期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 433, 期 23, 页码 -出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2021.167282
关键词
BirA*; miniTurbo; TurboID; proximity labeling; Csw/SHP2/PTPN11
资金
- Swedish Cancer Society [RHP CAN18/729, CAN18/834]
- Swedish Childhood Cancer Foundation [RHP:PR2019-0078]
- Swedish Research Council [RHP 2019-03914]
- Swedish Foundation for Strategic Research [RB13-0204]
- Goran Gustafsson Foundation [RHP2016]
- Ake Wiberg Foundation [GW M19-0561]
- Knut and Alice Wallenberg Foundation [KAW 2018.0057]
Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase with diverse functions in the central nervous system of Drosophila. By using BioID technology and CRISPR/Cas9, researchers identified molecules interacting with Alk and found that the next generation BioID proteins exhibit superior labeling speed and efficiency. LC-MS3-based screening revealed an extensive neuronal Alk proximitome and identified potential components of Alk signaling complexes.
Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS). To do this, we used CRISPR/Cas9 induced homology-directed repair (HDR) to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. Our results show that the next generation of BioID proteins (TurbolD and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of Alk(TurbolD) and Alk(miniTurbo) larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling. (C) 2021 The Author(s). Published by Elsevier Ltd.
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