function’’ in LRRK2 mutants with enhanced kinase activity [5,7,8]. LRRK2 protein can be modified posttranslationally (e.g. by phosphorylation) and forms a dimer in vitro and in vivo [9,10,11]. These alterations may play a critical role in regulating LRRK2 biochemical activities and cellular functions. However, until now little has been known about the normal function of LRRK2 or the pathogenic pathways mediated by PD-linked mutant LRRK2. Several studies show that the mutants of LRRK2 impair the outgrowth or maintenance of neurites in primary neuronal cultures, whereas reduced or abolished expression of LRRK2 has the opposite effect. In addition, inhibition of kinase activity seems to alleviate the toxic phenotype caused by LRRK2 mutants [8,12,13,14]. To elucidate the cellular pathway and pathogenic role of LRRK2 in PD, we investigated LRRK2 protein modifications and interactors in the brain. We show that LRRK2 is phosphorylated at multiple sites. Our study reveals that 14-3-3s bind LRRK2 and the binding depends on the phosphorylation of S935. Further- more, we show that protein kinase A (PKA) causes phosphoryla- tion of LRRK2 at S935 in vitro and in cell culture, implicating PKA pathway in regulating LRRK2 function. Finally, our study suggests that common PD mutations of LRRK2 impair phos- phorylation levels of S935 as well as14-3-3 binding. Our data, therefore, provide molecular insight into the regulation of LRRK2 and suggests a potential mechanism for LRRK2-mediated PD pathogenesis. Results Identification of phosphorylation sites in LRRK2 from mouse brain We previously reported the purification of FLAG-tagged LRRK2 protein from BAC transgenic mice [3]. For phosphorylation site identification, the purified LRRK2 protein was digested in-gel using various proteases and the resulting proteolytic peptides were analyzed by multiple mass spectrometer methods including MALDI-QqTOF, MALDI-ion trap (LCQ DECA XP), and nano-HPLC/ velos LTQ Orbitrap. The resulting MS/MS data were used to identify proteins and protein modifications. The results reveal 3 serine phosphoryla- tion sites (S910, 935 and 973) from tryptic peptides and 1 serine phosphorylation site (S912) in chymotryptic peptides of LRRK2, respectively (Figure 1A) (Figures S1, S2, S3 and S4). Interestingly, stoichiometry of all 4 serine phosphorylation appears high, as the ratios of MS/MS spectra for modified peptides versus unmodified peptides are all more than 30%. This result indicates the relative high probability of LRRK2 phosphorylation at these sites in the brain (Figures S1, S2, S3 and S4). In this initial study, we focused on the analysis of the high stoichiometry S935 phosphorylation. We developed an antibody raised against phosphorylated S935 (pS935) peptide. The anti- pS935 antibody detects a strong signal in purified FLAG-LRRK2 protein from BAC transgenic brains, while the signal is completely abolished upon the treatment with calf-intestinal alkaline phos- phatase (CIAP) (Figure 1B). The loss of phosphorylation at S935 with alkaline phosphatase treatment was also confirmed by mass spectrometric analysis (Figure S5). The antibody also detected pS935 signal in FLAG-LRRK2 protein isolated from transfected HEK-293T cells. In contrast, FLAG-LRRK2 mutant S935A, where serine 935 was replaced with alanine, was not recognized by this antibody, even though much more mutant protein (compared to wild type) was loaded in the gel (Figure 1C). We also examined the pS935 levels in purified FLAG-LRRK2 from different tissues and at different ages in the brain. The results show that FLAG- LRRK2 is phosphorylated at S935 in lung, spleen and kidney in Phosphorylation and 14-3-3 Binding of LRRK2 and PD addition to brain, and the pS935 levels relative to the total FLAG- LRRK2 protein amount are comparable among these tissues (Figure 1D). Moreover, the relative levels of pS935 do not change significantly at different ages in the brain (Figure 1E). The above results suggest that pS935 are maintained at a constant level under normal condition. Identification of 14-3-3s in LRRK2 protein complex and specific 14-3-3 isoforms as preferential LRRK2-binding proteins We sought to identify LRRK2-binding proteins in the brain by analyzing proteins that were affinity-isolated with FLAG-LRRK2 from BAC transgenic brain. We isolated only the proteins unique to the transgenic (compared to non-transgenic control) and performed mass spectrometry analysis. We identified several isoforms of 14-3-3 proteins, such as c, g, f and e (Figure 2A, arrow) (Figures S6, S7 and S8) that are specifically isolated by FLAG-LRRK2. Using commercial 14-3-3 isoform-specific anti- bodies, we found 14-3-3c, g, f as well as b, h in the immunoprecipitated products (Figure 2B). To further evaluate various 14-3-3 isoform binding to LRRK2, we co-expressed FLAG-LRRK2 with individual myc-tagged 14-3-3 isoforms in HEK-293T cells and tested their binding by co-immunoprecipi- tation (co-IP) analysis. The results indicated that, although all six isoforms co-IP with LRRK2, the c and g forms pulled down much more LRRK2 protein than did the other isoforms (Figure 2C). In a parallel experiment, we incubated each purified GST-14-3-3 isoform protein with BAC-FLAG-LRRK2 transgenic mouse brain lysate and, followed by a GST pull-down assay. The result again showed enhanced binding of 14-3-3c and g with LRRK2, compared to 14-3-3f, e and h (Figure 2D). The above data collectively suggests that LRRK2 prefers 14-3-3c and g as binding partners and that the binding is likely mediated through direct protein-protein interactions. Binding of 14-3-3 to LRRK2 depends on phosphorylation of S935 14-3-3 proteins are a family of conserved regulatory molecules that control various cellular functions by directly binding target signaling proteins, including kinases, phosphatases and receptors [15]. 14-3-3 binding was found frequently in a phospho-serine dependent manner and occurs in a consensus motif (RSXpSXP and RXY/FXpSXP) [16]. We next investigated whether pS935 of LRRK2 is relevant to the binding of 14-3-3 (although S935 surrounding sequence does not fit well with the known consensus motif). First, we transfected HEK-293T cells with plasmids containing Myc-14-3-3c and FLAG-LRRK2 or FLAG-LRRK2- S935A. We found that anti-Myc antibody co-immunoprecipitated 14-3-3c with FLAG-LRRK2 but not FLAG-LRRK2-S935A (Figure 3A). Reciprocal Co-IP assay using anti-FLAG antibody showed a similar result that 14-3-3 was pulled down only with LRRK2 wild type but not S935A mutant (Figure 3B). To further test the role of phophorylated S935 in binding, we incubated purified GST-14-3-3c and BAC-FLAG-LRRK2 transgenic mouse brain lysate in the presence of phospho-peptide QRHSNpSLGP (containing pS935) and non-phospho-peptide QRHSNSLGP (control) at various concentrations. GST-14-3-3c pulled down FLAG-LRRK2 in the absence of the peptides; however, adding the pS935-containing peptide effectively inhibited the co-precip- itation of GST-14-3-3c with FLAG-LRRK2 in a dosage- dependent manner. By contrast, the control peptide has no inhibitory effect toward the pull-down of GST-14-3-3c and FLAG-LRRK2 under the same conditions (Figure 3C). These PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17153