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

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 S(35) 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.