Tumour growth is represented as the fold change mean??SEM. All mice were weighed at the end of the treatments. SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1?mM [\32P]ATP in a 30?min 30C reaction (upper panel) followed by immunoblot analysis with the indicated antibodies (lower panel). Kinase reactions are presented as means??SD for triplicate reaction. kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1?mM [\32P]ATP in a 30?min 30C reaction (upper panel). Kinase reactions are presented as means??SD for triplicate reaction. Immunoprecipitates (IP) and lysates were analysed by immunoblot with the indicated antibodies. One\hour (1\h) treatment with the PDK1 inhibitor GSK2334470 (Najafov kinase assay as in (C). Kinase reactions are presented as means??SD for triplicate reaction. Immunoprecipitates (IP) and lysates (lower panel) were also subjected to immunoblot analysis with the indicated antibodies. ZR\75\1 cells were cultured in the absence or presence of 1 1?M MK\2206 for 5?days. Cells were then treated in Angiotensin 1/2 + A (2 – 8) the absence or presence of 0.1?M AZD8055 or 0.1?M rapamycin for 1?h. SGK3 was immunoprecipitated and subjected to kinase assay as in (C). Kinase reactions are presented as means??SD for triplicate reaction. The immunoprecipitates (IP) and lysates were analysed with the indicated antibodies. Open in a separate window Figure EV2 Further evidence that SGK3 activity induced by inhibition of PI3K/Akt is regulated by hVps34 ZR\75\1 cells were treated with 1?M MK\2206 for 5?days, and then, 1?h prior to cell lysis, cells were further treated with increasing doses of SAR405. ZR\75\1 cells cultured in serum in the absence of Akt inhibitor were treated for 1 h with the indicated concentrations of SAR405. The cell lysates were analysed by immunoblot using the indicated antibodies. ZR\75\1 cells were treated for 5?days with 1?M MK\2206 or 1?M GDC0941. One hour prior to lysis, the cells were incubated in the presence of absence of 0.3?M SAR405. SGK3 was immunoprecipitated from lysates and subjected to kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1?mM [\32P]ATP in a 30?min 30C reaction (upper panel). Kinase reactions are presented as means??SD for triplicate reaction. Immunoprecipitates (IP) and lysates were also analysed by immunoblot with the indicated antibodies. mTORC2 regulates activation of SGK3 downstream of hVps34 The identity of the hydrophobic motif kinase that phosphorylates SGK3 downstream of hVps34 has not been established. Since mTORC2 regulates activation of SGK1 (Garcia\Martinez & Alessi, 2008), we wished to explore Angiotensin 1/2 + A (2 – 8) whether mTORC2 also mediates SGK3 hydrophobic motif phosphorylation under conditions of prolonged treatment with Class I PI3K or Akt inhibitors. To achieve this, we generated ZR\75\1 cells in which the Rictor subunit of mTORC2 (Sarbassov (Fig?5B and Appendix?Fig?S1B). Open in a separate window Figure 5 14h selectively suppresses both the activity and activation of SGK3 by PDK1 and mTORC2 Chemical structure of the Sanofi\14h SGK inhibitor. IC50 values of Sanofi\14h SGK inhibitor on the indicated recombinant kinases. Protein kinase profiling undertaken against the Dundee panel of 140 protein kinases in the presence of 1?M Sanofi\14h at the International Centre for Protein Kinase Profiling. The result for each kinase is presented as a mean kinase activity of the reaction taken in triplicate relative to a control reaction where the inhibitors were omitted. Abbreviations and assay conditions are described at http://www.kinase-screen.mrc.ac.uk. ZR\75\1 cells were treated for 1?h with the indicated concentrations of 14h. The cell lysates were analysed by immunoblot Angiotensin 1/2 + A (2 – 8) analysis using the indicated antibodies. ZR\75\1 cells were treated for 1?h with the indicated concentrations of 14h. SGK3 was immunoprecipitated from cell lysates and subjected to kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1?mM [\32P]ATP in a 30?min 30C reaction (upper panel). Kinase reactions are presented as means??SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. The effect of the indicated concentration of 14h Rabbit Polyclonal to Keratin 17 on the ability of SGK3[S486E]\GST to be activated by PDK1 in the presence of PtdIns(3)P was assessed as described in Fig?4. 14h suppresses NDRG1 phosphorylation in cells For cellular experiments, we employed 14h, as it was the most potent SGK3 inhibitor (Fig?5B). Treatment of ZR\75\1 cells cultured in serum with increasing doses of 14h for 1?h resulted in a dose\dependent decrease in NDRG1 phosphorylation. NDRG1 phosphorylation was maximally suppressed at 1C3?M 14h, under conditions where Akt\specific substrate PRAS40 was not.