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. 2020 Mar 27;295(13):4194-4211.
doi: 10.1074/jbc.RA119.011265. Epub 2020 Feb 18.

Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1

Otto Kauko  1 , Susumu Y Imanishi  2 , Evgeny Kulesskiy  3 , Laxman Yetukuri  2 , Teemu Daniel Laajala  4 , Mukund Sharma  1 , Karolina Pavic  2 , Anna Aakula  2 , Christian Rupp  2 , Mikael Jumppanen  2 , Pekka Haapaniemi  2 , Luyao Ruan  5 , Bhagwan Yadav  3 , Veronika Suni  2 , Taru Varila  2 , Garry L Corthals  2 , Jüri Reimand  6 , Krister Wennerberg  3 , Tero Aittokallio  4 , Jukka Westermarck  7
Affiliations

Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1

Otto Kauko et al. J Biol Chem. .

Abstract

Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies.

Keywords: AURK inhibitor; LMNA; cancer; drug screening; nucleophosmin; phosphatase activation-responsive site (PARS); phosphatase inhibition-responsive site (PIRS); phosphoproteomics; protein phosphatase 2 (PP2A); systems biology.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
PPP2R1A or PAIP inhibition regulates largely nonoverlapping phosphosites. A, schematic presentation of PP2A complex and its activity regulation by subunit composition and by endogenous inhibitor proteins (PAIPs). Whereas B-subunits regulate substrate specificity of the trimeric PP2A complex, each PAIP (CIP2A, PME-1, and SET) regulates PP2A complex activity by a distinct mechanism. B, Western blot analysis of selectivity of siRNAs for the studied PP2A proteins in HeLa cells 72 h after siRNA transfection. C, PPPP2R1A or PAIP inhibition causes global shift in HeLa cell phosphoproteome. Shown are fold change distributions of representative replicates (top) and mean log 2-fold changes of all replicates (bottom). Fig. S1H shows fractions of differentially regulated phosphosites for each condition. D, Venn diagram shows the overlap between PPP2R1A and PAIP-regulated peptides at q <0.05. Total number of significantly-regulated peptides are shown in parentheses. E, analysis of targets of different kinases by phosphomotif antibodies arranged from the most PPP2R1A-responsive phosphoproteins (PIRS) to the most SET-responsive phosphoproteins (PARS) in descending order. F, unsupervised soft clustering analysis of the high-confidence phosphoproteome data into six clusters. Cluster membership percentage is indicated by a color scale, and representative peptides are listed inside the plots. Cluster centers (indicated by black dots) exhibit mainly either up-regulation (cluster 2) or down-regulation (clusters 3–6). Cluster 1 instead shows the pattern of phosphoregulation for peptides that respond to both PP2A inhibition and activation. Cluster membership of each clustered phosphopeptide is shown in Table S1.
Figure 2.
Figure 2.
Sequence context analysis of the phosphorylation sites differentially regulated by PPP2R1A or by PAIPs. A–C: PIRS, phosphatase inhibition responsive site; PARS, phosphatase activation responsive site. A, phosphopeptide abundances in control samples (no PP2A modulations) divided by corresponding protein abundances (quantified by ≥2 peptides in the sample without phosphopeptide enrichment) for each cluster. This information was available for 140, 179, 314, 292, 206, and 78 peptides for clusters 1–6, respectively. *, p < 0.05, and ***, p < 0.001, in Tukey-Kramer test for pairwise comparisons of mean values between clusters. B, analysis of the fraction of phosphothreonines of all phosphorylation sites in each cluster reveals significant enrichment in PIRS clusters having lower baseline phosphorylation based on A. Error bars represent 95% confidence intervals. **, p < 0.01, and ***, p < 0.001 in χ2 test. C, analysis of fraction of phosphosites with acidophilic p(S/T)XX(D/E) motif in each cluster shows association with PARS clusters. D, number of peptides with 1–4 phosphosites associated with each cluster.
Figure 3.
Figure 3.
Regulation of cellular processes and pathways. A, cellular processes and pathways enriched in proteins phosphoregulated by PP2A modulations. The majority of enriched cellular functions was associated predominantly with only one cluster, indicating that biological functions can also be roughly divided to be responsive to either PP2A inhibition or activation. B, five most significantly-enriched biological process GO terms based on phosphoproteomes significantly (q <0.05) regulated by CIP2A, PME-1, or SET.
Figure 4.
Figure 4.
PP2A regulates RNA-processing factors and canonical pathways. A, differentially-regulated phosphosites (q <0.05) on proteins in the enriched category “RNA processing and splicing” from g:Profiler analysis. At the level of significantly-regulated peptides, PPP2R1A and PAIPs regulate different splicing factors. B, schematic presentation of SET- and PPP2R1A-regulated phosphosites on NPM. Mut, region most commonly mutated in NPM resulting in nucleoplasmic localization of the protein. C, analysis of nuclear distribution of WT and indicated NPM mutants in transiently-transfected MDA–MB-231 cells. Phosphorylation mimicking D mutants display similar nucleoplasmic localization than known cancer mutants of NPM. Scale bar, 20 μm. D and E, differential association of selected canonical pathways (D), and kinase targets (E) with either PIRS or PARS clusters.
