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

Abstract

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)P2] binds the pleckstrin homology (PH) domain-containing proteins Akt and TAPP1. Type Iα inositol polyphosphate 4-phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P2, forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4-phosphatase colocalized with early and recycling endosomes. On growth factor stimulation, 4-phosphatase endosomal localization persisted, but in addition the 4-phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns(3)P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts from homozygous Weeble mice, which have a mutation in the type I 4-phosphatase, exhibited dilated early endosomes. 4-Phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP1 PH domain. The 4-phosphatase contains C2 domains, which bound PtdIns(3,4)P2, and C2-domain-deletion mutants lost PtdIns(3,4)P2 4-phosphatase activity, did not localize to endosomes or inhibit TAPP1 PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates phosphoinositide 3-kinase signals at distinct subcellular locations.

Loss-of-function of inositol polyphosphate-4-phosphatase reversibly increases the severity of allergic airway inflammation

“…INPP4A activity was measured based on indirect estimation of PtdIns(3)P; briefly, lung cytosols were prepared using mitochondria isolation kit (MITO-ISO1, Sigma Chemical Co.) and were incubated (37 °C for 1 h) with 2 µM PtdIns(3,4)P2 (substrate of INPP4A) in reaction buffer 48 and reaction products were further incubated with PtdIns(3)P detector protein, followed by transferring final products to PtdIns(3)P-coated microplate for competitive enzyme-linked immunosorbent assay (ELISA), and the final INPP4A activity was expressed as PtdIns(3)P in picomoles produced from PtdIns(3,4)P2. Calpain activity was performed using a calpain activity assay kit (BIOvision, Inc., Mountain View, CA, USA).…”

Genes involved in cell adhesion, cell motility and mitogenic signaling are altered due to HPV 16 E5 protein expression

“…PI3K activity generates phosphoinositides, which control cell survival, proliferation, migration and cellular trafficking (Cantley, 2002;Czech, 2003). Inositol polyphosphate-4-phosphatase is a regulator of PI3K, and it has been implicated in PI3K signaling at distinct subcellular junctions (Ivetac et al, 2005). Constitutive upregulation of cell-survival signaling mediated by PI3K/Akt has already been implicated in oncogenic transformation and tumor development, as it prevents apoptotic cell death during uncontrolled proliferation (Chang et al, 2003).…”

Mechanistic Basis of Differential Cellular Responses of Phosphatidylinositol 3,4-Bisphosphate- and Phosphatidylinositol 3,4,5-Trisphosphate-binding Pleckstrin Homology Domains

“…It has been reported that PtdIns(3,4,5)P 3 accumulation after cell stimulation is immediate and transient, whereas PtdIns(3,4)P 2 accumulation is slightly delayed and significantly more sustained in platelets (6,7). Also, unlike PtdIns(3,4,5)P 3 that is primarily present in the plasma membrane, PtdIns(3,4)P 2 may be localized at endosomes, intralumi-nal vesicles, endoplasmic reticulum, as well as the plasma membrane (8).…”