Phosphorylation and 14-3-3 Binding of LRRK2 and PD Figure 7. PKA Phosphorylation, 14-3-3 binding of LRRK2, and the effect of common familial mutations of LRRK2 in 14-3-3 binding. A schematic model showing PKA (or other kinase) phosphorylation of S910/S935, dimeric14-3-3 binding of LRRK2 at pS910/pS935 sites in wild type LRRK2; PD-linked mutations R1441G, Y1699C or G2019S abolishes or reduces phosphorylation of S910/S935 and impairs 14-3-3 binding. In addition, we propose that dimeric 14-3-3 bind to pS910 and pS935 in the same LRRK2 molecule and binding of 14-3-3 plays little role in LRRK2 dimer formation. doi:10.1371/journal.pone.0017153.g007 Materials and Methods Reagents Modified trypsin and EDTA-free protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Indianapolis, IN). Modified porcine trypsin and chymotrypsin were purchased from Promega Inc. (Madison, WI). LC/MS grade water and acetonitrile (ACN) were purchased from Honeywell Burdick & Jackson (Muekegon, MI). Protein inhibitor tablets and CIAP were purchased from Roche (Branchbury, NJ); FLAG-M2 beads, H89 and Forskolin (FSK) were purchased from Sigma (St. Louis, MO); Site-mutagenesis kit was purchased from Stratagene (Santa Clara, CA); NuPAGE Bis-Tris gels, MOP SDS running buffer, antioxidant, and lipofectamine 2000 kit were purchased from Invitrogen (Carlsbad, CA). GelCode Blue Stain Reagent, Tri- fluoroacetic acid and Tris (2-carboxyethyl)-phosphine hydrochlo- ride were purchased from Pierce (Rockford, IL). The following antibodies were used in this study: FLAG (Sigma, St. Louis, MO), Myc, pGSK-3-alph/beta (S21/9), b-actin and PKA C-alpha (Cell Signaling, Danvers, MA), 14-3-3 isoforms and HA (Santa Cruz, Ca), Phospho-S935 (developed with Cocalico Biological Inc. Reamstown, PA). PKA catalytic subunit was purchased from New England Biolabs (Ipswich, MA). Non-phospho- and Phospho- peptides (QRHSNSLGPC and QRHSNpSLGPC) were synthe- sized with acetylated N-termini and HPLC purified in The Rockefeller University Proteomics Resource Center. Transgenic mice and plasmids Mice were housed in the Center for Comparative Medicine at Mount Sinai School of Medicine. Handling procedures were in accordance with NIH guidelines and approved by the Institutional Animal Care and Use Committees of the institute (IACUC). The institution has Animal Welfare Assurance on file with the Office for laboratory Animal Welfare. The Assurance number is A3111- 01. BAC transgenic mice expressing FLAG-LRRK2-Wt and FLAG-LRRK2-G2019S were described previously [3,21]. The BAC mice expressing FLAG-LRRK2-R1441G or FLAG- LRRK2-K1906M were generated using the similar BAC modi- fication strategy. The following plasmid vectors were used to express LRRK2, 14-3-3 isoforms and LRRK2 fragment (800– 1000aa): 36FLAG and 36Myc plasmid were purchased from Sigma (St. Louis, MO). HA vector was purchased from Santa Cruz (Santa Clara, CA). GST vector was purchased from (GE Healthcare, Piscataway, NJ). Full-length LRRK2 cloning method was described in previous report. The mutations (S935A, R1441G, Y1699C, K1906M and G2019S) were generated by using the Quick-change site-directed mutagenesis kit. PLoS ONE | www.plosone.org 10 March 2011 | Volume 6 | Issue 3 | e17153