstrin homology domains with membranes toward a computational biochemistry of membrane recognition
    Journal of Molecular Biology, 2018
    Co-Authors: Fiona B Naughton, Antreas C Kalli, Mark S P Sansom
    Abstract:

    Pleckstrin homology (PH) domains mediate protein-membrane interactions by binding to Phosphatidylinositol Phosphate (PIP) molecules. The structural and energetic basis of selective PH-PIP interactions is central to understanding many cellular processes, yet the molecular complexities of the PH-PIP interactions are largely unknown. Molecular dynamics simulations using a coarse-grained model enables estimation of free-energy landscapes for the interactions of 12 different PH domains with membranes containing PIP2 or PIP3, allowing us to obtain a detailed molecular energetic understanding of the complexities of the interactions of the PH domains with PIP molecules in membranes. Distinct binding modes, corresponding to different distributions of cationic residues on the PH domain, were observed, involving PIP interactions at either the "canonical" (C) and/or "alternate" (A) sites. PH domains can be grouped by the relative strength of their C- and A-site interactions, revealing that a higher affinity correlates with increased C-site interactions. These simulations demonstrate that simultaneous binding of multiple PIP molecules by PH domains contributes to high-affinity membrane interactions, informing our understanding of membrane recognition by PH domains in vivo.

  • interactions of pleckstrin homology domains with membranes adding back the bilayer via high throughput molecular dynamics
    Structure, 2016
    Co-Authors: Eiji Yamamoto, Antreas C Kalli, Kenji Yasuoka, Mark S P Sansom
    Abstract:

    A molecular simulation pipeline for determining the mode of interaction of pleckstrin homology (PH) domains with Phosphatidylinositol Phosphate (PIP)-containing lipid bilayers is presented. We evaluate our methodology for the GRP1 PH domain via comparison with structural and biophysical data. Coarse-grained simulations yield a 2D density landscape for PH/membrane interactions alongside residue contact profiles. Predictions of the membrane localization and interactions of 13 PH domains reveal canonical, non-canonical, and dual PIP-binding sites on the proteins. Thus, the PH domains associate with the PIP molecules in the membrane via a highly positively charged loop. Some PH domains exhibit modes of interaction with PIP-containing membranes additional to this canonical binding mode. All 13 PH domains cause a degree of local clustering of PIP molecules upon binding to the membrane. This provides a global picture of PH domain interactions with membranes. The high-throughput approach could be extended to other families of peripheral membrane proteins.

  • association of peripheral membrane proteins with membranes free energy of binding of grp1 ph domain with Phosphatidylinositol Phosphate containing model bilayers
    Journal of Physical Chemistry Letters, 2016
    Co-Authors: Fiona B Naughton, Antreas C Kalli, Mark S P Sansom
    Abstract:

    Understanding the energetics of peripheral protein–membrane interactions is important to many areas of biophysical chemistry and cell biology. Estimating free-energy landscapes by molecular dynamics (MD) simulation is challenging for such systems, especially when membrane recognition involves complex lipids, e.g., Phosphatidylinositol Phosphates (PIPs). We combined coarse-grained MD simulations with umbrella sampling to quantify the binding of the well-explored GRP1 pleckstrin homology (PH) domain to model membranes containing PIP molecules. The experimentally observed preference of GRP1-PH for PIP3 over PIP2 was reproduced. Mutation of a key residue (K273A) within the canonical PIP-binding site significantly reduced the free energy of PIP binding. The presence of a noncanonical PIP-interaction site, observed experimentally in other PH domains but not previously in GRP1-PH, was also revealed. These studies demonstrate how combining coarse-grained simulations and umbrella sampling can unmask the molecular ...

  • interactions of phosphatase and tensin homologue pten proteins with Phosphatidylinositol Phosphates insights from molecular dynamics simulations of pten and voltage sensitive phosphatase
    Biochemistry, 2014
    Co-Authors: Antreas C Kalli, Isabel Devaney, Mark S P Sansom
    Abstract:

    The phosphatase and tensin homologue (PTEN) and the Ciona intestinalis voltage sensitive phosphatase (Ci-VSP) are both Phosphatidylinositol Phosphate (PIP) phosphatases that contain a C2 domain. PTEN is a tumor suppressor protein that acts as a phosphatase on PIP3 in mammalian cell membranes. It contains two principal domains: a phosphatase domain (PD) and a C2 domain. Despite detailed structural and functional characterization, less is known about its mechanism of interaction with PIP-containing lipid bilayers. Ci-VSP consists of an N-terminal transmembrane voltage sensor domain and a C-terminal PTEN domain, which in turn contains a PD and a C2 domain. The nature of the interaction of the PTEN domain of Ci-VSP with membranes has not been well established. We have used multiscale molecular dynamics simulations to define the interaction mechanisms of PTEN and of the Ci-VSP PTEN domains with PIP-containing lipid bilayers. Our results suggest a novel mechanism of association of the PTEN with such bilayers, i...

Wendy F Boss - One of the best experts on this subject based on the ideXlab platform.

