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Phosphoinositides and PIPs01:42

Phosphoinositides and PIPs

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Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ring. The inositol head group extends into the cytoplasm, where it is modified by adding phosphate groups to form phosphatidylinositol phosphates or PIPs.
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...
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IP3/DAG Signaling Pathway01:11

IP3/DAG Signaling Pathway

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Membrane lipids such as phosphatidylinositol (PI) are precursors for several membrane-bound and soluble second messengers. Specific kinases phosphorylate PI and produce phosphorylated inositol phospholipids. One such inositol phospholipids are the  phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], present in the inner half of the lipid bilayer. Upon ligand binding, GPCR stimulates Gq proteins to turn on phospholipase Cꞵ. Activated phospholipase Cꞵ cleaves PI(4,5)P2 and...
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PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

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The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a...
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The JAK-STAT Signaling Pathway01:20

The JAK-STAT Signaling Pathway

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Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as  SH2...
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Rab Proteins01:14

Rab Proteins

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Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
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Assembly of Signaling Complexes01:30

Assembly of Signaling Complexes

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Multiprotein signaling complexes are formed in a dynamic process involving protein-protein interactions at the cytoplasmic domain of transmembrane receptors or enzymatic and non-enzymatic proteins associated with the receptor. These complexes ensure the activation and propagation of intracellular signals that regulate cell functions.
Interaction domains in cell signaling
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Related Experiment Video

Updated: Jun 11, 2025

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
08:07

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Published on: July 26, 2019

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Identification of the GABARAP binding determinant in PI4K2A.

Yan Chen1, Barbara Barylko2, John P Eichorst1

  • 1School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, U.S.A.

Bioscience Reports
|September 30, 2024
PubMed
Summary
This summary is machine-generated.

GABARAP protein binds to PI4K2A via a specific motif, crucial for organelle transport and autophagosome-lysosome fusion. This interaction is isoform-specific and may involve phosphoinositide lipids.

Keywords:
ATG8GABARAPLIR motiffluorescence fluctuation spectroscopyphosphatidylinositol 4-kinase 2A

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Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • GABARAP, an ATG8 family protein, facilitates intracellular transport and is vital for autophagosome-lysosome fusion.
  • GABARAP interacts with Type II phosphatidylinositol 4-kinase, PI4K2A, an interaction critical for cellular processes.

Purpose of the Study:

  • To identify the specific GABARAP interaction motif (GIM) within PI4K2A.
  • To elucidate the molecular basis for GABARAP's specificity towards PI4K2A over PI4K2B.
  • To investigate the role of phosphoinositides in GABARAP-membrane interactions.

Main Methods:

  • Site-directed mutagenesis to identify the GABARAP interaction motif (GIM) in PI4K2A.
  • Analysis of GABARAP binding to PI4K2A and PI4K2B isoforms.
  • Biochemical assays to assess GABARAP binding to various phosphoinositides.

Main Results:

  • A 7-amino acid segment within the PI4K2A catalytic domain was identified as the GABARAP interaction motif (GIM).
  • The GIM is located in an exposed loop, explaining GABARAP's specific binding to PI4K2A and not PI4K2B.
  • Mutating the PI4K2A GIM abolished GABARAP binding and its recruitment to organelles.
  • GABARAP demonstrated binding to mono-phosphorylated phosphoinositides: PI3P, PI4P, and PI5P.

Conclusions:

  • The GABARAP interaction motif (GIM) in PI4K2A is essential for GABARAP binding and its function in intracellular transport.
  • The structural basis for GABARAP's isoform specificity has been defined.
  • Phosphoinositides may contribute to the membrane-binding interactions of GABARAP with proteins.