Skip to main content
SpringerLink
Log in

Cell Cycle–Specific Protein Phosphatase 1 (PP1) Substrates Identification Using Genetically Modified Cell Lines

  • Protocol
  • First Online:
Cell Cycle Control

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2740))

Abstract

The identification of protein phosphatase 1 (PP1) holoenzyme substrates has proven to be a challenging task. PP1 can form different holoenzyme complexes with a variety of regulatory subunits, and many of those are cell cycle regulated. Although several methods have been used to identify PP1 substrates, their cell cycle specificity is still an unmet need. Here, we present a new strategy to investigate PP1 substrates throughout the cell cycle using clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 genome editing and generate cell lines with endogenously tagged PP1 regulatory subunit (regulatory interactor of protein phosphatase one, RIPPO). RIPPOs are tagged with the auxin-inducible degron (AID) or ascorbate peroxidase 2 (APEX2) modules, and PP1 substrate identification is conducted by SILAC proteomic-based approaches. Proteins in close proximity to RIPPOs are first identified through mass spectrometry (MS) analyses using the APEX2 system; then a list of differentially phosphorylated proteins upon RIPPOs rapid degradation (achieved via the AID system) is compiled via SILAC phospho–mass spectrometry. The “in silico” overlap between the two proteomes will be enriched for PP1 putative substrates. Several methods including fluorescence resonance energy transfer (FRET), proximity ligation assays (PLA), and in vitro assays can be used as substrate validations approaches.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wang B, Wu H, Hu C et al (2021) An overview of kinase downregulators and recent advances in discovery approaches. Sig Transduct Target Ther 6:1–19. https://doi.org/10.1038/s41392-021-00826-7

    Article  CAS  Google Scholar 

  2. Novak B, Kapuy O, Domingo-Sananes MR, Tyson JJ (2010) Regulated protein kinases and phosphatases in cell cycle decisions. Curr Opin Cell Biol 22:801–808. https://doi.org/10.1016/j.ceb.2010.07.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Morgan DO (2007) The cell cycle: principles of control. New Science Press, London

    Google Scholar 

  4. Manning G, Whyte DB, Martinez R et al (2002) The protein kinase complement of the human genome. Science (1979) 298:1912–1934

    CAS  Google Scholar 

  5. Li X, Wilmanns M, Thornton J, Köhn M (2013) Elucidating human phosphatase-substrate networks. Sci Signal 6(275). https://doi.org/10.1126/scisignal.2003203

  6. Nasa I, Kettenbach AN (2018) Coordination of protein kinase and phosphoprotein phosphatase activities in mitosis. Front Cell Dev Biol 6:30

    Article  PubMed  PubMed Central  Google Scholar 

  7. Hochegger H, Takeda S, Hunt T (2008) Cyclin-dependent kinases and cell-cycle transitions: does one fit all? Nat Rev Mol Cell Biol 9:910–916. https://doi.org/10.1038/nrm2510

    Article  CAS  PubMed  Google Scholar 

  8. Bollen M, Gerlich DW, Lesage B (2009) Mitotic phosphatases: from entry guards to exit guides. Trends Cell Biol 19:531–541. https://doi.org/10.1016/j.tcb.2009.06.005

    Article  CAS  PubMed  Google Scholar 

  9. Olsen JV, Vermeulen M, Santamaria A et al (2010) Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 3:ra3. https://doi.org/10.1126/scisignal.2000475

    Article  CAS  PubMed  Google Scholar 

  10. Domingo-Sananes MR, Kapuy O, Hunt T, Novak B (2011) Switches and latches: a biochemical tug-of-war between the kinases and phosphatases that control mitosis. Philos Trans R Soc Lond Ser B Biol Sci 366:3584–3594. https://doi.org/10.1098/rstb.2011.0087

