PMID- 22723946 OWN - NLM STAT- MEDLINE DCOM- 20121212 LR - 20231105 IS - 1932-6203 (Electronic) IS - 1932-6203 (Linking) VI - 7 IP - 6 DP - 2012 TI - The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling. PG - e39132 LID - 10.1371/journal.pone.0039132 [doi] LID - e39132 AB - 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 (IKKalpha and IKKbeta) and IKK-related (IKKepsilon 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. FAU - Dzamko, Nicolas AU - Dzamko N AD - MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee, Scotland. n.dzamko@neura.edu.au FAU - Inesta-Vaquera, Francisco AU - Inesta-Vaquera F FAU - Zhang, Jiazhen AU - Zhang J FAU - Xie, Chengsong AU - Xie C FAU - Cai, Huaibin AU - Cai H FAU - Arthur, Simon AU - Arthur S FAU - Tan, Li AU - Tan L FAU - Choi, Hwanguen AU - Choi H FAU - Gray, Nathanael AU - Gray N FAU - Cohen, Philip AU - Cohen P FAU - Pedrioli, Patrick AU - Pedrioli P FAU - Clark, Kristopher AU - Clark K FAU - Alessi, Dario R AU - Alessi DR LA - eng GR - 1ZIAAG000944-04/PHS HHS/United States GR - MC_U127070193/MRC_/Medical Research Council/United Kingdom GR - G0700656/MRC_/Medical Research Council/United Kingdom GR - 089698/Wellcome Trust/United Kingdom GR - Wellcome Trust/United Kingdom GR - MC_U127084348/MRC_/Medical Research Council/United Kingdom GR - G1100713/MRC_/Medical Research Council/United Kingdom GR - ZIA AG000944/Intramural NIH HHS/United States GR - MC_G1000735/MRC_/Medical Research Council/United Kingdom PT - Journal Article PT - Research Support, N.I.H., Intramural PT - Research Support, Non-U.S. Gov't DEP - 20120618 PL - United States TA - PLoS One JT - PloS one JID - 101285081 RN - 0 (Antibodies, Monoclonal) RN - 0 (Cytokines) RN - 0 (Lipopeptides) RN - 0 (Myeloid Differentiation Factor 88) RN - 0 (Pam(3)CSK(4) peptide) RN - 0 (Toll-Like Receptors) RN - 452VLY9402 (Serine) RN - EC 2.7.1.- (Tbk1 protein, mouse) RN - EC 2.7.11.1 (Leucine-Rich Repeat Serine-Threonine Protein Kinase-2) RN - EC 2.7.11.1 (Lrrk2 protein, mouse) RN - EC 2.7.11.1 (Protein Serine-Threonine Kinases) RN - EC 2.7.11.10 (I-kappa B Kinase) SB - IM MH - Animals MH - Antibodies, Monoclonal/immunology/pharmacology MH - Cell Line MH - Cytokines/metabolism MH - Gene Expression Regulation/drug effects MH - Humans MH - I-kappa B Kinase/*metabolism MH - Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 MH - Lipopeptides/pharmacology MH - Macrophages/metabolism MH - Mice MH - Mice, Knockout MH - Myeloid Differentiation Factor 88/metabolism MH - Parkinson Disease/genetics/*metabolism MH - Phosphorylation MH - Protein Serine-Threonine Kinases/genetics/immunology/*metabolism MH - Serine/metabolism MH - *Signal Transduction MH - Toll-Like Receptors/agonists/*metabolism PMC - PMC3377608 COIS- Competing Interests: Our research Unit receives general funding from the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Janssen Pharmaceutica; Merck KgaA and Pfizer) for financial support. I can also confirm that this does not alter our adherence to all the PLoS ONE policies on sharing data and materials. EDAT- 2012/06/23 06:00 MHDA- 2012/12/13 06:00 PMCR- 2012/06/18 CRDT- 2012/06/23 06:00 PHST- 2012/02/15 00:00 [received] PHST- 2012/05/16 00:00 [accepted] PHST- 2012/06/23 06:00 [entrez] PHST- 2012/06/23 06:00 [pubmed] PHST- 2012/12/13 06:00 [medline] PHST- 2012/06/18 00:00 [pmc-release] AID - PONE-D-12-04578 [pii] AID - 10.1371/journal.pone.0039132 [doi] PST - ppublish SO - PLoS One. 2012;7(6):e39132. doi: 10.1371/journal.pone.0039132. Epub 2012 Jun 18.