Quantitative proteomics and phosphoproteomics of PP2A-PPP2R5D variants reveal deregulation of RPS6 phosphorylation via converging signaling cascades
2023; Elsevier BV; Volume: 299; Issue: 9 Linguagem: Inglês
10.1016/j.jbc.2023.105154
ISSN1083-351X
AutoresKali A. Smolen, Cinta M. Papke, Mark R. Swingle, Alla Musiyenko, Chenchen Li, E. Alan Salter, Ashley D. Camp, Richard E. Honkanen, Arminja N. Kettenbach,
Tópico(s)Cellular transport and secretion
ResumoGenetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients. Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients. Phosphoprotein phosphatase 2 regulatory protein 5D (PPP2R5D)-related intellectual disability (ID) and developmental delay disorder (OMIM#616355) is a syndrome characterized by mild to severe neurodevelopmental delay. The disorder, also known as Jordan's Syndrome, is characterized by ID, epilepsy, macrocephaly, autism-spectrum-disorder (ASD), hypotonia, and delayed motor skill development (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar, 2Loveday C. Tatton-Brown K. Clarke M. Westwood I. Renwick A. Ramsay E. et al.Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth.Hum. Mol. Genet. 2015; 24: 4775-4779Crossref PubMed Scopus (64) Google Scholar, 3Mirzaa G. Foss K. Nattakom M. Chung W.K. PPP2R5D-related neurodevelopmental disorder.in: Adam M.P. Ardinger H.H. Pagon R.A. Wallace S.E. Bean L.J.H. Stephens K. GeneReviews® [Internet]. University of Washington, Seattle; 1993–2020, Seattle, WA2019Google Scholar, 4Shang L. Henderson L.B.B. Cho M.T.T. Petrey D.S.S. Fong C.T.C.-T. Haude K.M.M. et al.De novo missense variants in PPP2R5D are associated with intellectual disability, macrocephaly, hypotonia, and autism.Neurogenetics. 2016; 17: 43-49Crossref PubMed Scopus (50) Google Scholar). PPP2R5D-related developmental disorder is autosomal dominant, arising from de novo germline missense mutations in the PPP2R5D gene (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar, 2Loveday C. Tatton-Brown K. Clarke M. Westwood I. Renwick A. Ramsay E. et al.Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth.Hum. Mol. Genet. 2015; 24: 4775-4779Crossref PubMed Scopus (64) Google Scholar, 5Fitzgerald T.W. Gerety S.S. Jones W.D. van Kogelenberg M. King D.A. McRae J. et al.Large-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228Crossref PubMed Scopus (804) Google Scholar). PPP2R5D encodes a regulatory subunit of type 2A serine/threonine phosphoprotein phosphatase (PP2A). Most PP2A phosphatases function as heterotrimeric holoenzymes, which are ubiquitously expressed. To generate each PP2A holoenzyme, a unique regulatory-targeting (B) subunit is assembled with a common core dimer consisting of a scaffolding (A) subunit and a catalytic (C) subunit (6Brautigan D.L. Protein Ser/Thr phosphatases--the ugly ducklings of cell signalling.FEBS J. 2013; 280: 324-345Crossref PubMed Scopus (181) Google Scholar, 7Brautigan D.L. Shenolikar S. Protein serine/threonine phosphatases: keys to unlocking regulators and substrates.Annu. Rev. Biochem. 2018; 87: 921-964Crossref PubMed Scopus (104) Google Scholar) (Fig. 1A). The A-C core dimer is expressed in most, if not all, human cells, and humans express two highly similar isoforms of both the C and A subunits (PPP2CA/PPP2CB and PPP2R1A/PPP2R1B, respectively). There are four families of B-subunits (B, B', B'', and B'''/Striatin), and each family has several members (Fig. 1A). Some B subunits are widely expressed, while the expression of others is restricted to a subset of cell types (8Janssens V. Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1595) Google Scholar). PPP2R5D encodes the delta isoform of the B'-family subunit and is called B'δ, B56δ, PR61, and PPP2R5D in the literature. PPP2R5D/B56δ is ubiquitously expressed, with slightly higher levels reported in the brain, breast, testis, and gastrointestinal tissue (9McCright B. Rivers A.M. Audlin S. Virshup D.M. The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm.J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 10Wilhelm M. Schlegl J. Hahne H. Gholami A.M. Lieberenz M. Savitski M.M. et al.Mass-spectrometry-based draft of the human proteome.Nature. 2014; 509: 582-587Crossref PubMed Scopus (1412) Google Scholar, 11Schmidt T. Samaras P. Frejno M. Gessulat S. Barnert M. Kienegger H. et al.ProteomicsDB.Nucleic Acids Res. 2018; 46: D1271-D1281Crossref PubMed Scopus (155) Google Scholar). In addition to each B-family having several isoforms, additional isoforms are generated by alternate splicing (8Janssens V. Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1595) Google Scholar, 12Virshup D.M. Protein phosphatase 2A: a panoply of enzymes.Curr. Opin. Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (295) Google Scholar). Therefore, although commonly referred to as PP2A in the literature, combinatorially, the PP2A family may include over a 100 unique holoenzymes (Fig. 1A). To date, over 20 germline mutations in the PPP2R5D coding region have been reported, generating pathogenic missense mutations of 13 amino acids (13Landrum M.J. Lee J.M. Benson M. Brown G.R. Chao C. Chitipiralla S. et al.ClinVar: improving access to variant interpretations and supporting evidence.Nucleic Acids Res. 2018; 46: D1062-D1067Crossref PubMed Scopus (2104) Google Scholar) (Fig. 1B and Table S1). Many pathogenic variants are charge reversal changes, in which a negatively charged acidic amino acid (e.g., glutamic acid, E) is mutated to a positively charged basic amino acid (e.g., lysine or arginine, K or R) due to a single base genomic alteration (e.g., glutamate (E), encoded by GAA or GAG, is converted to lysine (K), encoded by AAA or AAG). The most frequently observed pathogenic changes are p.Glu198Lys (E198K), p.Glu200Lys (E200K), p.Glu420Lys (E420K), and p.Asp251X (D251X, with X representing Ala, His, Val, or Tyr). Patients with E198K or E420K variants, both predicted to be "probably damaging" mutations using PolyPhen-2 (14Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. et al.A method and server for predicting damaging missense mutations.Nat. Methods. 2010; 7: 248-249Crossref PubMed Scopus (10006) Google Scholar), often display severe clinical symptoms (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar, 15Biswas D. Cary W. Nolta J.A. PPP2R5D-related intellectual disability and neurodevelopmental delay: a review of the current understanding of the genetics and biochemical basis of the disorder.Int. J. Mol. Sci. 2020; 21: 1286Crossref PubMed Scopus (24) Google Scholar). The E198 residue, its surrounding region, and much of the protein core are conserved between PPP2R5C/B56γ and PPP2R5D/B56δ (Fig. S1), and the crystal structure of a trimeric PP2A holoenzyme containing PPP2R5C (B56γ) has been solved (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar, 16Xu Y. Xing Y. Chen Y. Chao Y. Lin Z. Fan E. et al.Structure of the protein phosphatase 2A holoenzyme.Cell. 2006; 127: 1239-1251Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 17Cho U.S. Xu W. Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme.Nature. 2007; 445: 53-57Crossref PubMed Scopus (357) Google Scholar, 18Xu Y. Chen Y. Zhang P. Jeffrey P.D. Shi Y. Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation.Mol. Cell. 2008; 31: 873-885Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In the PPP2R5C structure, the amino acids corresponding to E197, E198, and E200 in PPP2R5D reside in an acidic loop between α-helices 3 and 4. This loop is in close contact with the catalytic metals contained in the C-subunit of the PP2A-core dimer (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar, 16Xu Y. Xing Y. Chen Y. Chao Y. Lin Z. Fan E. et al.Structure of the protein phosphatase 2A holoenzyme.Cell. 2006; 127: 1239-1251Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 17Cho U.S. Xu W. Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme.Nature. 2007; 445: 53-57Crossref PubMed Scopus (357) Google Scholar, 18Xu Y. Chen Y. Zhang P. Jeffrey P.D. Shi Y. Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation.Mol. Cell. 2008; 31: 873-885Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), suggesting a role in the regulation of catalytic activity (Fig. 1, C–E). The prevalence of pathogenic variants in the corresponding loop in PPP2R5D indicates that the acidic loop is important for holoenzyme function (Fig. 1, B–E). Homology models also suggest that other pathogenic variants (e.g., E197K, E200K, P201R, W207R, and E420K) are positioned on the surface, facing the active site of the catalytic subunit (Fig. 1, C–E) (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar, 4Shang L. Henderson L.B.B. Cho M.T.T. Petrey D.S.S. Fong C.T.C.-T. Haude K.M.M. et al.De novo missense variants in PPP2R5D are associated with intellectual disability, macrocephaly, hypotonia, and autism.Neurogenetics. 