Extracellular signal-regulated kinase 5 associates with casein kinase II to regulate GPIb-IX-mediated platelet activation via the PTEN/PI3K/Akt pathway.

Essentials The mechanisms of extracellular signal-regulated kinase 5 (ERK5) in GPIb-IX signaling are unclear. Function of ERK5 in GPIb-IX was tested using aggregation, western blotting, and mass spectrometry. The protein interacting with ERK5 in human platelets was identified as casein kinase II (CKII). ERK5 associates with CKII to regulate the activation of the PI3K/Akt pathway in GPIb-IX signaling. SUMMARY:Background The platelet glycoprotein (GP) Ib-IX complex plays essential roles in thrombosis and hemostasis. The mitogen-activated protein kinases (MAPKs) ERK1/2 and p38 have been shown to be important in the GPIb-IX-mediated signaling leading to integrin activation. However, the roles of the MAPK extracellular signal-regulated kinase 5 (ERK5) in GPIb-IX-mediated platelet activation are unknown. Objective To reveal the function and mechanisms of ERK5 in GPIb-IX-mediated platelet activation. Methods The functions of ERK5 in GPIb-IX-mediated human platelet activation were assessed using botrocetin/VWF, ristocetin/VWF, or platelet adhesion to von Willebrand factor (VWF) under shear stress in the presence of a specific inhibitor of ERK5. ERK5-associated proteins were pulled down from Chinese hamster ovary (CHO) cells transfected with HA-tagged-ERK5, identified by mass spectrometry, and confirmed in human platelets. Roles of ERK5-associated proteins in GPIb-IX-mediated platelet activation were clarified using specific inhibitors. Results The phosphorylation levels of ERK5 were significantly enhanced in human platelets stimulated with botrocetin/VWF or ristocetin/VWF. The ERK5 inhibitor XMD8-92 suppressed the second wave of human platelet aggregation induced by botrocetin/VWF or ristocetin/VWF and inhibited human platelet adhesion on immobilized VWF under shear stress. Casein kinase II (CKII) was identified as an ERK5-associated protein in human platelets. The CKII inhibitor TBB, similar to the ERK5 inhibitor XMD8-92, specifically restrained PTEN phosphorylation, therefore suppressing Akt phosphorylation in human platelets treated with botrocetin/VWF. Conclusion ERK5 associates with CKII to play essential roles in GPIb-IX-mediated platelet activation via the PTEN/PI3K/Akt pathway.

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