scholarly journals Endocytosis and vesicular trafficking of immune complexes and activation of phospholipase D by the human high-affinity IgG receptor requires distinct phosphoinositide 3-kinase activities

1999 ◽  
Vol 344 (2) ◽  
pp. 605-611 ◽  
Author(s):  
David J. GILLOOLY ◽  
Alirio J. MELENDEZ ◽  
Austin R. HOCKADAY ◽  
Margaret M. HARNETT ◽  
Janet M. ALLEN
Keyword(s):  
Phospholipase D ◽  
Membrane Trafficking ◽  
Immune Complexes ◽  
Initial Phase ◽  
Interferon Γ ◽  
Vesicular Trafficking ◽  
Regulatory Subunit ◽  
High Affinity ◽  
Late Endosomes ◽  
Phosphoinositide 3 Kinase

FcγRI, the human high-affinity IgG receptor, is responsible for the internalization of immune complexes and their subsequent targetting to the lysosomes for degradation. We show here that aggregation of FcγRI by surface immune complexes in interferon-γ-primed U937 cells causes the transient appearance of swollen vacuolar structures, probably swollen late endosomes, which disappear as the immune complexes are degraded. Wortmannin and LY294002, specific inhibitors of phosphoinositide 3-kinases (PI 3-kinases), delay the disappearance of these structures and also correspondingly inhibit degradation of FcγRI-mediated immune complexes. In addition these inhibitors delay the initial phase of FcγRI-mediated endocytosis of immune complexes and block the activity of FcγRI-stimulated phospholipase D, an enzyme that has previously been implicated in membrane-trafficking events. p85 is the regulatory subunit of PI 3-kinase. A p85-dependent PI 3-kinase was shown to be involved in the initial phase of FcγRI-mediated endocytosis, but not in the trafficking of immune complexes for degradation or the activation of phospholipase D. The results presented here show a role for a p85-independent PI 3-kinase in regulating the trafficking of FcγRI-mediated immune complexes, either directly or as a result of the activation of phospholipase D, and a distinct role for a p85-dependent PI 3-kinase isoform in the initial phases of FcγRI-mediated internalization of immune complexes.

scholarly journals The ATG5 Interactome Links Clathrin Vesicular Trafficking With The ATG8 Lipidation Machinery For Autophagosome Assembly

2019 ◽  
Author(s):  
Kiren Baines ◽  
Jon D. Lane
Keyword(s):  
Membrane Trafficking ◽  
Mode Of Delivery ◽  
Surface Expression ◽  
Vesicular Trafficking ◽  
Light Chains ◽  
Autophagic Flux ◽  
Autophagosome Formation ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Phosphoinositide 3 Kinase

ABSTRACTAutophagosome formation involves the sequential actions of conserved ATG family proteins that regulate the lipidation of the ubiquitin-like modifier ATG8 at the nascent isolation membrane. Although the molecular steps driving this process are well understood, the source of membranes supplied for the expanding autophagosome and their mode of delivery remain uncertain. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12∼ATG5 conjugate that is crucial for ATG8 lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins caused defects in autophagic flux in cell-based starvation assays. To determine whether the ATG12∼ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. Proteins whose surface expression increased contingent on autophagic capability included EPHB2, SLC12A4, and JAG1. Those whose surface expression was decreased included CASK, SLC27A4 and LAMP1. These data provide evidence for direct regulatory coupling between the ATG12∼ATG5 conjugate and the clathrin membrane trafficking system, and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery.

scholarly journals BECLIN1: Protein Structure, Function and Regulation

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1522
Author(s):  
Sharon Tran ◽  
W. Douglas Fairlie ◽  
Erinna F. Lee
Keyword(s):  
Protein Structure ◽  
Structure Function ◽  
Membrane Trafficking ◽  
Biochemical Properties ◽  
Class Iii ◽  
Form Class ◽  
Protein Interactome ◽  
Protein Functions ◽  
Phosphoinositide 3 Kinase ◽  
Shed Light

BECLIN1 is a well-established regulator of autophagy, a process essential for mammalian survival. It functions in conjunction with other proteins to form Class III Phosphoinositide 3-Kinase (PI3K) complexes to generate phosphorylated phosphatidylinositol (PtdIns), lipids essential for not only autophagy but other membrane trafficking processes. Over the years, studies have elucidated the structural, biophysical, and biochemical properties of BECLIN1, which have shed light on how this protein functions to allosterically regulate these critical processes of autophagy and membrane trafficking. Here, we review these findings and how BECLIN1’s diverse protein interactome regulates it, as well as its impact on organismal physiology.

scholarly journals Fission Yeast Rad26 Is a Regulatory Subunit of the Rad3 Checkpoint Kinase

2002 ◽  
Vol 13 (2) ◽  
pp. 480-492 ◽  
Author(s):  
Tom D. Wolkow ◽  
Tamar Enoch
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Complex Analysis ◽  
Kinase Activity ◽  
Regulatory Subunit ◽  
Kinase Activation ◽  
Checkpoint Proteins ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.