2016; 17: 43-49Crossref PubMed Scopus (50) Google Scholar, 15Biswas D. Cary W. Nolta J.A. PPP2R5D-related intellectual disability and neurodevelopmental delay: a review of the current understanding of the genetics and biochemical basis of the disorder.Int. J. Mol. Sci. 2020; 21: 1286Crossref PubMed Scopus (24) Google Scholar). The greater severity of symptoms observed in patients with the E198K variant compared to patients with the E200K variant may indicate a more significant role for E198 in PP2A function (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar, 4Shang L. Henderson L.B.B. Cho M.T.T. Petrey D.S.S. Fong C.T.C.-T. Haude K.M.M. et al.De novo missense variants in PPP2R5D are associated with intellectual disability, macrocephaly, hypotonia, and autism.Neurogenetics. 2016; 17: 43-49Crossref PubMed Scopus (50) Google Scholar, 15Biswas D. Cary W. Nolta J.A. PPP2R5D-related intellectual disability and neurodevelopmental delay: a review of the current understanding of the genetics and biochemical basis of the disorder.Int. J. Mol. Sci. 2020; 21: 1286Crossref PubMed Scopus (24) Google Scholar). Previous biochemical studies have reported that when expressed in vitro as a tagged fusion protein in HEK298T (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar), overexpression of PPP2R5D E198K increases the phosphorylation of PP2A-B56δ substrates, such as serine 9 of GSK-3β (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar). These observations are consistent with dominant-negative suppression of PPP2R5D-dependent PP2A activities (1Houge G. Haesen D. Vissers L.E.L.M. Mehta S. Parker M.J. Wright M. et al.B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability.J. Clin. Invest. 2015; 125: 3051-3062Crossref PubMed Scopus (84) Google Scholar). To decipher the molecular events associated with clinically relevant PPP2R5D variants, we adapted genomic editing methods to establish human cell lines recapitulating known pathogenic variants in PPP2R5D, allowing the study of how the expression and actions of endogenous variant proteins alter normal biological processes. Employing a fourth-generation single-base editing system (BE4-Gam), we previously generated heterozygous E420K variant cell lines (19Papke C.M. Smolen K.A. Swingle M.R. Cressey L. Heng R.A. Toporsian M. et al.A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth.J. Biol. Chem. 2021; 296100313Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). We reported that the endogenous E420K variant in HEK293 cells is associated with increased phosphorylation levels of several PPP2R5D substrates (19Papke C.M. Smolen K.A. Swingle M.R. Cressey L. Heng R.A. Toporsian M. et al.A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth.J. Biol. Chem. 2021; 296100313Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). However, the most common pathogenic variant (E198K) could not be generated using BE4-Gam without also causing nonsynonymous bystander mutations, likely because BE4-Gam has a multibase editing window and the desired c.592G>A mutation is located in a region surrounded by multiple cytosines encoding E197, D199, and E200. To overcome this technical challenge, we adapted a CRISPR-PRIME system, which utilizes a catalytically impaired Cas9 nickase (H840A mutant) fused to reverse transcriptase (RT). CRISPR-PRIME editing uses a synthetic prime editing guide RNA (pegRNA), which both specifies the target site and encodes a template encoding the desired edit (Figs. S2 and S3). The methods described herein proved useful for introducing the desired nucleotide change (c.592G>A: p.E198K) in the genome of HEK293 cells and can likely be adapted to generate cell lines with almost any single base change. To investigate changes in PP2A-PPP2R5D function in E198K heterozygous cells, we performed quantitative proteomic and phosphoproteomic analyses to determine protein and phosphorylation abundance changes in WT, E198K, and E420K HEK293 cells. Our studies reveal both unique and common deregulated phosphorylation events in PPP2R5D E198K and E420K heterozygous cell lines. Intriguingly, we identify increased phosphorylation of an activating threonine (T202) and tyrosine (Y204) on extracellular signal-regulated kinase (ERK) (p44/p42 MAPK), an activating threonine (T389/T412) in p70 ribosomal protein S6 kinase (p70S6K/RPS6KB1/2), activating serines (S221 and S380) of p90 ribosomal protein S6 kinase 1 (RSK1/RPS6KA1), and (S232 and S389) of p90 ribosomal protein S6 kinase 6 (RSK4/RPS6KA6) with a corresponding increase in the phosphorylation of a common substrate (RPS6) as shared in both variant cell