Phosphorylation and 14-3-3 Binding of LRRK2 and PD Figure 2. Identification of 14-3-3s as LRRK2 interaction proteins. (A) Coomassie blue staining showing the affinity-purified FLAG-LRRK2 and its binding proteins from FLAG-LRRK2 transgenic brain. The positions of the FLAG-LRRK2 and 14-3-3 bands are labeled with arrows. Wild-type mouse brain was used as the control for background proteins during purification. (B) Western blot analysis was performed to detect the presence of various 14-3-3 isoforms in association with the affinity purified FLAG-LRRK2 protein. Antibodies against specific isoforms of 14-3-3 were used. Wild type mouse tissue was used as the control. (C) FLAG-LRRK2 and different Myc-14-3-3 isoform plasmids were co-transfected into HEK-293T cells and Immunoprecipitation was performed using anti-Myc antibody, followed by the analysis of protein levels using indicated antibodies. (D) FLAG-LRRK2 plasmid was transfected into HEK-293T cells and transfected cell lysate was incubated with different purified GST-14-3-3 isoforms as indicated. The GST-pull down assay was assayed by Western blot analysis of the FLAG-LRRK2. doi:10.1371/journal.pone.0017153.g002 contrast to wild type 14-3-3, mutant K50E or V181D completely lost the binding to LRRK2 as shown in experiments that either use anti-FLAG or anti-Myc antibody for the co-IP (Figure 3 D, E), consistent with the notion that 14-3-3 binding depends on phospho-serine in LRRK2. To study the relationship of 14-3-3 binding and S935 phosphorylation, we transfected FLAG-LRRK2 in the absence or presence of 14-3-3 wild type or mutant K50E/V181D, which was previously shown as a dominant negative mutant (DN) that abolishes wild type14-3-3 binding to the target proteins [19]. The result showed that the relative pS935 levels, as measured with anti- pS935 staining over anti-FLAG staining, were significantly higher with co-transfection of Myc-14-3-3c and FLAG-LRRK2 than FLAG-LRRK2 transfection alone. In contrast, co-transfection of FLAG-LRRK2 with Myc-14-3-3 DN markedly reduced the relative pS935 levels of LRRK2 (Figure 3F). These results suggest that 14-3- 3 binding prevents the dephosphorylation of LRRK2 at S935. S935A mutation does not affect LRRK2 dimerization One of the important roles of 14-3-3 binding is to provide structural support for the target protein dimer formation via the self-dimerization of 14-3-3 proteins [20]. Since LRRK2 is known to form dimers in vitro and in vivo [9,10,11], we next investigated whether 14-3-3 interaction of LRRK2 is important for the dimerization of LRRK2. HEK-293T cells were co-transfected with HA-tagged LRRK2 and FLAG-LRRK2 or FLAG-LRRK2 S935A mutant. The reciprocal co-IP experiments show that FLAG-LRRK2 wild type and FLAG-LRRK2 S935A mutant did PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e17153