Substitution of Ser171 to Glu (SET-S171E) in SET compromised its inhibitory effect on PP2A.

<p>Substitution of Ser171 to Glu (SET-S171E), which mimics phosphorylated SET at Ser171 residue, reduced its inhibitory effect on PP2A compared with Ser171 to Ala substitution (SET-S171A) or wild-type SET did. (A) The phosphatase activity of PP2A (37 µU) under the presence of indicated concentration of recombinant SET was monitored by using ³²P-labeled MBP as a substrate. After SET proteins and PP2A were preincubated for 15 min at 4°C, ³²P-MBP was added and incubated for 10 min at 37°C. The reaction mixture was separated with SDS-PAGE. The inhibitory effect of SET proteins on PP2A activity was visualized by exposing the SDS-PAGE gel with PP2A-treated ³²P-MBP to X-ray film. The data shown are representative results from three independent experiments with similar results. (B) The relative PP2A activity to the radioactivity of ³²P-MBP without SET (assigned to be 1) was calculated from the radioactivity of MBP bands in the dried gel shown in (A). Values represent the means ±SD from three independent experiments.