  • the n terminal membrane occupation and recognition nexus domain of arabidopsis Phosphatidylinositol Phosphate kinase 1 regulates enzyme activity
    Journal of Biological Chemistry, 2007
    Co-Authors: Yang Ju Im, Imara Y Perera, Amanda J Davis, Eva Johannes, Nina S Allen, Wendy F Boss
    Abstract:

    Abstract The type I B family of Phosphatidylinositol Phosphate kinases (PIPKs) contain a characteristic region of Membrane Occupation and Recognition Nexus (MORN) motifs at the N terminus. These MORN motifs are not found in PIPKs from other eukaryotes. To understand the impact of the additional N-terminal domain on protein function and subcellular distribution, we expressed truncated and full-length versions of AtPIPK1, one member of this family of PIPKs, in Escherichia coli and in tobacco cells grown in suspension culture. Deletion of the N-terminal MORN domain (amino acids 1–251) of AtPIPK1 increased the specific activity of the remaining C-terminal peptide (ΔMORN) >4-fold and eliminated activation by phosphatidic acid (PtdOH). PtdOH activation could also be eliminated by mutating Pro396 to Ala (P396A) in the predicted linker region between the MORN and the kinase homology domains. AtPIPK1 is product-activated and the MORN domain binds PtdIns(4,5)P2. Adding back the MORN peptide to ΔMORN or to the PtdOH-activated full-length protein increased activity ∼2-fold. Furthermore, expressing the MORN domain in vivo increased the plasma membrane PtdInsP kinase activity. When cells were exposed to hyperosmotic stress, the MORN peptide redistributed from the plasma membrane to a lower phase or endomembrane fraction. In addition, endogenous PtdInsP kinase activity increased in the endomembrane fraction of hyperosmotically stressed cells. We conclude that the MORN peptide can regulate both the function and distribution of the enzyme in a manner that is sensitive to the lipid environment.

  • characterization and comparative analysis of arabidopsis Phosphatidylinositol Phosphate 5 kinase 10 reveals differences in arabidopsis and human Phosphatidylinositol Phosphate kinases
    FEBS Letters, 2005
    Co-Authors: Imara Y Perera, Amanda J Davis, Dia Galanopoulou, Wendy F Boss
    Abstract:

    Abstract Arabidopsis Phosphatidylinositol Phosphate(PtdInsP) kinase 10 (AtPIPK10; At4g01190) is shown to be afunctional enzyme of the subfamily A, type I AtPtdInsP kinases.It is biochemically distinct from AtPIPK1 (At1g21980), the onlyother previously characterized AtPtdInsP kinase which is of theB subfamily. AtPIPK10 has the same K m , but a 10-fold lowerV max than AtPIPK1 and it is insensitive to phosphatidic acid.AtPIPK10 transcript is most abundant in inflorescence stalksand flowers, whereas AtPIPK1 transcript is present in all tissues.Comparative analysis of recombinant AtPIPK10 and AtPIPK1with recombinant HsPIPKI a reveals that the Arabidopsis en-zymes have roughly 200- and 20-fold lower V max /K m , respec-tively. These data reveal one explanation for the longstandingmystery of the relatively low Phosphatidylinositol-(4,5)-bisphos-phate:Phosphatidylinositol-4-Phosphate ratio in terrestrialplants. 2005 Published by Elsevier B.V. on behalf of the Federation ofEuropean Biochemical Societies.Keywords: Phosphatidylinositol Phosphate kinase; Lipidkinase; Arabidopsis1. IntroductionBiochemical characterization of the human PtdInsP kinaseshas provided a clear basis for understanding the regulation ofPhosphatidylinositol-(4,5)-bisPhosphate (PtdIns(4,5)P

Sergio Grinstein - One of the best experts on this subject based on the ideXlab platform.

  • an electrostatic switch displaces Phosphatidylinositol Phosphate kinases from the membrane during phagocytosis
    Journal of Cell Biology, 2009
    Co-Authors: Gregory D Fairn, Richard A. Anderson, Koji Ogata, Roberto J Botelho, Philip D Stahl, Pietro De Camilli, Tobias Meyer, Shoshana J Wodak, Sergio Grinstein
    Abstract:

    Plasmalemmal Phosphatidylinositol (PI) 4,5-bisPhosphate (PI4,5P2) synthesized by PI 4-Phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-γ prevented the disappearance of PI4,5P2 and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.

  • Phosphatidylinositol 4 5 bisPhosphate hydrolysis directs actin remodeling during phagocytosis
    Journal of Cell Biology, 2005
    Co-Authors: Cameron C Scott, Roberto J Botelho, Philip D Stahl, Wendy Dobson, Natasha Coadyosberg, Philippe Chavrier, David A Knecht, Colin M Heath, Sergio Grinstein
    Abstract:

    The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of Phosphatidylinositol-4,5-bisPhosphate (PI(4,5)P2) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P2 hydrolysis or increased PI(4,5)P2 generation by overexpression of Phosphatidylinositol Phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P2 dictates the remodeling of actin necessary for completion of phagocytosis.

  • localized biphasic changes in Phosphatidylinositol 4 5 bisPhosphate at sites of phagocytosis
    Journal of Cell Biology, 2000
    Co-Authors: Roberto J Botelho, Richard A. Anderson, Tobias Meyer, Mary N Teruel, Renee Dierckman, Alan Wells, John D York, Sergio Grinstein
    Abstract:

    Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of Phosphatidylinositol-4,5-bisPhosphate (4,5-PIP2) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP2 accompanied by recruitment of type Iα Phosphatidylinositol Phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP2 correlated with mobilization of phospholipase Cγ (PLCγ) and with the localized formation of diacylglycerol. The presence of 4,5-PIP2 and active PLCγ at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP2 is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

Nick V. Grishin - One of the best experts on this subject based on the ideXlab platform.