Figure 5.
Figure 5.
Differential spatial distribution of PP2A-modulated targets. A, subcellular localizations of PPP2R1A and PAIPs, retrieved from RRID:SCR_006710. PPP2R1A: CAB018599: U-2 OS, image 2; CIP2A: HPA039570: U-2 OS, image 1; PME-1: CAB004541: U-2 OS, image 1; SET: HPA063683: U-2 OS, image 1. Scale bar, 10 μm. B, analysis of nuclear proteins in each cluster demonstrates association between PARS and nuclear localization of the target protein (clusters 4–6). Based on lower frequency of nuclear proteins in cluster 1 & 2, it is apparent that cytoplasmic proteins are under higher dephosphorylation activity than nuclear proteins. C, fraction of nuclear proteins in the proteins with differentially-regulated phosphopeptides for each condition. A and C, **, p < 0.01, and ***, p < 0.001, in χ2 test. D, differentially-regulated phosphosites on proteins in the enriched category Nuclear envelope organization. Proximity ligation assay of PPP2R1A and Lamin A/C protein association at nuclear lamina is shown in the inset; with PPP2R1A siRNA as a PLA staining specificity control. Red dot indicates close proximity of PPP2R1A and Lamin A/C proteins. Scale bar, 10 μm. E, Chromosome organization indicates an example of chromatin-associated protein complexes regulated dominantly by nuclear PP2A inhibitors SET and PME-1. Inset shows Western blot analysis of DNMT1 Ser-714 phosphorylation from A549 cells transfected with SET siRNA for 72 h. * denotes unspecific band. F, schematic presentation of the observed differential cytoplasm–nuclear association of phosphorylation patterns of PP2A targets. Based on data, highly-phosphorylated peptides in clusters 4–6 that are most responsive to SET and PME-1 inhibition are enriched in nucleus. Highly-phosphorylated peptides associated with cluster 5 & 6 were enriched on CK2 targets. Cytoplasmic peptides instead were more frequently found to be regulated by CIP2A and to be associated with low phosphate occupancy and PKC target sequences. Together, these results indicate for a gradient where cytoplasmic PP2A is more weakly inhibited (e.g. by CIP2A) than nuclear PP2A (e.g. by SET and PME-1).
Figure 6.
Figure 6.
Effects of PPP2R1A and PAIPs in global drug responses. A, drug-sensitivity scores (ΔDSS) of 238 cancer drugs in HeLa cells are ranked by correlation to the dephosphorylation activity index derived from differentially-regulated peptides for each gene, which is represented as the top color bar in A and as a red line in B–G. Correlation rank demonstrates that drugs can be ranked according to the dependence of their response on the type of PP2A modulation. Significant enrichment of selected drug groups based on their dependence is shown in the right panel. B, average ΔDSS over all 238 drugs demonstrating that on average drugs follow the dephosphorylation activity (from S1H) in their efficacy. C–G, selected drug responses for indicated drugs in PP2A-modulated HeLa cells. Shown is mean ± S.D. of response to different drugs targeting the indicated mechanism. The cellular sensitivity for Aurora kinase (AURK) inhibitors (C) and JAK2 inhibitors (D) followed closely the dephosphorylation activity by the same PP2A modulations. HDAC (E) and BET (F) inhibitors constitute prototypic examples of drug classes for which PPP2R1A inhibition drove resistance, whereas the PAIP inhibition did not result in drug sensitization. G, ATP-competitive mTOR inhibitors show paradoxical dependence where both PPP2R1A and SET inhibition sensitize to drug action. Rapalogs show differential dependence from ATP competitive inhibitors.
Figure 7.
Figure 7.
Summary scheme of phosphoregulation rules by PP2A modulations. Based on the results, most phosphosites and cellular processes appear to be sensitive to either phosphatase inhibition or activation. In contrast, only few processes show characteristics of bidirectional regulation. The preferable amino acid context for PIRS and PARS sites is also depicted. PIRS and PARS processes show differential spatial distribution between the cytoplasm and nucleus. Based on these results, efficient pharmacological interference with different cellular processes requires understanding whether those processes are either under PIRS or PARS dominance. As an example, phosphatase activation would mostly affect nuclear processes, whereas cytoplasmic processes would be most amenable to modulation by phosphatase inhibition.
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