A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade
| Autor: | Lidia Hermida-Nogueira, Irene Izquierdo, Serena Lacerenza, Roberto Pinto-Llorente, Vivian de los Ríos, Luis A. Morán, Ángel García, María Isabel Loza, Johannes A. Eble, Joaquín Abián, Vanessa Casas, María N. Barrachina, J I Casal, Eduardo Domínguez, Montserrat Carrascal |
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| Přispěvatelé: | Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas, Universidade de Santiago de Compostela. Departamento de Farmacoloxía, Farmacia e Tecnoloxía Farmacéutica, Ministerio de Economía y Competitividad (España), Xunta de Galicia, European Commission, Barrachina, Maria N. [0000-0002-1701-6899], Hermida-Nogueira, Lidia [0000-0001-7858-6046], Casas, Vanessa [0000-0001-7438-0323], Eble, Johannes A. [0000-0001-9156-2137], de los Ríos, Vivian [0000-0001-5582-6879], Domínguez, Eduardo [0000-0003-3359-5867], Loza, María Isabel [0000-0003-4730-0863], Casal, J. Ignacio [0000-0003-1085-2840], Carrascal, Montserrat [0000-0002-0205-2176], Barrachina, Maria N., Hermida-Nogueira, Lidia, Casas, Vanessa, Eble, Johannes A., de los Ríos, Vivian, Domínguez, Eduardo, Loza, María Isabel, Casal, J. Ignacio, Carrascal, Montserrat |
| Rok vydání: | 2020 |
| Předmět: |
Adult
Blood Platelets Male Platelets 0301 basic medicine Platelet Aggregation Proteome Syk CLEC-2 signaling 030204 cardiovascular system & hematology Thromboxane A2 Young Adult 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Tyrosinephosphoproteome Humans Lectins C-Type Platelet activation Phosphorylation Tyrosine Phosphotyrosine Membrane Glycoproteins Kinase Hematology Middle Aged Phosphoproteins Platelet Activation 3. Good health Cell biology Adenosine Diphosphate Kinetics Adenosine diphosphate 030104 developmental biology chemistry Tyrosine phosphoproteome Calcium Female Signal transduction Signal Transduction Proto-oncogene tyrosine-protein kinase Src |
| Zdroj: | Thrombosis and Haemostasis Minerva. Repositorio Institucional de la Universidad de Santiago de Compostela instname Digital.CSIC. Repositorio Institucional del CSIC |
| ISSN: | 2567-689X 0340-6245 |
| DOI: | 10.1055/s-0039-3400295 |
| Popis: | 15 p.-7 fig.-2 tab. 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. This study was supported by the Spanish Ministryof Economy and Competitiveness (MINECO) [grant No. SAF2016-79662-R], co-funded by the European Regional Develop-ment Fund (ERDF); and the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia [ED431C2018/21; predoctoral grant Plan I2C 2014; and CentroSingular 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 andinnovation programme under the Marie Skłodowska-Curiegrant agreement No 766118. J.A.E. is supported by DeutscheForschungsgemeinschaft [DFG grant: EB177/13-1] |
| Databáze: | OpenAIRE |
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