Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase

Abstract

Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known. Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases. The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking. Immunof luorescence analysis of 3T3-L1 adipocytes with antisera against p170͞EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin. In vitro, p170͞EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production. Thus, p170͞EEA1 may define a family of proteins that mediate the regulatory effects of 3-phosphoinositides on membrane trafficking in yeast and mammalian cells.

Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

“…FYVE domains in proteins such as EEA1 and Hrs bind to PI 3-P, a product of some PI 3-kinase enzymes, most notably hVPS34 (36). Treatment of HeLa or BHK cells with the PI 3-kinase inhibitor, wortmannin, leads to an almost complete redistribution of EEA1 from membranes to the cytosol (16,20). We similarly measured Hrs distribution between particulate and cytosolic fractions, prepared from cells treated or untreated with wortmannin.…”

A Hrs binding protein having a Src homology 3 domain is involved in intracellular degradation of growth factors and their receptors

“…Treatment of cells with phosphatidylinositol 3-OH kinase (PI(3)K) inhibitor wortmannin causes the dissociation of EEA1 from early endosomes (Patki et al 1997). A mutation in the ring FYVE domain of EEA1 that interferes with zinc binding causes the cytosolic localization of EEA1 (Stenmark et al 1996).…”

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

“…EEA-1 localizes to early endosomal membranes, by an interaction between its FYVE domain, PtdIns(3)P, and Rab5-GTP (Stenmark et al, 1996). Treatment of cells with 100 nM wortmannin blocks PtdIns(3)P synthesis on early endosomes, leading to EEA-1 dissociation (Patki et al, 1997), and the early endosomes become dilated, looking like large, hollow rings (Simonsen et al, 1998).…”