lines (20Ruvinsky I. Meyuhas O. Ribosomal protein S6 phosphorylation: from protein synthesis to cell size.Trends Biochem. Sci. 2006; 31: 342-348Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). RPS6 plays a key role in global protein synthesis, ribosomal biogenesis, maturation of pre-rRNA, and translation (21Bohlen J. Roiuk M. Teleman A.A. Phosphorylation of ribosomal protein S6 differentially affects mRNA translation based on ORF length.Nucleic Acids Res. 2021; 49: 13062-13074Crossref PubMed Scopus (27) Google Scholar, 22Yi Y.W. You K.S. Park J.S. Lee S.G. Seong Y.S. Ribosomal protein S6: a potential therapeutic target against cancer?.Int. J. Mol. Sci. 2021; 23: 48Crossref PubMed Scopus (36) Google Scholar). RPS6 has also been implicated in several other cellular processes including apoptosis, the response to DNA damage, cellular proliferation and migration, and more (20Ruvinsky I. Meyuhas O. Ribosomal protein S6 phosphorylation: from protein synthesis to cell size.Trends Biochem. Sci. 2006; 31: 342-348Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). Yet, while it is well-established that RPS6 is indispensable, the precise role of RPS6 phosphorylation has yet to be elucidated (22Yi Y.W. You K.S. Park J.S. Lee S.G. Seong Y.S. Ribosomal protein S6: a potential therapeutic target against cancer?.Int. J. Mol. Sci. 2021; 23: 48Crossref PubMed Scopus (36) Google Scholar). Although the dysregulation of the upstream regulatory mechanisms differs between the E198K and E420K variant cell lines, inhibition of mTORC1 with rapamycin or of p70S6K with LY2584702 reduces RPS6 phosphorylation in both variant cell lines. These results suggest inappropriate mTORC1 activation may be a common deregulation in PPP2R5D-related neurodevelopmental disorders. Thus, further investigation with rapamycin analogs, such as Everolimus, which has proven safe and effective in treating neuropsychiatric disorders (e.g., tuberous sclerosis complex syndromes (TSC)), as well as LY2584702 treatment, which has been shown helpful to patients with ASD and fragile X, may be warranted to evaluate their potential as therapeutic options for patients with the most common PPP2R5D variants (23Bhattacharya A. Mamcarz M. Mullins C. Choudhury A. Boyle R.G. 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Swingle M.R. Cressey L. Heng R.A. Toporsian M. et al.A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth.J. Biol. Chem. 2021; 296100313Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). To address this key question, we employed PE3b, a third-generation prime editor (Figs. S2 and S3), to introduce a single transition (c.592G>A) into the genome of HEK293 cells, generating E198K cell lines containing the most common pathogenic PPP2R5D variant, for comparison with a previously generated E420K line (26Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (2248) Google Scholar). For PRIME editing, we designed a pegRNA that positions a nicking Cas9 mutant (nCas9) to target the to-be-edited strand at a specific genomic locus and also encodes the desired edit via a RT template and primer binding site contained within its sequence. We synthesized a plasmid expressing both the pegRNA and a secondary nicking sgRNA containing a guide sequence designed to target nicking of the unedited strand only after the pegRNA/RT–mediated single strand edit was made. Upon expression of the PRIME editor system, the sense strand 5′ of the E198 coding sequence is nicked in the PPP2R5D exon 5 (pegRNA nick in Fig. S2), and the K198 mutation is introduced via reverse transcription. Following flap resolution, the sgRNA selectively guides nicking of the unedited antisense strand in edit-containing duplexes (sgRNA nick in Fig. S2) to stimulate mismatch repair using the edited strand as the template. We used electroporation to introduce the plasmid into the cells. The cells were grown for 48 h to ensure cell replication had occurred, which made the mutation permanent in the genome. The cells were then single-cell sorted, and the emerging colonies were screened for the desired c.592G>A base change in a single allele (E198K-heterozygous) by Sanger sequencing (Fig. S3). To ensure the clonal cell lines with the desired mutation were homogenous, each confirmed E198K line was single-cell sorted two additional times before further analysis. Complete genomic exon sequencing of parental, E198K-het-, and E420K-het-variant lines was performed to detect potential off-target editing and