    Article  CAS  Google Scholar 

  11. Bouchoux C, Uhlmann F (2011) A quantitative model for ordered Cdk substrate dephosphorylation during mitotic exit. Cell 147:803–814. https://doi.org/10.1016/j.cell.2011.09.047

    Article  CAS  PubMed  Google Scholar 

  12. Hendrickx A, Beullens M, Ceulemans H et al (2009) Docking motif-guided mapping of the interactome of protein phosphatase-1. Chem Biol 16:365–371. https://doi.org/10.1016/J.CHEMBIOL.2009.02.012

    Article  CAS  PubMed  Google Scholar 

  13. Moorhead GBG, Trinkle-Mulcahy L, Ulke-Lemée A (2007) Emerging roles of nuclear protein phosphatases. Nat Rev Mol Cell Biol 8:234–244. https://doi.org/10.1038/nrm2126

    Article  CAS  PubMed  Google Scholar 

  14. Bollen M, Peti W, Ragusa MJ, Beullens M (2010) The extended PP1 toolkit: designed to create specificity. Trends Biochem Sci 35:450–458. https://doi.org/10.1016/j.tibs.2010.03.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Trinkle-Mulcahy L, Sleeman JE, Lamond AI (2001) Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells. J Cell Sci 114:4219–4228. https://doi.org/10.1242/JCS.114.23.4219

    Article  CAS  PubMed  Google Scholar 

  16. Egloff MP, Johnson DF, Moorhead G et al (1997) Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1. EMBO J 16:1876–1887. https://doi.org/10.1093/EMBOJ/16.8.1876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ceulemans H, Bollen M (2004) Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiol Rev 84:1–39. https://doi.org/10.1152/PHYSREV.00013.2003/ASSET/IMAGES/LARGE/9J0140278009.JPEG

    Article  CAS  PubMed  Google Scholar 

  18. Wakula P, Beullens M, Ceulemans H et al (2003) Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1. J Biol Chem 278:18817–18823. https://doi.org/10.1074/jbc.M300175200

    Article  CAS  PubMed  Google Scholar 

  19. Heroes E, Lesage B, Görnemann J et al (2013) The PP1 binding code: a molecular-lego strategy that governs specificity. FEBS J 280:584–595. https://doi.org/10.1111/j.1742-4658.2012.08547.x

    Article  CAS  PubMed  Google Scholar 

  20. Cori GT, Green AA (1943) Crystalline muscle phosphorylase: II. Prosthetic group. J Biol Chem 151:31–38. https://doi.org/10.1016/S0021-9258(18)72111-X

    Article  CAS  Google Scholar 

  21. Shimizu H, Ito M, Miyahara M et al (1994) Characterization of the myosin binding subunit of smooth muscle myosin phosphatase. J Biol Chem 269:30407–30411

    Article  CAS  PubMed  Google Scholar 

  22. Shirazi A, Iizuka K, Fadden P et al (1994) Purification and characterization of the mammalian myosin light chain phosphatase holoenzyme. The differential effects of the holoenzyme and its subunits on smooth muscle. J Biol Chem 269:31598–31606

    Article  CAS  PubMed  Google Scholar 

  23. Alessi D, MacDougall LK, Sola MM et al (1992) The control of protein phosphatase-1 by targeting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur J Biochem 210:1023–1035. https://doi.org/10.1111/J.1432-1033.1992.TB17508.X

    Article  CAS  PubMed  Google Scholar 

  24. Okubo S, Ito M, Takashiba Y et al (1994) A regulatory subunit of smooth muscle myosin bound phosphatase. Biochem Biophys Res Commun 200:429–434. https://doi.org/10.1006/bbrc.1994.1467

    Article  CAS  PubMed  Google Scholar 

  25. Pato MD, Kerc E (1985) Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards. J Biol Chem 260:12359–12366. https://doi.org/10.1016/S0021-9258(17)39033-6

