Archives

  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • br Program in Biochemistry and Structural Biology Cell and

    2019-09-23

    
    1Program in Biochemistry and Structural Biology, Cell and Developmental Biology, and Molecular Biology, Weill Cornell Medicine Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA
    2Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
    3Molecular Pharmacology Program, Memorial Sloan
    Kettering Cancer Center,
    4Lead Contact
    *Correspondence:
    [email protected]
    This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
    phosphatase 2A (PP2A), is unknown. PP2A is a major source of serine/threonine phosphatase activity in eukaryotic cells. In the PP2A heterotrimer, a catalytic subunit (PP2A-Ca/b) and a ÔÔscaffoldingÕÕ subunit (PP2A-Aa/b) are targeted to substrates by four evolutionarily conserved families of regulatory subunits. PP2A inactivation has been previously linked to tumorigenesis with the discovery that the SV40 small t antigen blocks the binding of PP2A-Aa/b to regulatory subunits (Pallas et al., 1990), leading to cellular transformation (Chen et al., 2004). Potentially similar perturbations in PP2A have been found to positively correlate with WGD in tumors. These include homozygous deletion of PPP2R2A, which encodes the B55a regulatory subunit, and heterozygous missense mutations in PPP2R1A, which encodes the a isoform of the scaffolding subunit PP2A-A, that accounts for 90% of total PP2A-A (Zhou et al., 2003). Hotspot mutations in PP2A-Aa are expected to prevent B55 and B56 regulatory subunit binding (Cho and Xu, 2007; Xu et al., 2006, 2008) and thus likely decrease the functionality of PP2A-Aa. Indeed, functional inactivation of PP2A by a recurrent P179R mutation in PPP2R1A has been recently implicated as a driver of tumorigenesis in high-grade endometrial carcinoma (Taylor et al., 2019). In other studies, over-expression of certain hotspot PP2A-Aa mutants in tissue culture AUY922 (NVP-AUY922) has been observed to alter phospho-signaling (Haesen et al., 2016; Jeong et al., 2016). However, the impact of PP2A-Aa missense mutations with respect to WGD has not been examined.
    Here we examine the impact of two prevalent hotspot mutations in PPP2R1A, P179R and R183W, on PP2A holoenzyme assembly and Þnd that a reduction in protein-protein interactions predominates and is shared between the two mutants. We then focus on the P179R mutation in PPP2R1A, as this mutation is linked to tumorigenesis (Taylor et al., 2019). When introduced into one allele of endogenous PPP2R1A, the P179R mutation reduces PP2A holoenzyme assembly and intracellular targeting of PP2AB56 in mitosis. Strikingly, we Þnd that these changes are sufÞcient to increase centrosome clustering after WGD, possibly in part through factors at the cell cortex that position centrosomes. Moreover, overexpression of wild-type PP2A-Aa partially rescues centrosome clustering, suggesting that this phenotype arises from a decrease in PP2A activity in mitosis.
    RESULTS
    Characterization of Prevalent Cancer-Associated PP2A-Aa Missense Mutations
    PPP2R1A is most frequently mutated in uterine cancers (Figure 1A), and to explore the cellular impact of the two most frequent PPP2R1A missense mutations (Figure 1B), we generated retinal pigment epithelial (RPE-1) hTERT cell lines expressing GFP-tagged PP2A-Aa wild-type (WT), P179R, or R183W. Each construct was expressed at 30%Ð40% of the level of endogenous PP2A-Aa/b (Figure 1C). Using quantitative mass spectrometry, we compared the composition of PP2A complexes isolated from stable isotope labeling by amino acids in cell culture (SILAC)-labeled cells by immunoprecipitation of WT or mutant GFP-PP2A-Aa. The P179R mutation signiÞcantly reduced PP2A-Aa binding to four B56 regulatory subunits (B56a/ PPP2R5A, B56b/PPP2R5B, B56d/PPP2R5D, and B56ε/PPP2R5E). Accordingly, the binding of proteins that associate with PP2A-Aa via B56 subunits including GEF-H1 (ARHGEF2), Liprina1 (PPFIA1) (Hertz et al., 2016), and MTCL1 (Hyodo et al., 2016) was similarly reduced (Figure 1D). The P179R mutation also signif-icantly reduced binding to the B55d/PPP2R2D regulatory subunit (Figure 1D). The binding of STRN regu-latory subunits (STRN, STRN3, and STRN4), a B00 regulatory subunit (PPP2R3A), and PP2A-C (PPP2CA, PPP2CB) was unaffected (Figure 1D). The R183W mutant had an overall similar decrease in protein-protein interactions (Figure 1E). Notably, although several proteins were observed to have increased binding to a PP2A-Aa mutant, none were shared between the two mutants (Figure 1F). Thus the major impact of both P179R and R183W mutations is to reduce protein-protein interactions.