br A Heterozygous P R Mutation in PP A Aa
A Heterozygous P179R Mutation in PP2A-Aa Impacts PP2A Holoenzyme Assembly in Human Cells
To examine if a heterozygous PP2A-Aa missense mutation is sufÞcient to impact PP2A functionality, we introduced a P179R mutation into one allele of endogenous PPP2R1A in RPE-1 cells. The P179R mutation was selected because it is the most prevalent missense mutation in uterine tumors, which have the highest incidence of PP2A-Aa alterations (Cerami et al., 2012). We used adeno-associated virus-mediated gene tar-geting (Berdougo et al., 2009) to introduce a C to G mutation in exon Þve of PPP2R1A (Figure 2A) and iso-lated two independent heterozygous clones (Figure 2B). The mutation did not alter the levels of PP2A-Aa or PP2A-Aa/b (Figure 2C). Similarly, PP2A-Aa immunoprecipitates from WT and PP2A-AaP179R/+ Cefepime had equivalent levels of PP2A-C (Figure S1A) and phosphatase activity (Figure S1B). By contrast, we observed near-2-fold reductions in PP2A-Aa association with B56g, d, and ε (Figures 2D, 2E, and S1C) and B55a
Figure 1. Quantitative Proteomic Characterization of Cancer-Associated PP2A-Aa Mutations
(C) Western blot analysis of cells expressing GFP-PP2Aa-WT or indicated mutants. Solid line indicates intervening lanes have been removed.
(D and E) GFP immunoprecipitates from isotopically labeled RPE-1 cells expressing GFP-PP2A-Aa (WT, P179R, or R183W) were analyzed by mass spectrometry. Volcano plots with the mean log2 fold-change of proteins bound to mutant versus GFP-PP2Aa-WT against Ðlog10 p value. 2-fold change (vertical dashed lines); p < 0.05 (horizontal dashed lines); red and blue circles indicate PP2A regulatory and catalytic subunits respectively.
(F) Heatmap of proteins with signiÞcant changes in association. Green to red gradient represents the mean log2 fold-change. X, protein not detected.
(Figures S1D and S1E). Consistent with a decrease in PP2AB56 holoenzyme levels, intracellular targeting of both PP2A-Aa and B56a to the centromere or kinetochore was reduced in PP2A-AaP179R/+ cells (Figures 2F and 2G). Collectively, these results indicate that a heterozygous P179R mutation in PP2A-Aa is sufÞcient to alter the level of a subset of PP2A holoenzymes.
PP2A-AaP179R/+ Cells Suppress Multipolar Cell Division after Cytokinesis Failure
PPP2R1A mutations are enriched in tumors that experience WGD (Zack et al., 2013). Given that the levels of PP2AB55 and PP2AB56 holoenzymes were reduced in PP2A-AaP179R/+ cells, and that each of these holoen-zyme families contribute to cell division (Craney et al., 2016; Espert et al., 2014; Foley et al., 2011; Kitajima et al., 2006; Lee et al., 2017; Nijenhuis et al., 2014; Schmitz et al., 2010), we considered the possibility that the P179R mutation in PPP2R1A might impact cells in mitosis following WGD. To test this, we induced one round of cytokinesis failure (Yang et al., 2008) by treating cells with cytochalasin D or B, inhibitors of actin polymerization (Cooper, 1987), or with blebbistatin, a myosin II inhibitor (Straight et al., 2003). Binucleate cells were imaged live during the ensuing mitosis (Figure 3A). To avoid a G1 cell-cycle arrest triggered by cytokinesis failure (Andreassen et al., 2001), we treated Tp53+/+ cells with p38 inhibitor SB 203580 (Cuenda et al., 1995) or utilized Tp53 / cells. Almost 30% of WT cells treated with cytochalasin D underwent multipolar cell divisions. Strikingly, PP2A-AaP179R/+ cells exhibited 4-fold reduction in the frequency of multipolar cell divisions compared with WT cells (7% in clone a and 8% in clone b) (Figures 3B and 3C). PP2A-AaP179R/+ cells also underwent fewer multipolar cell divisions when cytokinesis failure was induced with cytochalasin B or blebbistatin (Figure 3C). Similar results were observed in Tp53 / PP2A-AaP179R/+ cells (Figure 3D). Notably, whereas the expression of PP2A-Aa -P179R in WT cells had no impact on the outcome of cell division, the expression of GFP-PP2A-Aa-WT in PP2A-AaP179R/+ cells partially rescued the cell division phenotype (Figures 3E and 3F), suggesting that the observed phenotype is likely due to haploinsufÞciency.