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Phosphoproteomic evaluation of pharmacological inhibition of leucine‐ rich repeat kinase 2 reveals significant off‐target effects of LRRK ‐2‐ IN ‐1
Author(s) -
Luerman Gregory C.,
Nguyen Chuong,
Samaroo Harry,
Loos Paula,
Xi Hualin,
HurtadoLorenzo Andres,
Needle Elie,
Stephen Noell G.,
Galatsis Paul,
Dunlop John,
Geoghegan Kieran F.,
Hirst Warren D.
Publication year - 2014
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.12483
Subject(s) - phosphorylation , kinase , biology , neurite , lrrk2 , microbiology and biotechnology , leucine rich repeat , biochemistry , mutation , gene , in vitro
Genetic mutations in leucine‐rich repeat kinase 2 ( LRRK 2) have been linked to autosomal dominant Parkinson's disease. The most prevalent mutation, G2019S, results in enhanced LRRK 2 kinase activity that potentially contributes to the etiology of Parkinson's disease. Consequently, disease progression is potentially mediated by poorly characterized phosphorylation‐dependent LRRK 2 substrate pathways. To address this gap in knowledge, we transduced SH ‐ SY 5Y neuroblastoma cells with LRRK 2 G2019S via adenovirus, then determined quantitative changes in the phosphoproteome upon LRRK 2 kinase inhibition ( LRRK 2‐ IN ‐1 treatment) using stable isotope labeling of amino acids in c ulture combined with phosphopeptide enrichment and LC ‐ MS / MS analysis. We identified 776 phosphorylation sites that were increased or decreased at least 50% in response to LRRK 2‐ IN ‐1 treatment, including sites on proteins previously known to associate with LRRK 2. Bioinformatic analysis of those phosphoproteins suggested a potential role for LRRK 2 kinase activity in regulating pro‐inflammatory responses and neurite morphology, among other pathways. In follow‐up experiments, LRRK 2‐ IN ‐1 inhibited lipopolysaccharide‐induced tumor necrosis factor alpha ( TNF α) and C‐X‐C motif chemokine 10 (CXCL10) levels in astrocytes and also enhanced multiple neurite characteristics in primary neuronal cultures. However, LRRK 2‐ IN ‐1 had almost identical effects in primary glial and neuronal cultures from LRRK 2 knockout mice. These data suggest LRRK 2‐ IN ‐1 may inhibit pathways of perceived LRRK 2 pathophysiological function independently of LRRK 2 highlighting the need to use multiple pharmacological tools and genetic approaches in studies determining LRRK 2 function.Genetic mutations in leucine‐rich repeat kinase 2 (LRRK2) have been linked to Parkinson's disease, potentially contributing to the pathogenesis through increased kinase activity acting via phosphorylation‐dependent LRRK2 substrate pathways. To determine the potential pathways, we performed SILAC combined with phosphopeptide enrichment and LC‐MS/MS analysis +/‐ LRRK2 inhibitor. Bioinformatic analysis of those phosphoproteins suggested a potential role for LRRK2 kinase activity in regulating pro‐inflammatory responses and neurite morphology, among other pathways.