IDENTIFYING CANDIDATE SUBSTRATES OF THE LEUCINE RICH REPEAT KINASE 2 BY MASS SPECTROMETRY-BASED PROTEOMICS
William Edelman, Leo Pallanck, Judit Villen.
University of Washington, Seattle, WA.
Mutations in the human leucine-rich repeat kinase 2 (LRRK2) contribute to genetic forms of Parkinson’s disease (PD) in people and related phenotypes in model organisms. However, it is not yet known what the biologically relevant substrates are for LRRK2, and to what extent they play in cell signaling. In this work, we ask: what are the in vivo effectors of LRRK2 phosphorylation in a fly model of PD? We applied phosphopeptide enrichment, peptide fractionation, and quantitative mass-spectrometry to interrogate the neuronal proteome for direct and indirect effectors of LRRK2. We used 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 revealed candidate substrates for in vivo activity. Additionally, we presented an in vitro kinase reaction of LRRK2 on peptides to discover direct sites of LRRK2 phosphorylation on specific proteins. We identified over 15,000 unique peptides covering over 4,000 proteins extracted from fly heads. In this work, we applied fractionation methods to extract high-confidence proteome and phosphoproteome changes in response to expressing human WT-LRRK2. These proteins may play a role in signaling mediated by LRRK2 and provide insights into the mechanisms of LRRK2-related Parkinsonian phenotypes.