Phospho-inositide-dependent kinase 1 regulates signal dependent translation in megakaryocytes and platelets

Abstract

Background: Regulated protein synthesis is essential for megakaryocyte (MK) and platelet functions, including platelet production and activation. PDK1 (phosphoinositide-dependent kinase 1) regulates platelet functional responses and has been associated with circulating platelet counts. Whether PDK1 also directly regulates protein synthetic responses in MKs and platelets, and platelet production by MKs, remains unknown.

Objective: To determine if PDK1 regulates protein synthesis in MKs and platelets.

Methods: Pharmacologic PDK1 inhibitors (BX-795) and mice where PDK1 was selectively ablated in MKs and platelets (PDK1^-/- ) were used. PDK1 signaling in MKs and platelets (human and murine) were assessed by immunoblots. Activation-dependent translation initiation and protein synthesis in MKs and platelets was assessed by probing for dissociation of eIF4E from 4EBP1, and using m7-GTP pulldowns and S³⁵ methionine incorporation assays. Proplatelet formation by MKs, synthesis of Bcl-3 and MARCKs protein, and clot retraction were employed for functional assays.

Results: Inhibiting or ablating PDK1 in MKs and platelets abolished the phosphorylation of 4EBP1 and eIF4E by preventing activation of the PI3K and MAPK pathways. Inhibiting PDK1 also prevented dissociation of eIF4E from 4EBP1, decreased binding of eIF4E to m7GTP (required for translation initiation), and significantly reduced de novo protein synthesis. Inhibiting PDK1 reduced proplatelet formation by human MKs and blocked MARCKs protein synthesis. In both human and murine platelets, PDK1 controlled Bcl-3 synthesis. Inhibition of PDK1 led to complete failure of clot retraction in vitro.

Conclusions: PDK1 is a previously unidentified translational regulator in MKs and platelets, controlling protein synthetic responses, proplatelet formation, and clot retraction.

Keywords: platelet activation; platelets; protein translation; signal transduction; thrombosis.

Figure 1:. PDK1 regulates protein synthesis in CD34⁺ derived, cultured, human megakaryocytes.

A. CD34+ megakaryocytes were left alone or treated with BX-795 (1μM) from culture day 11 – 13. Cells were collected on Day 13 and adhered to fibrinogen in media that contains S³⁵ methionine for 2 hours. Cyclohexamide (Cyclo) was used as a positive control. Protein synthesis was measured using scintillation counts. B. On culture day 13, CD34+ megakaryocytes were adhered to a fibrinogen coated surface for 60 min in the presence or absence of BX-795 (1μM). Cell lysates were immunoprecipitated with 4EBP1 and samples were probed for eIF4E using an anti-eIF4E mouse mAb. C. CD34+ megakaryocytes were left alone or adhered to fibrinogen (60 min) in the presence or absence of BX-795. Cells were lysed and incubated with m7 GTP- agarose beads. The beads were eluted to collect the eIF4E protein. Samples were analyzed for eIF4E binding using western blot (**P < 0.05) (***P < 0.01).

Figure 2:. Inhibition of PDK1 effects translation control pathways.

A. Day 13 CD34+ megakaryocytes were incubated with or without BX-795 (1μM) under adherent or non-adherent conditions on fibrinogen. CD34+ megakaryocyte proteins were separated by SDS-PAGE and probed for phosphorylation of p-Akt (T308), extracellular signal-regulated kinase 1/2 (p-ERK1/2 (T202/Y204), p-4EBP1 (T37) and p-eIF4E (S209). The immunoblots are representative of three independent experiments. (**P < 0.05). B. Day 5 bone marrow derived mouse megakaryocytes from wild type or platelet specific PDK1 knockout mice were analyzed for p-Akt (T308), extracellular signal-regulated kinase 1/2 (p-ERK1/2 (T202/Y204), p-4EBP1 (T37) and p-eIF4E (S209) under adherent or non-adherent conditions on fibrinogen. The western blots shown are representative of three independent experiments. (****P < 0.0001).

Figure 3:. PDK1 inhibition regulates proplatelet formation and MARCKs expression.