    Article  CAS  PubMed  Google Scholar 

  26. Gratecos D, Fischer EH, Detwiler TC, Hurd S (1977) Rabbit muscle phosphorylase phosphatase. 1. Purification and chemical properties. Biochemistry 16:4812–4817. https://doi.org/10.1021/BI00641A009/ASSET/BI00641A009.FP.PNG_V03

    Article  CAS  PubMed  Google Scholar 

  27. Vagnarelli P, Ribeiro S, Sennels L et al (2011) Repo-man coordinates chromosomal reorganization with nuclear envelope reassembly during mitotic exit. Dev Cell 21:328–342. https://doi.org/10.1016/J.DEVCEL.2011.06.020

    Article  CAS  PubMed  Google Scholar 

  28. Wu D, De Wever V, Derua R et al (2018) A substrate-trapping strategy for protein phosphatase PP1 holoenzymes using hypoactive subunit fusions. J Biol Chem 293:15152–15162. https://doi.org/10.1074/jbc.RA118.004132

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Huguet F, Gokhan E, Foster HA et al (2022) Repo-Man/protein phosphatase 1 SUMOylation mediates binding to lamin A and serine 22 dephosphorylation. Open Biol 12:220017. https://doi.org/10.1098/rsob.220017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Carrara M, Sigurdardottir A, Bertolotti A (2017) Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors. Nat Struct Mol Biol 24:708. https://doi.org/10.1038/NSMB.3443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Fedoryshchak RO, Přechová M, Butler AM et al (2020) Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme. elife 9:e61509. https://doi.org/10.7554/eLife.61509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Huet G, Rajakylä EK, Viita T et al (2013) Actin-regulated feedback loop based on Phactr4, PP1 and cofilin maintains the actin monomer pool. J Cell Sci 126:497–507. https://doi.org/10.1242/jcs.113241

    Article  CAS  PubMed  Google Scholar 

  33. Morris JH, Knudsen GM, Verschueren E et al (2014) Affinity purification–mass spectrometry and network analysis to understand protein-protein interactions. Nat Protoc 9:2539. https://doi.org/10.1038/NPROT.2014.164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chang IF (2006) Mass spectrometry-based proteomic analysis of the epitope-tag affinity purified protein complexes in eukaryotes. Proteomics 6:6158–6166. https://doi.org/10.1002/PMIC.200600225

    Article  CAS  PubMed  Google Scholar 

  35. Westermarck J, Ivaska J, Corthals GL (2013) Identification of protein interactions involved in cellular signaling. Mol Cell Proteomics 12:1752. https://doi.org/10.1074/MCP.R113.027771

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Budayeva HG, Cristea IM (2014) A mass spectrometry view of stable and transient protein interactions. Adv Exp Med Biol 806:263. https://doi.org/10.1007/978-3-319-06068-2_11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Schwinn MK, Steffen LS, Zimmerman K et al (2020) A simple and scalable strategy for analysis of endogenous protein dynamics. Sci Rep 10:1–14. https://doi.org/10.1038/s41598-020-65832-1

    Article  CAS  Google Scholar 

  38. Moriya H (2015) Quantitative nature of overexpression experiments. Mol Biol Cell 26:3932–3939. https://doi.org/10.1091/mbc.E15-07-0512

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lackner DH, Carré A, Guzzardo PM et al (2015) A generic strategy for CRISPR-Cas9-mediated gene tagging. Nat Commun 6:10237. https://doi.org/10.1038/ncomms10237

    Article  CAS  PubMed  Google Scholar 

  40. Roux KJ, Kim DI, Raida M, Burke B (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196:801–810. https://doi.org/10.1083/jcb.201112098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Trinkle-Mulcahy L (2019) Recent advances in proximity-based labeling methods for interactome mapping. F1000Res 8(135). https://doi.org/10.12688/F1000RESEARCH.16903.1

  42. Kim DI, Jensen SC, Noble KA et al (2016) An improved smaller biotin ligase for BioID proximity labeling. Mol Biol Cell 27:1188–1196. https://doi.org/10.1091/mbc.E15-12-0844

