Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Abstract

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-l cells. The IC50 was "40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the Endocytosis occurs in most eukaryotic cells and is essential for uptake of nutrients, downregulation of cell surface receptors, and maintenance of cell homeostasis (1). Generally there are two types of endocytosis: receptor-mediated endocytosis and fluid-phase endocytosis. Receptor-mediated endocytosis can be either a constitutive process (e.g., the process mediated by transferrin receptor) or a ligand-induced event (e.g., the downregulation of activated platelet-derived growth factor receptor). The internalization of cell surface receptors occurs at specific areas (coated pits) that are coated with clathrin molecules. Fluid-phase endocytosis is a constitutive cellular process that internalizes liquids and solutes from extracellular environments. After internalization, fusion between endosomal vesicles occurs with delivery of endocytosed contents into a population of early endosomes where they are further sorted to other intracellular destinations. Receptor-mediated endocytosis and fluid-phase endocytosis possibly share a common machinery at the early endosome fusion step. For example, the well-characterized small GTPase RabS regulates membrane fusion between early endosomes that can be derived from either receptor-mediated endocytosis or fluid-phase endocytosis (2-5).In the past several years, there has been an explosive discovery of molecules that control distinct membrane trafficking steps. Among the newest additions are phosphatidylinositol (PI) 3-kinases and their lipid products: phosphatidylinositol 3-phosphate (PI-3-P), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. The first suggestion of PI 3-kinase involvement came from the study of VPS34p, a yeast protein essential for protein targeting to the yeast vacuole and vacuole morphogenesis (6). VPS34p shares sequence homology with the catalytic subunit (p110) of a mammalian PI 3-kinase and indeed exhibits PI 3-kinase activity (7). PI 3-kinase is also involved in IgE-mediated histamine secretion (8) and insulinregulated glucose transport (9-11). PI 3-kinase is necessary for the downregulation (endocytosis) of the activated platelet-derived growth factor receptor (12). Although a detailed mechanism has not been established, these lines of evidence strongly suggest the involvement of PI 3-kinase in several aspects of intracellular membrane trafficking.In this study, we address the importance of PI 3-kinase activity in constitutive endocytosis and endosome fusion. A very useful tool in the study of PI 3-kinases is the fungal metabolite wortman...

Intracellular trafficking of nonviral vectors

“…101 Trafficking was affected by the phosphatidylinositol-3-OH kinase (PI3K) inhibitors, wortmannin and LY294002, as PI3K contributes to vesicle fusion. 102 Cytochalasin B, which inhibits F-actin assembly, slightly increased uptake. Cytochalasin B blocks noncoated pit formation and phagocytosis but not clathrin-mediated endocytosis.…”

From receptor to transporter: insulin signalling to glucose transport

“…The wortmannin inhibited exit of recycling from prelysosomal compartments is known to depend on Rab 9 [128]. Similarly, in a cell-free endosome fusion assay system addition of constitutively active Rab5 overcame the wortmannin inhibitory effect on fusion [129].…”

The Type Iα Inositol Polyphosphate 4-Phosphatase Generates and Terminates Phosphoinositide 3-Kinase Signals on Endosomes and the Plasma Membrane

“…PtdIns(3)P is synthesized on early endosomes by the wort- mannin-sensitive PI3-kinase hVPS34, which is recruited to endosomal membranes by Rab5GTP (Christoforidis et al, 1999). Several studies have proposed that PtdIns(3)P regulates early endosomal fusion, because inhibition of PI3-kinase activity by using the inhibitor wortmannin is associated with early endosomal dilatation and inhibition of in vitro early endosomal fusion (Jones and Clague, 1995;Li et al, 1995;Jones et al, 1998). EEA-1 localizes to early endosomal membranes, by an interaction between its FYVE domain, PtdIns(3)P, and Rab5-GTP (Stenmark et al, 1996).…”