A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade

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-0039-3400295C-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 signalosomeThis 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]S

Primeiro levantamento de mosca das frutas (Diptera: Tephritidae) e diversidade de parasitoides entre frutos de myrtaceae em todo o Estado da Bahia, Brasil

The objective of this study was to evaluate the diversity of fruit fly (Diptera: Tephritidae) species that use myrtaceous fruit, particularly guava, as hosts in several localities in the state of Bahia and to determine the infestation rates, pupal viability rates, and fruit fly-parasitoid associations. Sampling of myrtaceous fruit was carried out in 24 municipalities in different regions in the state of Bahia. Four fruit fly species, Anastrepha fraterculus, Anastrepha zenildae, Anastrepha sororcula, and Ceratitis capitata were obtained from the collected fruit. Three parasitoid species (Hymenoptera: Braconidae) emerged from Anastrepha larvae/pupae, Doryctobracon areolatus, Utetes anastrephae, and Asobara anastrephae. Doryctobracon areolatus emerged from A. fraterculus, A. sororcula and A. zenildae; Utetes anastrephae emerged from A. fraterculus and A. zenildae; and Asobara anastrephae emerged from A. fraterculus. Fruit fly and myrtaceous fruit associations are reported for the first time in several municipalities in the state of Bahia. A. zenildae was found infesting Syzygium malaccense for the first time in Brazil