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kubitz L, Bitsch S, Zhao X et al (2022) Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells. Commun Biol 5:657. https://doi.org/10.1038/s42003-022-03604-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hall MP, Unch J, Binkowski BF et al (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7:1848. https://doi.org/10.1021/CB3002478

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Dixon AS, Schwinn MK, Hall MP et al (2016) NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem Biol 11:400–408. https://doi.org/10.1021/ACSCHEMBIO.5B00753/ASSET/IMAGES/CB-2015-007533_M004.GIF

    Article  CAS  PubMed  Google Scholar 

  46. Claes Z, Jonkhout M, Crespillo-Casado A, Bollen M (2019) The antibiotic robenidine exhibits guanabenz-like cytoprotective properties by a mechanism independent of protein phosphatase PP1:PPP1R15A. J Biol Chem 294:13478–13486. https://doi.org/10.1074/jbc.RA119.008857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lalonde S, Ehrhardt DW, Loqué D et al (2008) Molecular and cellular approaches for the detection of protein–protein interactions: latest techniques and current limitations. Plant J 53:610–635. https://doi.org/10.1111/J.1365-313X.2007.03332.X

    Article  CAS  PubMed  Google Scholar 

  48. Rizzo MA, Davidson MW, Piston DW (2009) Fluorescent protein tracking and detection: applications using fluorescent proteins in living cells. Cold Spring Harb Protoc 4(12). https://doi.org/10.1101/PDB.TOP64

  49. Gibson TJ, Seiler M, Veitia RA (2013) The transience of transient overexpression. Nat Methods 10:715–721. https://doi.org/10.1038/nmeth.2534

    Article  CAS  PubMed  Google Scholar 

  50. Voeltz GK, Prinz WA, Shibata Y et al (2006) A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124:573–586. https://doi.org/10.1016/J.CELL.2005.11.047

    Article  CAS  PubMed  Google Scholar 

  51. Harder Z, Zunino R, McBride H (2004) Sumo1 conjugates mitochondrial substrates and participates in mitochondrial fission. Curr Biol 14:340–345. https://doi.org/10.1016/J.CUB.2004.02.004

    Article  CAS  PubMed  Google Scholar 

  52. Qian J, Beullens M, Lesage B, Bollen M (2013) Aurora B defines its own chromosomal targeting by opposing the recruitment of the phosphatase scaffold repo-man. Curr Biol 23:1136–1143. https://doi.org/10.1016/j.cub.2013.05.017

    Article  CAS  PubMed  Google Scholar 

  53. Cheng YL, Chen RH (2010) The AAA-ATPase Cdc48 and cofactor Shp1 promote chromosome bi-orientation by balancing Aurora B activity. J Cell Sci 123:2025–2034. https://doi.org/10.1242/JCS.066043

    Article  CAS  PubMed  Google Scholar 

  54. Peggie MW, MacKelvie SH, Bloecher A et al (2002) Essential functions of Sds22p in chromosome stability and nuclear localization of PP1. J Cell Sci 115:195–206. https://doi.org/10.1242/JCS.115.1.195

    Article  CAS  PubMed  Google Scholar 

  55. Pedelini L, Marquina M, Ariño J et al (2007) YPI1 and SDS22 proteins regulate the nuclear localization and function of yeast type 1 phosphatase Glc7. J Biol Chem 282:3282–3292. https://doi.org/10.1074/jbc.M607171200

    Article  CAS  PubMed  Google Scholar 

  56. Eiteneuer A, Seiler J, Weith M et al (2014) Inhibitor-3 ensures bipolar mitotic spindle attachment by limiting association of SDS22 with kinetochore-bound protein phosphatase-1. EMBO J 33:2704–2720. https://doi.org/10.15252/EMBJ.201489054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Lakowicz JR (2006) Principles of fluorescence spectroscopy, pp 1–954. https://doi.org/10.1007/978-0-387-46312-4/COVER