spontaneous somatic mutations that could have occurred during repeated single-cell sorting, as previously described (19Papke C.M. Smolen K.A. Swingle M.R. Cressey L. Heng R.A. Toporsian M. et al.A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth.J. Biol. Chem. 2021; 296100313Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Cell lines with off-target or enriched somatic mutations in protein-coding regions were discarded. Both mass spectrometry (MS) and Western blot analysis of total lysates from the parental (WT), the E198K, and E420K cell lysates revealed similar PPP2R5D expression (Figs. 1F, S4 and S5). Furthermore, the amount of PPP2R2A, PPP2R5A, and the PP2A scaffolding subunit were not significantly changed (Fig. S4). However, by Western blotting, we detected a slight increase in the levels of PP2AC (Fig. S4). To determine if the expression of PPP2R5D variants affects PP2A holoenzyme stability or assembly, we immunoprecipitated endogenous PPP2R5D and probed the precipitates for PPP2R5D and PP2AC (Figs. 1F and S5). We found that in cells expressing PPP2R5D variants, the amount of catalytic subunit that co-IPs with PPP2R5D was reduced by 25.97 ± 7.5% and 56.1 ± 1.1% for the E198K and E420K variants, respectively (Figs. 1F and S5). We also compared the phosphatase activity of PP2A holoenzymes containing WT or variant PPP2R5D (Fig. 1G) using an established fluorescent assay that employs 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a substrate (27Choy M.S. Swingle M. D'Arcy B. Abney K. Rusin S.F. Kettenbach A.N. et al.PP1:Tautomycetin complex reveals a path toward the development of PP1-specific inhibitors.J. Am. Chem. Soc. 2017; 139: 17703-17706Crossref PubMed Scopus (38) Google Scholar, 28Chattopadhyay D. Swingle M.R. Salter E.A. Wood E. D'Arcy B. Zivanov C. et al.Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C.Biochem. Pharmacol. 2016; 109: 14-26Crossref PubMed Scopus (28) Google Scholar, 29Ni L. Swingle M.S. Bourgeois A.C. Honkanen R.E. High yield expression of serine/threonine protein phosphatase type 5, and a fluorescent assay suitable for use in the detection of catalytic inhibitors.Assay Drug Dev. Technol. 2007; 5: 645-653Crossref PubMed Scopus (22) Google Scholar, 30Wegner A.M. McConnell J.L. Blakely R.D. Wadzinski B.E. An automated fluorescence-based method for continuous assay of PP2A activity.Methods Mol. Biol. 2007; 365: 61-69PubMed Google Scholar). For these studies, we immunoprecipitated endogenous PPP2R5D using a surplus of a previously characterized PPP2R5D-specific antibody that recognizes a near C-terminal epitope unique to PPP2R5D (19Papke C.M. Smolen K.A. Swingle M.R. Cressey L. Heng R.A. Toporsian M. et al.A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth.J. Biol. Chem. 2021; 296100313Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). We observed dephosphorylation of DiFMUP in all samples. When normalized to the amount of PPP2R5D immunoprecipitated, less activity was observed for both PPP2R5D variants. These results are consistent with the reduced levels of catalytic subunit in PPP2R5D IPs generated from the variant cell lines. When normalized to the amount of PP2A catalytic subunit detected in the IPs, the phosphatase activity was similar between WT, E198K, and E420K samples. Together, our data is consistent with the reduction in phosphatase activity observed in the IPs of variant cell extracts arising from reduced retention or reduced incorporation of PP2AC into PPP2R5D variant holoenzymes. Analysis of growth rates using a cell proliferation assay, in which cells were plated at equal density and the number of cells were counted over consecutive days, revealed that PPP2R5D variants alter cell growth. When compared to WT HEK293 cells, we observed slower rates of growth in cells heterozygous for PPP2R5D E198K or E420K, with a significant reduction in growth in E198K and E420K variant cells observed by 96 h and 48 h, respectively (Fig. 1H). To obtain an unbiased assessment of how the E198K and E420K PPP2R5D variants affect cellular signaling, we employed quantitative proteomics and phosphoproteomics using the tandem mass tag (TMT)-MS3 approach, as reported previously (19Papke C.M. Smolen K.A. Swingle M.R. Cressey L. Heng R.A. Toporsian M. et al.A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth.J. Biol. Chem. 2021; 296100313Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). HEK293 WT, E198K, and E420K
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