Characterisation 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 PI-3 kinase

We describe a inhibitor of Vps34, termed VPS34-IN1 (25 nM IC50) that does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of Class I and II PI3Ks. VPS34-IN1 induces a rapid dose dependent dispersal of a specific PtdIns(3)P binding probe from endosome membranes. We explored whether SGK3, the only protein kinase known to interact specifically with PtdIns(3)P, might be controlled by Vps34. Mutations disrupting PtdIns(3)P-binding, ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop and hydrophobic-motif residues. VPS34-IN1 induced a rapid ~55% loss of SGK3 phosphorylation. 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 that do not inhibit Vps34, suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~85%. This data suggests SGK3 is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34. The second though the conversion of Class I PI3K product, PtdIns(3,4,5)P3 to PtdIns(3)P, via the sequential actions of the PtdIns 5-phosphatases (SHIP1/2) and PtdIns 4-phosphatase (INPP4B). 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 ...

Source: BJ Signal - September 1, 2014 Category: Biochemistry Authors: R Bago, N Malik, M J Munson, A Prescott, P Davies, E M Sommer, N Shpiro, R Ward, D Cross, I G Ganley, D R Alessi Tags: BJ ChemBio Source Type: research