The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling.

Nicolas Dzamko,Li Tan,Nathanael Gray,Francisco Inesta-Vaquera,Hwanguen Choi,Jiazhen Zhang,Huaibin Cai,Kristopher Clark,Chengsong Xie,Philip Cohen,Patrick Pedrioli,Dario R Alessi,Simon Arthur,Ted M Dawson

doi:10.1371/journal.pone.0039132

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR) pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs) or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKβ) and IKK-related (IKKε and TBK1) kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser910 and Ser935 as pharmacodynamic biomarkers.

Highlights

Mutations in the gene encoding the protein kinase leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson’s disease [1,2]
Recent studies have demonstrated that LRRK2 is highly expressed in immune cells, in particular, macrophages and Blymphocytes [13,14,15]
We investigated the effect of Toll-Like Receptor (TLR) agonists on LRRK2 phosphorylation in RAW264.7 macrophage cells

Summary

Introduction

Mutations in the gene encoding the protein kinase LRRK2 (leucine rich repeat kinase 2) cause autosomal dominant Parkinson’s disease [1,2]. The most common mutation replaces glycine 2019 with a serine within the magnesium-binding DYG motif (DFG motif in most other kinases) of the kinase domain, thereby increasing LRRK2 activity 3-fold [6,7]. This indicates that inhibitors of LRRK2 activity might be of therapeutic benefit for the treatment of Parkinson’s disease. Little is known about the physiological role of LRRK2 and to date no properly validated substrates have been identified

Methods

Results

Conclusion