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. Author manuscript; available in PMC: 2020 Mar 20.
Published in final edited form as: Oncogene. 2019 Sep 20;39(3):703–717. doi: 10.1038/s41388-019-1012-2

Figure 2. Aα R183W alters the PP2A interactome and decreases PP2A activity.

Figure 2.

(A) AP-MS analysis of V5 tagged wild type or R183W Aα protein from SW620 cells. Data is represented as a dot plot: the color of the circle indicates the spectral count values, circle size displays the relative abundance of preys (interacting proteins) between baits (WT and R183W mutant Aα protein, finally, the colored edge of the circles indicates the FDR or confidence of the interaction. Statistical analysis performed by SAINT and visualization performed using ProHits-viz (n=3). (B and C) Co-immunoprecipitation of V5-tagged wild type or R183W Aα protein to determine binding of select PP2A family members from SW620 cells (B) and M/R cells (C). (D and E) Quantification of relative binding of PP2A subunit family members from co-immunoprecipitation of V5-tagged wild type or R183W Aα protein in SW620 (D) or M/R (E) cells. Binding normalized to V5 levels and plotted relative to wild type Aα, n=3 (Multiple T-tests, with Holm-Sidak method of correction for multiple comparisons: p-values: *<0.05, **≤0.01, ***≤0.001, ****≤0.0001). (F) PP2A activity assay of WT (purple) and mutant R183W (red) Aα holoenzymes. Briefly, V5-tagged WT and R183W Aα protein was co-immunoprecipitated and the resulting IPs, including co-immunopreciptated proteins, were incubated for 15 minutes with increasing concentrations of DiFMUP and analyzed for activity by fluorescence, data presented as the mean ± SD (n=3). (G) Immunoblot analysis of select PP2A subunits from cytoplasmic and nuclear fractionation of cell lysates from M/R cells expressing V5-tagged wild type or R183W Aα protein, showing that localization of the mutant protein and subsequent subunits is unchanged relative to wild type. A representative immunoblot is shown, (n=3). (H) Free energy surface (FES) plot of the WT Aα subunit, as calculated from Well Tempered-metadynamics simulations. The two minima have been labelled as A and B. The representative structure of the clustered conformations in the minima has been illustrated. In the minimum, the side chain of R183 makes interactions with the side chain of N211. This interaction is facilitated by an ion pair interaction between R182 and D215. (I) Free energy surface (FES) plot of the R183W mutant Aα subunit, as calculated from Well Tempered-metadynamics simulations. The deepest minimum has been labelled as A. The representative structure of the clustered conformations in the minima has been illustrated. In the minimum, the side chain of R183W mutant makes interactions with the backbone carbonyl oxygen atom of S219. The loss of interactions as a result of the mutation pushes the A subunit into a conformation that is not observed in the WT R183 PP2A Aα subunit.