Identification of PtdIns(3,4)P₂ effectors in human platelets using quantitative proteomics

After decades in PtdIns(3,4,5)P₃'s shadow, PtdIns(3,4)P₂ has now emerged as a bona fide regulator of important cellular events, including endocytosis and cell migration. New understanding of PtdIns(3,4)P₂'s cellular roles has been possible via novel approaches to observe and quantify cellular PtdIns(3,4)P₂ dynamics, alongside methods to target the kinases and phosphatases governing phosphoinositide turnover. Despite this, the mechanisms by which PtdIns(3,4)P₂ orchestrates its cellular roles remain more poorly understood, most notably because, to date, few PtdIns(3,4)P₂ effectors have been identified. Here, we develop and apply an affinity-proteomics strategy to conduct a global screen for PtdIns(3,4)P₂ interactors in human platelets; a primary cell type with striking PtdIns(3,4)P₂ accumulation. Through an integrated approach, coupling affinity capture of PtdIns(3,4)P₂-binding proteins to both label-free and isobaric tag-based quantitative proteomics, we identify a diverse PtdIns(3,4)P₂ interactome. Included are long-established PtdIns(3,4)P₂-binding proteins such as PLEKHA1, PLEKHA2, AKT and DAPP1, and a host of potentially novel effectors, including MTMR5, PNKD, RASA3 and GAB3. The PtdIns(3,4)P₂ interactome shows an enrichment of pleckstrin homology (PH) domain-containing proteins, and through bioinformatics and array analyses we characterise the PH domain of MTMR5 and define its phosphoinositide selectivity. The interactome is also diverse in function, including several proteins known to support protein trafficking and cytoskeletal mobilisation. Such proteins have the ability to drive key platelet events, and to fulfil recently-defined roles for PtdIns(3,4)P₂ in a wider range of cell types. Moreover, this study will serve as a valuable resource for the future characterisation of effector-driven PtdIns(3,4)P₂ function.