  58. Förster T (2012) Energy migration and fluorescence. J Biomed Opt 17:11002. https://doi.org/10.1117/1.JBO.17.1.011002

    Article  Google Scholar 

  59. Algar WR, Hildebrandt N, Vogel SS, Medintz IL (2019) FRET as a biomolecular research tool – understanding its potential while avoiding pitfalls. Nat Methods 16:815–829. https://doi.org/10.1038/s41592-019-0530-8

    Article  CAS  PubMed  Google Scholar 

  60. Trinkle-Mulcahy L, Chusainow J, Lam YW et al (2007) Visualization of intracellular PP1 targeting through transiently and stably expressed fluorescent protein fusions. Methods Mol Biol 365:133–154. https://doi.org/10.1385/1-59745-267-X:133

    Article  PubMed  Google Scholar 

  61. de Los Santos C, Chang CW, Mycek MA, Cardullo RA (2015) FRAP, FLIM, and FRET: detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy. Mol Reprod Dev 82:587. https://doi.org/10.1002/MRD.22501

    Article  PubMed Central  Google Scholar 

  62. Xue Y, Ren J, Gao X et al (2008) GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy. Mol Cell Proteomics 7:1598–1606. https://doi.org/10.1074/MCP.M700574-MCP200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Iakoucheva LM, Radivojac P, Brown CJ et al (2004) The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res 32:1037–1049. https://doi.org/10.1093/nar/gkh253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Blom N, Gammeltoft S, Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites1 1Edited by F. E. Cohen. J Mol Biol 294:1351–1362. https://doi.org/10.1006/jmbi.1999.3310

    Article  CAS  PubMed  Google Scholar 

  65. Ingrell CR, Miller ML, Jensen ON, Blom N (2007) NetPhosYeast: prediction of protein phosphorylation sites in yeast. Bioinformatics 23:895–897. https://doi.org/10.1093/bioinformatics/btm020

    Article  CAS  PubMed  Google Scholar 

  66. Tang Y-R, Chen Y-Z, Canchaya CA, Zhang Z (2007) GANNPhos: a new phosphorylation site predictor based on a genetic algorithm integrated neural network. Protein Eng Des Sel 20:405–412. https://doi.org/10.1093/protein/gzm035

    Article  CAS  PubMed  Google Scholar 

  67. Wang Y, Liu Z, Cheng H et al (2014) EKPD: a hierarchical database of eukaryotic protein kinases and protein phosphatases. Nucleic Acids Res 42:D496–502. https://doi.org/10.1093/NAR/GKT1121

    Article  PubMed  Google Scholar 

  68. Liberti S, Sacco F, Calderone A et al (2013) HuPho: the human phosphatase portal. FEBS J 280:379–387. https://doi.org/10.1111/J.1742-4658.2012.08712.X

    Article  CAS  PubMed  Google Scholar 

  69. Duan G, Li X, Köhn M (2015) The human DEPhOsphorylation database DEPOD: a 2015 update. Nucleic Acids Res 43:D531. https://doi.org/10.1093/NAR/GKU1009

    Article  CAS  PubMed  Google Scholar 

  70. Mitchell CJ, Kim MS, Zhong J et al (2016) Unbiased identification of substrates of protein tyrosine phosphatase ptp-3 in C. elegans. Mol Oncol 10:910. https://doi.org/10.1016/J.MOLONC.2016.03.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Feng S, Zhou H, Dong H (2019) Using deep neural network with small dataset to predict material defects. Mater Des 162:300–310. https://doi.org/10.1016/j.matdes.2018.11.060

    Article  Google Scholar 

  72. Zhao W (2017) Research on the deep learning of the small sample data based on transfer learning. AIP Conf Proc 1864. https://doi.org/10.1063/1.4992835

