Phosphatidylinositol-3-OH kinases are Rab5 effectors

Abstract

hosphatidylinositol-3-OH kinases (PI(3)Ks) constitute a family of lipid-modifying enzymes that are involved in signal transduction, cytoskeletal organization and membrane transport 1 . Three different classes of PI(3)K have been described, which differ in their specificities for phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate substrates, as well as in their regulation. PI(3)Ks are needed for receptor trafficking in the endocytic pathway in mammalian cells and for transport from the Golgi to the vacuole in yeast (reviewed in ref. 1). The docking and fusion of early endosomes, which is regulated by the small GTPase Rab5 (ref. 2), requires PI(3)K activity 3 . In addition, PI(3)Ks regulate the recruitment to membranes of EEA1, an effector of the small GTPase Rab5 and a core component of the docking and fusion machinery 4,5 that binds phosphatidylinositol-3-phosphate (PtdIns(3)P) through a FYVEfinger motif 6 . We have recently identified over 20 cytosolic proteins that interact with the active form of Rab5 (ref. 5). Here we report the identification of two distinct PI(3)Ks, hVPS34 and p85α-p110β, among these cytosolic proteins. We suggest a new mechanism by which Rab5 can modify its membrane environment by coupling the local production of phosphoinositides to the selective recruitment of Rab5 effector proteins.We used matrix-assisted laser-desorption-ionization mass spectrometry (MALDI-MS) to identify the proteins purified on the basis of a specific interaction with the active form of Rab5 (ref. 5). Surprisingly, among these proteins we identified the regulatory subunit of class-I PI(3)Ks, p85α, and confirmed its identity by western blotting (Fig. 1a). We therefore performed PI(3)K enzymatic assays to demonstrate the presence of catalytic activity in the Rab5-GTPγS column eluate. PI(3)K activity was markedly detected in this eluate and was inhibited >90% by 100 nM wortmannin (Fig. 1b). The activity was more than 100 times greater than the activity in the Rab5-GDP column eluate, and 900-fold greater than the background activity (Fig. 1b). The enrichment was ~10-fold higher than the corresponding value reported for Ras and PI(3)K 7 . PI(3)K therefore efficiently interacts with, and is recruited by, Rab5.Given the presence of a class-I-PI(3)K regulatory subunit in the Rab5-GTPγS eluate, we would expect the catalytic subunit to be either p110α or p110β (ref. 1). By using isoform-specific antibodies we established that p110β, but not p110α, was present in the eluate P Figure 1 The PI(3)Ks p85α-p110β and p150-hVPS34 bind specifically to a Rab5-GTPγS affinity column. a, Identification of p85α in the Rab5-GTPγS column eluate. Eluates from GST-Rab5-GDP or GST-Rab5-GTPγS affinity columns were analysed by SDS-PAGE, silver staining and MALDI analysis (left) or immunoblotting (right). b, PI(3)K-activity assay on eluates from GST-Rab5-GDP or GST-Rab5-GTPγS affinity columns, using phosphatidylinositol as a substrate, in the absence or presence of 100 nM wortmannin (WM); and quantifica...

A specific function for phosphatidylinositol 3-kinase α (p85α-p110α) in cell survival and for phosphatidylinositol 3-kinase β (p85α-p110β) in de novo DNA synthesis of human colon carcinoma cells

“…Finally this report con®rms speci®city among type I PI3Ks as previously suggested (Christoforidis et al, 1999;Deora et al, 1998;Hill et al, 2000;Roche et al, 1998Roche et al, , 1994b. The reason for speci®city is not known yet but one can envision a distinct intracellular localization between a and b.…”

A phosphoinositide conversion mechanism for exit from endosomes

“…These steps thus help to restrict PI(4,5)P 2 and PI(3,4)P 2 to the plasma membrane (Erdmann, Mao et al 2007). To assure dephosphorylation of PI(4,5)P 2 (Verstreken, Koh et al 2003;Erdmann, Mao et al 2007) and PI(3,4)P 2 as well as synthesis of PI(3)P (Christoforidis, Miaczynska et al 1999;Murray, Panaretou et al 2002) these PI 5-phosphatases and PI 4-phospatases are included in a Rab5 complex comprising its GEF rabaptin-5-rabex-5 and class III PI 3-kinase Vps34. Recently, the PI(4)P 4-phosphatase Sac2 was implicated in endocytic trafficking and recycling, also an early endosomal Rab5 effector that associates with OCRL and INPP4B (Hsu, Hu et al 2015;Nakatsu, Messa et al 2015).…”

“…The early endosome is highly enriched in PI(3)P. Maintenance of these high levels was attributed to the class III PI 3-kinase Vps34, an early endosomal Rab5-effector that synthesizes PI(3)P from PI de novo (Shin, Hayashi et al 2005). While PI(3)P synthesis by Vps34 is required for generation of the endosomal compartment (Christoforidis, Miaczynska et al 1999), its depletion is required for exit from endosomes, most notably for endosomal recycling. Here, a local burst of MTM1-mediated PI(3)P depletion on endosomes, initiated upon PI4K2α-dependent recruitment, is essential for cargo exit from early endosomes.…”

A pathway for phagosome maturation during engulfment of apoptotic cells

“…VPS-34 is the nematode homologue of the PI-3-kinase Vps34, and is a known Rab5 effector 54 involved in maturation of endosomes into acidic lysosomes. DYN-1 is the nematode homologue of mammalian dynamin, and belongs to a family of large GTPases that have been linked to both organization of the actin cytoskeleton 55 Ã 58 and internalization of antibody-opsonized cells 59 .…”