not differ in pulling down together with HA-LRRK2 (Figure 4A, B). Considering S935A mutant of LRRK2 is defective in phosphorylation at 935 and 14-3-3 binding, this result suggest that 14-3-3 binding to LRRK2 does not play a critical role in LRRK2 dimer formation. Rather, the dimerization of LRRK2 is perhaps determined by the sequence outside 14-3-3 binding region [9,10,11]. Common familial PD mutations of LRRK2 cause a reduction in pS935 levels and 14-3-3 binding Binding of 14-3-3 with various kinases plays an important role in regulating downstream signaling [15,19]. Although the LRRK2- mediated signaling pathway has not been clearly elucidated, we sought to investigate whether common PD mutations of LRRK2, including G2019S, R1441G and Y1699C, affect S935 phosphor- ylation and/or 14-3-3 binding of LRRK2. First, we examined the pS935 levels in the PD-linked mutants of LRRK2 expressed in HEK-293T cells. Compared to wild type LRRK2, mutants R1441G and Y1699C isolated from cells have markedly reduced pS935 levels; mutant G2019S and kinase-deficient mutant K1906M did not change significantly in pS935 levels (Figure 5A). However, when we assayed pS935 levels in LRRK2 wild type or mutant proteins isolated from transgenic brains [21], G2019S mutant showed a modest but significant decrease in pS935 levels as compared to LRRK2 wild type (Figure 5B). R1441G mutant from transgenic brain again exhibited a marked reduction of pS935 levels, whereas K1906M had little change in pS935 levels in the brain (Figure 5B). Next, we examined 14-3-3 binding with LRRK2 variants in transfected cells. We transfected HEK-293T cells with Myc-14-3-3c and FLAG-LRRK2 wild type or various mutant. Compared to wild type LRRK2, all three mutants (S935A, R1441G and G2019S) exhibited reduced co-immunoprecipitation with Myc- 14-3-3c, suggesting that these mutants are impaired in 14-3-3 binding. But the degree of impairment in these mutants differs, with S935A affected the most (,90% reduction) and G2019S the least (,20%) (Figure 5C). We further evaluated the alteration of PD mutants in 14-3-3 binding in vitro. We incubated purified GST-14-3-3c proteins with Phosphorylation and 14-3-3 Binding of LRRK2 and PD extracts from HEK-293T cells transfected with FLAG-LRRK2 wild type or various mutants, followed by analysis of FLAG- LRRK2 protein levels pulled down by GST-14-3-3c. The results showed that binding of 14-3-3 to mutant R1441G or Y1699C is markedly reduced as compared to wild type LRRK2, whereas 14- 3-3 binding is affected little in G2019S or K1906M mutant (Figure 5D). The above results indicate a general effect of the above PD-linked mutations on the phosphorylation of S935 and 14-3-3 binding: the reduction of pS935 levels and 14-3-3 binding caused by R1441G and Y1699C is consistent in various assays; while the inhibitory effect of G2019S is modest and assay- dependent. The disparity in G2019S results may reflect the different sensitivity of the various assay systems. It is noteworthy that the K1906M mutation did not alter the levels of pS935 or 14- 3-3 binding of LRRK2 in all assays, suggesting that the LRRK2 kinase activity is not primarily responsible for the phosphorylation of S935 (Figure 5A, B, D). Protein kinase A (but not LRRK2 kinase) causes phosphorylation of S935 in vitro and in cell culture As LRRK2 kinase activity is unlikely the cause for the phosphorylation of S935 on its own (Figure 5), we sought to identify other possible kinases that may be responsible for phosphorylation of S935. A previous study showed that cAMP- dependent protein kinase (PKA) could phosphorylate LRRK2 in vitro but without any knowledge about the phosphorylation sites [22]. We therefore performed kinase assay with purified PKA catalytic subunit and GST fusion protein containing LRRK2 fragment (800–1000aa). The result revealed that PKA phosphor- ylates S935 as detected with anti-pS935 antibody and that the phosphorylation is enhanced with increased input of PKA amount (Figure 6A). In addition, the phosphorylation of S935 is inhibited by PKA inhibitor H89 [23] (Figure 6B). Next, we tested whether or not PKA mediates phosphorylation of S935 in cells. Co- expression of FLAG-LRRK2 and PKA catalytic domain resulted in a significant increase in pS935 levels as compared to that with the transfection of FLAG-LRRK2 alone. Again, the phosphory- lation levels are enhanced with increasing transfected PKA Figure 4. S935A mutation does not affect LRRK2 dimerization. (A, B) FLAG-LRRK2 WT or S935A mutant construct was co-transfected with HA- LRRK2 constructs. The Co-IP assay was performed using either anti-HA (A) or anti-FLAG (B) antibody, followed by Western blot analysis using the indicated antibodies. doi:10.1371/journal.pone.0017153.g004 PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e17153