RT Journal Article
T1 A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade
A1 Izquierdo Berjano, Irene
A1 Núñez Barrachina, María
A1 Hermida Nogueira, Lidia
A1 Casas, Vanessa
A1 Morán Lara, Luis Arturo
A1 Lacerenza, Serena
A1 Pinto-Llorente, Roberto
A1 Eble, Johannes A.
A1 Ríos, Vivian de los
A1 Domínguez Medina, Eduardo
A1 Loza García, María Isabel
A1 Casal, José Ignacio
A1 Carrascal, Montserrat
A1 Abián, Joaquín
A1 García Alonso, Ángel
K1 Platelets
K1 CLEC-2 signaling
K1 Tyrosine phosphoproteome
AB C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr525+526) and Src(p-Tyr419), and for PLCγ2 activity (p-Tyr759). We demonstrated that Syk phosphorylation at Tyr525+526 also happens in the presence of ADP and TXA2 inhibitors, which is not the case for Src-pTyr419 and PLCγ2-pTyr759. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca2+ mobilization assays confirmed the relevance of ADP and TXA2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome
PB Thieme Publishing Group
SN 0340-6245
YR 2020
FD 2020
LK http://hdl.handle.net/10347/20681
UL http://hdl.handle.net/10347/20681
LA eng
NO Thromb Haemost 2020; 120(02): 262-276. DOI: 10.1055/s-0039-3400295
NO This is an Accepted Manuscript of an article published by Thieme Publishing Group in Thrombosis and Haemostasis on 04 January 2020, available online at https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-0039-3400295
NO This study was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) [grant No. SAF2016-79662-R], co-funded by the European Regional Development Fund (ERDF); and the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia [ED431C 2018/21; predoctoral grant Plan I2C 2014; and Centro Singular de investigación de Galicia accreditation 2016-2019, ED431G/05], co-funded by the European Regional Development Fund (ERDF). The study also received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766118. J.A.E. is supported by Deutsche Forschungsgemeinschaft [DFG grant: EB177/13-1]
DS Minerva
RD 24 abr 2026