The type Iα inositol polyphosphate 4-phosphatase generates and terminates phosphoinositide 3-kinase signals on endosomes and the plasma membrane (original) (raw)

2005, Molecular Biology of The Cell

Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate (PtdIns(3)P) on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate (PtdIns(3,4)P 2 ) binds the PH domain-containing proteins Akt and TAPP-1. Type Iα inositol polyphosphate 4phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P 2 forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4phosphatase co-localised with early and recycling endosomes. Upon growth factor stimulation 4-phosphatase endosomal localization persisted, but in addition the 4phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns3-P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts (MEFs) from homozygous Weeble mice, which have a mutation in the type I 4phosphatase, exhibited dilated early endosomes. 4-phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP-1 PH domain. The 4-phosphatase contains C2 domains which bound PtdIns(3,4)P 2 and C2-domain-deletion mutants lost PtdIns(3,4)P 2 4-phosphatase activity, did not localize to endosomes, or inhibit TAPP-1-PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates PI 3-kinase signals at distinct subcellular locations. asparagine and ligated into the KpnI site of pTrcHisC (pTrcHis-D693N). N-terminal GST-tandem C2 domains were PCR amplified and subcloned into the pGEX-KG HindIII site. 4-phosphatase (aa 305-939) was PCR amplified and subcloned into the KpnI site of pTrcHisC, or into the HindIII site of pGEX-KG (pTrcHis-4ptase∆C2AB and pGEX-KG-4ptase∆C2AB). Constructs were transformed into BL21 E.coli, incubated to an OD 600 0.6, and protein induced with isopropyl β-D-thiogalactopyranoside (1 mM final). Pelleted bacteria were resuspended in 50 mM Tris, pH 8, 1M NaCl, protease inhibitor cocktail (Roche) lysed by 3 x sonication 30 sec on ice, pelleted 19,000 x g (15 min) and purified by Ni 2+ affinity chromatography. Ni-NTA resin (2 ml) (Scientifix), pre-equilibrated with lysis buffer, was added to the cleared bacter ial lysate and rocked at 4°C 1 hr, pelleted at 700 x g for 5 min, washed, proteins eluted with 10 ml 75 mM imidazole, 50 mM Tris, pH 8, 1 M NaCl and concentrated using a Vivaspin 10,000 MW cut off (Vivascience). 2 ml glutathione Sepharose resin, pre-equilibrated with PBS, was added to the cleared bacterial lysate, rocked at 4°C for 1 hr, pelleted at 500 xg for 5 min, washed with ice cold PBS, and eluted with 10 mM glutathione, 50 mM Tris pH 8.