Abstract A04: Characterization of VPS34-IN1, a specific inhibitor of Vps34 reveals that the phosphatidylinositol 3-phosphate binding SGK3 protein kinase is regulated by class III PI-3 kinase

Abstract

Abstract The Vps34 Class III phosphoinositide 3-kinase (PI3K) phosphorylates phosphatidylinositol (PtdIns) 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 and FYVE domains. Here 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 PI3K. Administration of VPS34-IN1 to cells, induces a rapid dose dependent dispersal of GFP-2xFYVEHrs , a specific PtdIns(3)P binding probe, from endosome membranes. Previous studies revealed that serum and glucocorticoid regulated kinase-3 (SGK3) possesses a PtdIns(3)P binding N-terminal PX domain. SGK3 was first identified as a novel isoform of SGK1 that played a role in enabling the IL-3-dependent survival of hematopoietic cells. We explored whether SGK3, the only protein kinase known to interact specifically with PtdIns(3)P, might be controlled by Vps34. We utilised U2OS Flp/In inducible cell line to find that the mutations disrupting PtdIns(3)P-binding, ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop (PDK1 site) and hydrophobic motif (mTOR site) residues. Also, treatment of cells with VPS34-IN1 induced a rapid ∼50-60% loss of SGK3 phosphorylation and activity. Furthermore, a Class I PI3K inhibitor (GDC-0941) that does not inhibit Vps34, suppressed SGK3 activity by ∼30-40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ∼80-90% similar to treatment with an mTOR inhibitor (AZD8055). This data suggest that SGK3 phosphorylation and hence activity is mainly controlled by the activity of PI3K Class I and Class III and the regulation of SGK3 activity through Vps34 may provide a prosurvival signal and a resistance mechanism during the treatment of cancer cells with PI3K Class I inhibitors. Citation Format: Ruzica Bago, Eeva Sommer, Nazma Malik, Michael J. Munson, Alan R. Prescott, Paul Davies, Shpiro Natalia, Ian G. Ganley, Dario R. Alessi. Characterization of VPS34-IN1, a specific inhibitor of Vps34 reveals that the phosphatidylinositol 3-phosphate binding SGK3 protein kinase is regulated by class III PI-3 kinase. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A04.