A. CD34+ megakaryocytes are treated with BX795 (1μM) from day 11 – 13. Cells are collected on Day 13 and adheared to fibrinogen coated plates and incubated over night at 37⁰C. The cells are then treated with phalloidin dye and observed for proplatelet formation using confocal microscopy. The number of proplatelet forming cells were counted and graphs are plotted. B. CD34+ megakaryocytes are treated with BX795 (1μM) from day 11 – 13. Cells are collected on Day 13 and adheared to fibrinogen for 2 hours. Proteins were separated by using SDS-PAGE and probed from MARCKs protein. The immunoblot is representative of three independent experiments (****P < 0.05). C. Day 5 bone marrow derived mouse megakaryocytes from wild type or platelet specific PDK1 knockout mice were analyzed for expression of MARCKs proteins under adherent or non-adherent conditions on fibrinogen using SDS-PAGE. The western blots shown are representative of three independent experiments. (*P < 0.05).

Figure 4:. PDK1 regulates translation control pathways in human and murine platelets.

A. Washed human platelets were untreated or pretreated with the PDK1 inhibitor BX-795 (1μM) for 5 min, followed by stimulation with 2-methylthio-ADP (2MeSADP) (50 nM) under stirring for different time points. Western blots were then probed for phosphorylated Akt (T308), ERK1/2 (T202/Y204), 4EBP1(T37), and eIF4E (S209). The western blots shown are representative of three independent experiments. B. Washed wild type (WT) or PDK1 null murine platelets (PDK1−/−) were stimulated with 2-methylthio-ADP (2MeSADP) (50 nM) under stirring for 5 min. Western blots were then probed for phosphorylated Akt (T308), ERK1/2 (T202/Y204), 4EBP1(T37), and eIF4E (S209). The western blots shown are representative of three independent experiments. Graphs represent mean ± standard error of the mean from at least three different experiments (*P < 0.05).

Figure 5:. PDK1 regulates eIF4E activation and binding to an mRNA cap homologue in activated platelets.

A, B Washed human platelets were untreated or pretreated with the PDK1 inhibitor BX-795 (1μM) for 5 min, followed by stimulation with 2-methylthio-ADP (2MeSADP) (50nM) under stirring condition for 15 min. The platelets were lysed and part of the lysate was separated and used as loading control before adding m7 GTP-Sepharose beads. The platelet lysate was incubated with m7 GTP-Sepharose beads. The beads were eluted to collect the eIF4E protein. Samples were analyzed for eIF4E binding using western blot analysis. This experiment is a representation of three experiments (**P < 0.05). C. Washed human platelets were left alone or pre-treated with the GSK3β inhibitor SB216763, followed by stimulation with 2MeSADP. Cell lysates were immunoprecipitated with 4EBP1 and samples were probed for total eIF4E using an anti-eIF4E mouse mAb. D. Washed human platelets were left alone or pre-treated with the GSK3β inhibitor SB216763 for 5 min, followed by stimulation with 2MeSADP (50 nM) for 5 min. Western blots were then probed for phosphorylated 4EBP1(T37) and eIF4E (S209). The western blots shown are representative of three independent experiments (* P< 0.05).

Figure 6:. PDK1 regulates Bcl-3 protein synthesis and clot-retraction in activated platelets.

A. Washed human platelets were untreated or pretreated with the PDK1 inhibitor BX-795 (1μM) for 5 min, followed by stimulation with 2-methylthio-ADP (2MeSADP) (50nM) under stirring. Western blots were then probed for total Bcl-3 protein. Beta actin was used as loading control. The western blots shown are representative of three independent experiments. Graphs represent mean ± standard error of the mean from at least three different experiments (****P < 0.05). B. Washed wild type (WT) or PDK1 knockout (PDK1−/−) murine platelets were stimulated with 2-methylthio-ADP (2MeSADP) (50nM) under stirring for 5 min. Western blots were then probed total Bcl-3. The western blots shown are representative of three independent experiments. Graphs represent mean ± standard error of the mean from at least three different experiments (****P < 0.05). C. Washed human platelets were incubated with BX-795 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.

Figure 7:

Model representation of PDK1 regulation of translation in megakaryocytes and platelets.