Journal: Biochemical Journal
Article Title: 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization
Figure Lengend Snippet: Ser 910 and Ser 935 phosphorylation mediate binding of LRRK2 to 14-3-3 ( A ) Endogenous LRRK2 was immunoprecipitated (IP) with anti-LRRK2-(100–500) (S348C) antibody from Swiss 3T3 cells and FLAG–LRRK2 was immunoprecipitated with anti-FLAG–agarose from stable inducible T-REx HEK-293 cells. Immunoprecipitates were subjected to electrophoresis on a 4–12% Novex SDS/polyacrylamide gel and stained with Colloidal Blue. The gel is representative of several experiments. LRRK2 tryptic peptides were subjected to LC-MS/MS on an LTQ-Orbitrap mass spectrometer. M, molecular-mass marker. ( B ) Phospho-peptides identified by LTQ-Orbitrap MS shown in tabular format. Observed mass ( m / z ) and predicted mass (M) are shown, and the site of phosphorylation and peptide sequence are identified. The number of experiments evaluated ( N ) is indicated at the top of the column and the number of times, in total, the phosphorylated peptide was identified is indicated. ( C ) Domain structure of LRRK2 is presented to scale, with amino acid residues indicating domain boundaries indicated. Positions of identified phosphorylation sites are shown. LRR, leucine-rich repeat. ( D ) The indicated phosphorylation sites identified in ( A ) and ( B ) were mutated to an alanine residue and transiently expressed in HEK-293 cells. LRRK2 was immunoprecipitated with anti-FLAG–agarose and equal amounts of each protein were probed with FLAG (total) and the ability to directly bind 14-3-3 was assessed in an overlay assay. 14-3-3 and Hsp90 co-immunoprecipitation (Co-IP) was determined by immunoblotting the immunoprecipitates with the indicated antibodies. Kinase activity was assayed against 30 μM Nictide and specific activity was determined by correcting incorporation of phosphate for protein levels in the immunoprecipitate by quantitative immunoblot using the Odyssey system and is presented as c.p.m./absorbance units (cpm/LICOR AU). The data are the average for duplicate experiments that were repeated four separate times with similar results. ( E ) Streptavidin–agarose was conjugated to a biotinylated di-phosphorylated peptide encompassing Ser 910 (pS910) and Ser 935 (pS935) and incubated in the presence or absence of λ phosphatase in the presence or absence of the EDTA phosphatase inhibitor. The agarose beads were then incubated with HEK-293 cell lysates and interaction of 14-3-3 was assessed after beads were extensively washed and subjected to 14-3-3 immunoblot analysis. ( F ) The indicated forms of FLAG–LRRK2 were expressed in HEK-293 cells by transient transfection. Post-transfection (36 h), these were immunoprecipitated with anti-FLAG antibody and immunoblotted with phospho-specific antibodies against Ser 910 (S357C) and Ser 935 (S814C). Direct binding of immunoprecipitates to 14-3-3 was also assessed by a 14-3-3 overlay assay and co-immunoprecipitation of 14-3-3 and Hsp90 was assessed by immunoblotting with the respective antibodies. Unt., untransfected. ( G ) LRRK2 was immunoprecipitated from tissues of wild-type male C57BL/6 mice and immunoblotted for Ser 910 and Ser 935 phosphorylation and 14-3-3 binding was assessed by overlay assay as in ( F ). ( H ) Multiple sequence alignment of LRRK2 from Homo sapiens (NP_940980), Pan troglodytes (XP_001168494), Mus musculus (NP_080006), Rattus norvegicus (XP_235581), Bos taurus (XP_615760), Canis lupus familiaris (XP_543734) and Gallus gallus (XP_427077). Positions of the phosphorylated residues Ser 910 and Ser 935 are indicated. Identical residues are indicated in grey. ( I ) Sequence comparison of residues surrounding the Ser 910 and Ser 935 phosphorylation sites of human LRRK2.
Article Snippet: The HPLC system was coupled to a linear ion-trap–orbitrap hybrid mass spectrometer (LTQ-Orbitrap XL, Thermo Fisher Scientific) via a nanoelectrospray ion source (Proxeon Biosystems) fitted with a 5 cm Picotip FS360-20-10 emitter.
Techniques: Binding Assay, Immunoprecipitation, Electrophoresis, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Marker, Sequencing, Overlay Assay, Co-Immunoprecipitation Assay, Activity Assay, Incubation, Transfection, Mouse Assay