Trafficking of interleukin 2 and transferrin in endosomal fractions of T lymphocytes

1994 ◽  
Vol 107 (5) ◽  
pp. 1289-1295 ◽  
Author(s):  
V. Duprez ◽  
M. Smoljanovic ◽  
M. Lieb ◽  
A. Dautry-Varsat
Keyword(s):  
Horseradish Peroxidase ◽  
Interleukin 2 ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Recycling Endosomes ◽  
High Affinity ◽  
Human T Cell ◽  
Late Endosomes ◽  
Discontinuous Sucrose Gradient ◽  
Acidic Organelles

The T lymphocyte growth factor interleukin 2 binds to surface high-affinity receptors and is rapidly internalized and degraded in acidic organelles. The alpha and beta chains of high-affinity interleukin 2 receptors are internalized together with interleukin 2. To identify the intracellular pathway followed by interleukin 2, we have compared the subcellular distribution of interleukin 2, transferrin and a fluid-phase marker, horseradish peroxidase, in the human T cell line IARC 301.5. Transferrin was used as a marker of early and recycling endosomes, and horseradish peroxidase to probe for the whole endocytic pathway. Fractionation of intracellular organelles on a discontinuous sucrose gradient showed that internalized interleukin 2 is initially mostly found in compartments with similar densities to transferrin, e.g. early and recycling endosomes. The kinetics of entry and exit of interleukin 2 from such organelles was much slower than that of transferrin. Later on, interleukin 2 is predominantly found in dense lysosome-containing fractions. Very little, if any, interleukin 2 was found in fractions corresponding to late endosomes containing horseradish peroxidase. These results suggest that, after endocytosis, interleukin 2 enters early or recycling endosomes before it reaches dense lysosomes.

scholarly journals Differential signal transduction, membrane trafficking, and immune effector functions mediated by FcγRI versus FcγRIIa

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 318-327 ◽  
Author(s):  
Xilei Dai ◽  
Manikandan Jayapal ◽  
Hwee Kee Tay ◽  
Renji Reghunathan ◽  
Gen Lin ◽  
...  
Keyword(s):  
Antigen Presentation ◽  
Mhc Class Ii ◽  
Membrane Trafficking ◽  
Immune Complexes ◽  
Antigen Processing ◽  
Fc Receptors ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Leukocyte ◽  
Differential Signal ◽  
Proinflammatory Mediators

Abstract Receptors for the fragment crystallizable region of immunoglobulin-G (FcγRs) play an important role in linking the humoral and cellular arms of the immune response. In this study, we present a comprehensive functional comparison of 2 human Fc-receptors, FcγRI and FcγRIIa. Activation of FcγRI results in a novel signaling cascade that links phospholipase D1 to sphingosine kinase-1 in U937 cells and primary human monocytes. This induces the expression of proinflammatory mediators and is associated with trafficking of immune complexes into human leukocyte antigen-DM positive antigen-processing compartments coupled with improved MHC class II–mediated antigen presentation to T lymphocytes. In contrast, activation of FcγRIIa elicits signaling through phospholipase Cγ1, resulting in increases in intracellular calcium, activation of nicotinamide adenine dinucleotide phosphate-oxidative burst, and differential membrane trafficking combined with impaired antigen presentation and proinflammatory cytokine expression. These data provide a mechanistic insight into the disparate activities associated with Fc receptors in immunity, namely, reinforcement of immune responses through stimulation of proinflammatory signaling and antigen presentation, versus the maintenance of immunologic homeostasis through the noninflammatory clearance of immune complexes.

Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase

1996 ◽  
Vol 109 (2) ◽  
pp. 289-300 ◽  
Author(s):  
T.R. Jackson ◽  
I.J. Blader ◽  
L.P. Hammonds-Odie ◽  
C.R. Burga ◽  
F. Cooke ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Specific Inhibitor ◽  
Morphological Differentiation ◽  
Sympathetic Neuron ◽  
Neuronal Morphology ◽  
Regulatory Subunit ◽  
Membrane Reorganization ◽  
Phosphoinositide 3 Kinase

Application of nerve growth factor (NGF) to PC12 cells stimulates a programme of physiological changes leading to the development of a sympathetic neuron like phenotype, one aspect of which is the development of a neuronal morphology characterised by the outgrowth of neuritic processes. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: firstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphological responses to NGF, the formation of actin filament rich microspikes and subsequent neurite outgrowth. This correlated with wortmannin inhibition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) and phosphatidylinositol(3,4)bisphosphate (PtdIns(3,4)P2) production and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the overexpression of a mutant p85 regulatory subunit of the phosphoinositide 3-kinase, which cannot interact with the catalytic p110 subunit, also substantially inhibited the initiation of NGF-stimulated neurite outgrowth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outgrowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP3, is a necessary second messenger for the cytoskeletal and membrane reorganization events which occur during neuronal differentiation.

Sign in / Sign up

Export Citation Format

Share Document