PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses

Abstract

3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

Keywords: Kinases; platelet pharmacology; signal transduction.

Figure 1. BX compounds inhibit agonist-induced Akt, PRAS40 and GSK3β phosphorylation

Washed, aspirin-treated platelets were incubated with the indicated concentrations of PDK1 inhibitors for 5 min at 37°C prior to stimulation with 200 μM AYPGKF for 2 min at 37°C All experimental conditions had a final concentration of 0.1 % DMSO. A) Representative Western blot of three independent experiments. The phosphorylation of Akt Thr308 (A), Akt Ser473 (B), PRAS40 Thr246 (C) and GSK3β Ser9 (D) was determined by SDS-PAGE/Western blotting as described in Methods [BX795 (solid lines) or BX912 (hatched lines]. The graphs represent quantification of phosphorylation (ratio phosphorylated/total) expressed as a percentage of the signal obtained with AYPGKF without inhibitors, (mean ± SEM, n=3). *p<0.05.

Figure 2. BX795 inhibits agonist-induced phosphorylation of Akt, GSK3β and PRAS40

Washed, aspirin-treated platelets were incubated with vehicle or 1 μM BX795 for 5 min prior to stimulation with 250 μM AYPGKF. A) Representative Western blot of three independent experiments. The phosphorylation of Akt Thr308 (A), Akt Ser473 (B), GSK3β Ser9 (C) and PRAS40Thr246 (D)was determined by SOS-PAGE/Western blotting as described in Methods (vehicle (solid lines) or 1 μM BX795 (hatched lines)]. Phospshorylation is expressed as a ratio of phosphorylated protein/total Akt. The graphs are representative of three independent experiments.

Figure 3. PDK1-mediated phosphorylation of Akt on Thr308 is essential for its activity

Washed, aspirin-treated platelets were incubated with vehicle or BX795 for 5 min prior to stimulation with 200 μM AYPGKF for 2 min. Total Akt was immunoprecipitated from detergent lysates, and in vitro activity was assessed using a GSK3β fusion protein as substrate as described in Methods (A). Activity is expressed as a percentage of signal obtained with AYPGKF in the absence of BX795. (B) (n=3, mean ± SEM). *p<0.05.

Figure 4. BX795 inhibits agonist-induced aggregation, ATP secretion and thromboxane formation

Washed platelets without (A) or with (B) aspirin pre-treatment were incubated with BX795 for 5 min prior to activation with 100nM 2-MeSADP. Aggregation and ATP secretion were monitored in a Chronolog lumi-aggregometer. C) Aspirin-treated platelets were pre-incubated with vehicle (−) or 1 μM BX795 (+) for 5 min prior to activation with AYPGKF. Aggregation and ATP secretion were monitored as above. D) Non-aspirin treated platelets were treated as in C and activated with collagen. Aggregation and secretion were monitored as above. Results of all aggregations and secretions are representative of three independent experiments. E and F) Aspirin-free platelets were treated as in C and activated with 100 nM 2-MeSADP or 5 μg/ml collagen for 3.5 min. Samples were prepared for thromboxane analysis as described in Methods (mean ± SEM, n=3). *p<0.05.

Figure 5. BX795 does not inhibit PAR4-induced PKC activity

Washed, aspirin-treated platelets were incubated with vehicle, BX795 or 10μM GF109203X for 5 min at 37°C prior to stimulation with 200 μM AYPGKF for 2 min. Samples were analysed by SDS-PAGEAWestern blotting and probed with a phospho-(Ser) PKC substrate antibody (A) or a phospho-threonine antibody (B) as described in Methods. Results are representative of three independent experiments.

Figure 6. BX795 inhibits clot retraction

Washed, aspirin-treated platelets were incubated with vehicle or BX795 for 5 min prior to initiating clot retraction as described in Methods. Photographs were taken at the times indicated. Data are representative of three independent experiments.