FIGURE 2.

PPM1A and PPM1B modulate HTT Ser-421 phosphorylation status downstream of D2R activation. A, phylogenetic tree of the human PP2C family displaying PPM1A and PPM1B (underlined) as a discrete subfamily. The PP2C proteins tested in this study are indicated in black, and non-tested ones are in gray italics. Sequences were aligned using ClustalW Gonnet matrix. B–D and F, HEK293T cells were transiently transfected with 2 μg of HA-D2R and 2 μg of HTTN660-GFP along with 2 μg of the indicated phosphatase. 48 h after transfection, cells were stimulated by 20 μm apomorphine (Apo) for 45 min. Then cells were lysed, and the phospho-Ser-421-specific signal was detected by Western blot. The results presented in F establish that PPM1A dephosphorylates HTT Ser-421 in an extent identical to what is achieved by PPM1B. E, graphic representation of the structural alignment of the Homo sapiens PPM1A (1a6q, orange) and PPM1B (2p8e, pale blue) obtained by the MatchAlign command in PyMOL (amino acids 3–305). The resulting alignment generated a root mean square deviation of 0.307. In red are the manganese and phosphate, respectively, with which these phosphatases have been co-crystallized. All data presented here are derived from 4–7 sets of independent experiments.