Phosphorylation and 14-3-3 Binding of LRRK2 and PD LRRK2 variant levels were analyzed by Western blot analysis with the indicated antibodies and quantified using LI-COR and Odyssey system. The data was analyzed by One-way ANOVA (** P,0.01). Data are presented as mean value (6 SEM) from three independent experiments. (D) FLAG-LRRK2-WT, S935A, R1441G, Y1699C, G2019S or K1906M mutant plasmid was transfected individually into HEK-293T cells. Sepharose 4B beads-conjugated GST- 14-3-3c protein was incubated with different transfected cell lysates containing LRRK2-Wt or various mutants. Western blot analysis was performed to determine the LRRK2 variant amount after the GST pull-down assay. The results were quantified as described above. The ratio of pulled down LRRK2 and input LRRK2 was measured and analyzed by One-Way ANOVA (** P,0.01). Data are presented as mean value (6 SEM) from three independent experiments. doi:10.1371/journal.pone.0017153.g005 plasmid amount (Figure 6C). Phosphorylation of GSK, a known substrate of PKA, is used for the positive control of PKA activity in this assay. Furthermore, we used PKA activator forskolin (FSK) to treat cells expressing FLAG-LRRK2. The result showed that administration of FSK at 10 mM caused a significant increase in pS935 levels of LRRK2 likely as a result of enhanced PKA activity as indicated by the increase in pGSK levels (Figure 6D). Finally, in an in vitro kinase assay as described above (Figure 6A), we found that, while purified PKA directly phosphorylates S935 in GST- LRRK2 fragment, purified FLAG-LRRK2 kinase does not cause any change in pS935 levels in GST-LRRK2 fragment (Figure 6E), despite the active kinase activity of FLAG-LRRK2 towards generic substrate MBP (data not shown) [3]. This result further demonstrates that LRRK2 kinase is not responsible for pS935, whereas PKA is a potential upstream kinase that can cause the phosphorylation of LRRK2 at S935. Discussion Our study identifies serial molecular events related to the regulation of LRRK2: multiple novel phosphorylation sites (S910, S912, S935 and S973, all in the N-terminal region), potential phosphorylation of S935 by PKA, and pS935-dependent 14-3-3 binding of LRRK2. These events may represent an important part of a signaling pathway in regulating LRRK2 cellular function under physiological conditions. Furthermore, we show that the phosphorylation status of S935 and 14-3-3 binding of LRRK2 is impaired by the most common familial mutations including G2019S, R1441G and Y1699C. This result provides a new avenue for the study of pathogenic mutations of LRRK2 in PD. Interestingly, while our study was in final stage of submission, Nichols and Alessi’s group reported the phosphorylation of LRRK2 at S910, S935 and S973, and pS910/pS935-dependent 14-3-3 binding of LRRK2. Their study indicated a nearly identical role of S910 and S935 in 14-3-3 binding [24,25]. Furthermore, they examined 41 known LRRK2 mutations and found that five out of six pathogenic mutations (except G2019S) have markedly reduced phosphorylation of S910/S935 and thereby disrupted 14- 3-3 binding of LRRK2. Therefore, our study and theirs independently demonstrate the phosphorylation-dependent bind- ing of 14-3-3 to LRRK2, which may play a critical role in regulating LRRK2 function in vivo; PD-linked mutations impair 14-3-3 interaction with LRRK2 and thus disturb the regulation of LRRK2 by 14-3-3. Our study also provides additional insight into the functional relevance of the LRRK2-14-3-3 interaction. In particular, our results indicate that the most common mutation G2019S also causes a reduction of pS935 levels in LRRK2 from transgenic brain, despite little effect of G2019S seen in LRRK2 protein isolated from cell culture. Our study has further characterized the specific isoforms of 14-3-3 binding of LRRK2 and shown the relevance of 14-3-3 binding to the dimer formation of LRRK2. Thus, there are seven isoforms of 14-3-3 proteins and many 14-3- 3 target proteins only bind to selective isoforms of 14-3-3 [26,27,28]. Although several isoforms 14-3-3 were identified through our mass spectrometric analysis, our data suggest that LRRK2 binds preferentially to c and g form (both are abundant in the brain). This information is important for future biochemical and structural analyses of LRRK2-14-3-3 interac- tions. We have also sought to investigate whether 14-3-3 is a potential substrate of LRRK2, because previous studies had shown that 14-3-3 is a phosphoprotein [29,30]. Our data indicated that purified LRRK2 efficiently phosphorylates generic substrate MBP but not 14-3-3 (data not shown). In addition, because a body of evidence has indicated an important role of 14- 3-3 in assisting target protein dimerization [19], we tested whether 14-3-3 regulates LRRK2 dimer formation. Our data reveal that the LRRK2 mutant defective in phosphorylation of S935/14-3-3 binding is not affected in dimer formation. The result suggests that 14-3-3 does not play a critical role in dimerization of LRRK2; rather, dimerization of LRRK2 is mediated primarily by the sequence outside of the S910/S935 sites [9,10,11]. This result, along with the previous report, is consistent with a model that homo or hetero-dimeric 14-3-3 bind to two separate, neighboring phosphorylation sites (e.g. pS910 and S935) on the same LRRK2 molecule, and disruption of either phosphorylation site would destabilize dimeric 14-3-3 binding of LRRK2 (Figure 7) [25]. We have also examined the potential role of 14-3-3 binding in directing LRRK2 subcellular localization. However, overexpres- sion of wild type LRRK2 or S935A mutant results in largely diffuse localization of each protein in COS-7 or HeLa cells, thus no obvious difference was observed between the wild type and S935A mutant of LRRK2 (our unpublished results). This result, however, is in contrast to the recent report that S935A mutant was associated with punta localization in COS-7 cells [24,25]. It is unclear what causes the discrepancy of the subcellular localization of LRRK2 mutants in the two studies, despite that 14-3-3 binding of S935A mutant LRRK2 was impaired in both studies. An important question that remains to be answered is whether 14-3-3 binding represents a mechanism for LRRK2 trafficking or distribution at different subcompartments of neurons. Finally, our study reveals a potential link of PKA kinase to the phophorylation of LRRK2 at S935, therefore adding a new member to the list of known substrates of PKA that are also 14-3-3 targets [31,32,33,34]. One such an example is the PKA-mediated phosphorylation of S413 and 14-3-3 binding of pS413 in RIM1alpha, which is an active zone protein important for synaptic transmission [35,36]. Future study should investigate in detail whether PKA as well as other kinase activities are responsible for phosphorylation of S910, S912, S935 or S973. Furthermore, previous evidence indicated that the effect of dopamine is largely mediated through cAMP/PKA signaling cascade [37,38]. Given the observation of dopamine transmission deficits in several LRRK2 mutant rodent models [21,39,40,41], the potential connection of PKA-LRRK2 signaling indicates an attractive cellular pathway for the future dissection of the molecular mechanism underlying LRRK2 mutations in the pathogenesis of PD. PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e17153