IDENTIFYING CANDIDATE SUBSTRATES OF THE LEUCINE-RICH REPEAT KINASE 2 (LRRK2) BY MASS SPECTROMETRY-BASED PROTEOMICS
William Edelman, Leo Pallanck, Judit Villen.
University of Washington, Seattle, WA.
Mutations in human leucine-rich repeat kinase 2 (LRRK2) contribute to genetic forms of Parkinsonâ€™s disease (PD) in people and related phenotypes in model organisms. However, the biologically relevant substrates for LRRK2 are not yet known, and to what extent they play a role in cell signaling. In this work, we ask: what are the in vivo effectors of LRRK2 phosphorylation in a fly model of PD? We apply phosphopeptide enrichment, peptide fractionation, and quantitative mass-spectrometry to interrogate the neuronal proteome for direct and indirect effectors of LRRK2. We use a transgenic fly model to express wild-type human LRRK2 (hLRRK2-WT) or a hyperactive kinase mutant of human LRRK2 (hLRRK2-mutant) throughout the brain of the fly. We compared differences in protein abundance and phosphorylation of proteins. Increased phosphorylation on specific peptide sequences reveals candidate substrates for in vivo activity. Additionally, we present an in vitro kinase reaction of LRRK2 on peptides to discover direct sites of LRRK2 phosphorylation on specific proteins. The intersection of in vivo substrate candidates and in vitro substrates provides a list of potential bona-fide, biologically relevant substrates. These proteins may play a role in the signaling mediated by LRRK2 and provide insights into the mechanisms of LRRK2-related Parkinsonian phenotypes.