Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase

摘要

The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3) P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3) P -binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25?nM IC50 in?vitro , but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3) P -binding probe from endosome membranes, within 1?min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3) P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3) P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50–60% loss of SGK3 phosphorylation within 1?min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3) P -binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80–90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3) P . The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5) P 3 into PtdIns(3) P , via the sequential actions of the PtdIns 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate 4-phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity, in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K.Abbreviations: 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; DMEM, Dulbecco's modified Eagle's medium; EEA1, early endosome antigen 1; HRP, horseradish peroxidase; IGF, insulin-like growth factor; INPP4B, inositol polyphosphate 4-phosphatase type II; IP1, inositol phosphate; ITC, isothermal titration calorimetry; mTOR, mammalian target of rapamycin; mTORC, mammalian target of rapamycin complex; NDRG1, N-Myc downstream-regulated gene-1; PDK1, phosphoinositide-dependent kinase 1; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PRAS40, proline-rich Akt substrate 40 kDa; PtdIns, phosphatidylinositol; PX, Phox homology; SGK3, serum- and glucocorticoid-regulated kinase-3; SHIP1/2, Src homology 2-domain-containing inositol phosphatase 1/2; TSC2, tuberous sclerosis complex 2; Vps34, vacuolar protein sorting 34; VSV-G, vesicular stomatitis virus glycoprotein