Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson’s Disease Xianting Li1, Qing Jun Wang2¤, Nina Pan1, Sangkyu Lee3, Yingming Zhao3, Brian T. Chait2, Zhenyu Yue1* 1 Department of Neurology and Neuroscience, Mount Sinai School of Medicine, New York, New York, United States of America, 2 Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, United States of America, 3 Ben May Department for Cancer Research, The University of Chicago, Chicago, Illinois, United States of America Abstract Background: Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson’s disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown. Methodology/Principal Findings: Using affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms c and g) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2. Conclusions/Significance: LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2- linked PD. Citation: Li X, Wang QJ, Pan N, Lee S, Zhao Y, et al. (2011) Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson’s Disease. PLoS ONE 6(3): e17153. doi:10.1371/journal.pone.0017153 Editor: Tsuneya Ikezu, Boston University School of Medicine, United States of America Received December 10, 2010; Accepted January 20, 2011; Published March 1, 2011 Copyright: 2011 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the US National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) NS061152 (Z.Y.), NS060809 (Z.Y.), RNS055683A (Z.Y.), Michael J. Fox Foundation (Z.Y.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: zhenyu.yue@mssm.edu ¤ Current address: Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America Introduction Parkinson’s disease (PD) is a major human neurodegenerative disease clinically characterized by the motor function deficits as well as non-motor impairment. The most prominent neuropath- ological features are the loss of midbrain dopaminergic neurons and deposit of intracellular Lewy Body consisting mainly of a- synuclein. The pathogenic mechanism of PD remains largely undefined. However, the recent identification of genetic mutations that are associated with familial PD provides an entry to uncover cellular and molecular pathways that lead to the disease. Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) have been linked to the most common familial form and some sporadic forms of PD [1,2]. Because many LRRK2 mutation carriers exhibit typical PD symptoms indistinguishable from idiopathic PD cases, it is hypothesized that an understanding of the biology and pathophysiology of LRRK2 will provide new opportunities to develop effective treatments of PD. LRRK2 is a large complex protein of 280 kD containing two important enzymatic domains, serine/threonine kinase and ROC GTPase; it also carries several conserved protein motifs including leucine-rich repeats (LRR), the C-terminal of ROC (COR) domain and a WD40 repeat. Previous biochemical analysis demonstrates the kinase and GTPase activity of LRRK2, and these activities are apparently altered by several PD-linked mutations [3,4,5,6], leading to the hypothesis of ‘‘gain-of- PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17153