mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected]

Abstract

Protein kinase B (PKB, Akt) is a Ser/Thr kinase involved in the regulation of cell survival, proliferation, and metabolism and is activated by dual phosphorylation on Thr(308) in the activation loop and Ser(473) in the hydrophobic motif. It plays a contributory role to platelet function, although little is known about its regulation. In this study, we investigated the role of the mammalian target of rapamycin complex (mTORC)-2 in Akt regulation using the recently identified small molecule ATP competitive mTOR inhibitors PP242 and Torin1. Both PP242 and Torin1 blocked thrombin and insulin-like growth factor 1-mediated Akt Ser(473) phosphorylation with an IC(50) between 1 and 5 nm, whereas the mTORC1 inhibitor rapamycin had no effect. Interestingly, PP242 and Torin1 had no effect on Akt Thr(308) phosphorylation, Akt1 activity, and phosphorylation of the Akt substrate glycogen synthase kinase 3β, indicating that Ser(473) phosphorylation is not necessary for Thr(308) phosphorylation and maximal Akt1 activity. In contrast, Akt2 activity was significantly reduced, concurrent with inhibition of PRAS40 phosphorylation, in the presence of PP242 and Torin1. Other signaling pathways, including phospholipase C/PKC and the MAPK pathway, were unaffected by PP242 and Torin1. Together, these results demonstrate that mTORC2 is the kinase that phosphorylates Akt Ser(473) in human platelets but that this phosphorylation is dispensable for Thr(308) phosphorylation and Akt1 activity.

FIGURE 1.

mTOR inhibitors selectively inhibit phosphorylation of Akt Ser⁴⁷³ in human platelets. Washed platelets were incubated with vehicle (0.2% DMSO) or the indicated concentrations of PP242 (A and C) and Torin1 (B and D) for 15 min prior to stimulation with 0.1 unit/ml thrombin for 15 min (A and B) or 100 nm IGF-1 for 3 min (C and D). The phosphorylation of the indicated proteins was analyzed by SDS-PAGE/Western blotting of immunoprecipitates (p70S6K) or whole cell lysates (Akt, GSK3, pleckstrin, ERK, p38 MAPK). Membranes were stripped and reprobed for α-tubulin as a loading control. Results are representative of three independent experiments.

FIGURE 2.

mTOR inhibitors selectively inhibit phosphorylation of Akt Ser⁴⁷³ in primary adipocytes. Washed adipocytes were incubated with vehicle (0.2% DMSO) or the indicated concentrations of PP242 (A) and Torin1 (B) for 15 min prior to stimulation with 100 nm insulin for 15 min. The phosphorylation of the indicated proteins was analyzed by SDS-PAGE/Western blotting of whole cell lysates. Membranes were stripped and reprobed for α-tubulin as a loading control. Results are representative of three independent experiments.

FIGURE 3.

mTORC2-mediated Akt Ser⁴⁷³ phosphorylation is dispensable for phosphorylation of GSK3β, but not PRAS40. Washed platelets were incubated with vehicle (0.2% DMSO) or the indicated compounds for 15 min prior to stimulation with 0.1 unit/ml thrombin for 15 min (A) or 100 nm IGF-1 for 3 min (B). Alternatively, primary adipocytes were incubated with vehicle (0.2% DMSO) or with the indicated compounds for 15 min prior to stimulation with 100 nm insulin for 15 min (C). The phosphorylation of the indicated proteins was analyzed by SDS-PAGE/Western blotting of immunoprecipitates (p70S6K, A) or whole cell lysates (Akt, GSK3, PRAS40, pleckstrin, ERK, p38 MAPK). Membranes were stripped and reprobed for α-tubulin as a loading control. Results are representative of three independent experiments. The bar graphs represent quantification of Thr²⁴⁶ phosphorylation of PRAS40 (ratio phosphorylated/total) expressed as a percentage of the signal obtained with thrombin (A), IGF-1 (B) and insulin (C) alone (means ± S.E. (error bars), n = 3).

FIGURE 4.

mTORC2-mediated Akt Ser⁴⁷³ phosphorylation is not required for Akt1 activity in human platelets. Washed platelets were incubated with vehicle (0.2% DMSO) or the indicated compounds for 15 min prior to stimulation with 0.1 unit/ml thrombin for 15 min (A and B) or 100 nm IGF-1 for 3 min (C and D). Alternatively, primary adipocytes were incubated with vehicle (0.2% DMSO) or with the indicated compounds for 15 min prior to stimulation with 100 nm insulin for 15 min (E and F). Akt1 (A, C, and E) and Akt2 (B, D, and F) were immunoprecipitated (IP) from detergent lysates and phosphorylation of Thr³⁰⁸ and Ser⁴⁷³ analyzed by SDS-PAGE/Western blotting (i) and in vitro activity analyzed using the peptide substrate, RPRAATF (ii). Activity results are expressed as a percentage of the signal obtained with thrombin (A and B), IGF-1 (C and D), and insulin (E and F) alone (n = 3, mean ± S.E. (error bars); **, p < 0.001).

FIGURE 5.

mTOR activity does not contribute to platelet aggregation. Washed platelets (2 × 10⁸/ml) were incubated with the indicated concentrations of PP242 (A), Torin1 (B), or 200 nm rapamycin (C) for 15 min prior to stimulation of PAR1 with 0.7 μm SFLLRN. The resulting aggregation was recorded for 5 min and is expressed as percent aggregation. Results are representative of three independent experiments.