Fig 2: The A-subunit enhances PP2A methylation and interaction with LCMT-1.(A) Influence of the A-subunit, mini-A, and PTPA on PP2A methylation, tested by addition of these proteins in a stoichiometric amount to PP2Ac, prior to determination of the kinetics of PP2A methylation. Representative results were shown with mean ± SEM calculated from triplicate (upper figure panel). Statistical analysis of 4–12 independent experiments was performed and the average Km ± std. dev. and the P value were calculated and summarized in the table below. (B) Binding affinity between LCMT-1, preloaded with SAH, and unmethylated, methylated PP2A core enzyme or free PP2Ac, measured by ITC.

Fig 3: The A-subunit and PTPA synergistically maintain PP2A methylation in mammalian cells.Lysates of HeLa cells with the A-subunit and/or PTPA knockdown were analyzed by western blot prior to or after NaOH treatment using an antibody that specifically recognizes the unmethylated PP2Ac. The level of the unmethylated PP2Ac was determined by the ratio of the signal prior to and after NaOH treatment. The experiments were repeated four times; a representative result was shown with the calculated mean ± St. dev. and P value.

Fig 4: Pedigrees and identified PTPA variants. A and B show the pedigrees of Families 1 and 2, respectively. Filled symbols indicate individuals affected by parkinsonism; unfilled symbols indicate unaffected individuals; arrows denote index cases. AAO = age at onset; ref/var = heterozygous genotype for the corresponding variant; var/var = homozygous variant genotype; ref/ref = wild-type (reference) genotype. (C) PTPA gene schematic representation, including relative positions of the variants identified in this study annotated according to NM_178001. (D) 3D molecular rendering of PTPA, with the relative positions of the variants and the ATP binding and PP2A-C binding sites.

Fig 5: Electrostatic interactions between the A-subunit and LCMT-1.(A) Structural model of the PP2A core enzyme-LCMT-1 complex built from the structure of the PP2Ac-LCMT-1 complex and the A-subunit with morphed conformation. The electrostatic potential for the A-subunit (left panel) and LCMT-1 (right panel) is shown. The corresponding regions on the A-subunit and LCMT-1 for potential electrostatic interactions are circled (i and ii). The A-subunit residues that directly contact LCMT-1 in the model, Arg183 and Arg258, are shown. See also Movie S1. (B) The effect of the A-subunit mutations to Arg183 and Arg258 on methylation of the PP2A core enzyme. Results from 5–7 independent experiments were summarized and the average Km ± std. dev. and the P value were calculated and shown. (C) FRET assay measured changes in the distance between the N- and C-termini of the A-subunit in the core enzyme prior to and after addition of an excess molar amount of LCMT-1 or a stoichiometric amount of PR70 (108–575). Representative results were shown with mean ± SEM calculated from triplicate.