  73. Chaudhari M, Thapa N, Ismail H et al (2021) DTL-DephosSite: deep transfer learning based approach to predict dephosphorylation sites. Front Cell Dev Biol 9:662983. https://doi.org/10.3389/FCELL.2021.662983/FULL

    Article  PubMed  PubMed Central  Google Scholar 

  74. Holder J, Mohammed S, Barr FA (2020) Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit. elife 9:1–33. https://doi.org/10.7554/ELIFE.59885

    Article  Google Scholar 

  75. Touati SA, Kataria M, Jones AW et al (2018) Phosphoproteome dynamics during mitotic exit in budding yeast. EMBO J 37(10). https://doi.org/10.15252/EMBJ.201798745

  76. Hoermann B, Kokot T, Helm D et al (2020) Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of phosphatases PP1 and PP2A. Nat Commun 11:1–20. https://doi.org/10.1038/s41467-020-17334-x

    Article  CAS  Google Scholar 

  77. Godfrey M, Touati SA, Kataria M et al (2017) PP2ACdc55 phosphatase imposes ordered cell-cycle phosphorylation by opposing threonine phosphorylation. Mol Cell 65:393–402.e3. https://doi.org/10.1016/J.MOLCEL.2016.12.018

    Article  PubMed  PubMed Central  Google Scholar 

  78. Cundell MJ, Hutter LH, Bastos RN et al (2016) A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit. J Cell Biol 214:539. https://doi.org/10.1083/JCB.201606033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Hein JB, Hertz EPT, Garvanska DH et al (2017) Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis. Nat Cell Biol 19:1433–1440. https://doi.org/10.1038/ncb3634

    Article  CAS  PubMed  Google Scholar 

  80. McCloy RA, Parker BL, Rogers S et al (2015) Global phosphoproteomic mapping of early mitotic exit in human cells identifies novel substrate dephosphorylation motifs. Mol Cell Proteomics 14:2194–2212. https://doi.org/10.1074/MCP.M114.046938

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Waldman T, Kinzler KW, Vogelstein B (1995) p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer 55:5187–5190

    CAS  Google Scholar 

  82. Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT (2016) Rapid protein depletion in human cells by IAA-inducible degron tagging with short homology donors. Cell Rep 15:210–218. https://doi.org/10.1016/J.CELREP.2016.03.001

    Article  CAS  PubMed  Google Scholar 

  83. Hung V, Udeshi ND, Lam SS et al (2016) Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11:456–475. https://doi.org/10.1038/nprot.2016.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Nishimura K, Fukagawa T, Takisawa H et al (2009) An IAA-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6:917–922. https://doi.org/10.1038/nmeth.1401

    Article  CAS  PubMed  Google Scholar 

  85. ten Have S, Boulon S, Ahmad Y, Lamond AI (2011) Mass spectrometry-based immuno-precipitation proteomics – the user’s guide. Proteomics 11:1153. https://doi.org/10.1002/PMIC.201000548

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We want to thank Dr Florentin Huguet (Brunel University London, Vagnarelli Lab) for substantial contribution toward the development and validation of the presented method. The Vagnarelli Lab is supported by the Wellcome Trust Investigator award 210742/Z/18/Z to Paola Vagnarelli.

Author information

Author notes

  1. Authors Dorothee C Kommer and Konstantinos Stamatiou have equally contributed to this chapter.

Authors and Affiliations

  1. College of Health, Medicine and Life Science, Brunel University London, London, UK

    Dorothee C Kommer, Konstantinos Stamatiou & Paola Vagnarelli

Authors
  1. Dorothee C Kommer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Konstantinos Stamatiou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paola Vagnarelli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paola Vagnarelli .

Editor information

Editors and Affiliations

  1. Université de Montpellier, Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), CNRS UMR 5237, Montpellier Cedex 5, France

    Anna Castro

  2. Université de Montpellier, Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), CNRS UMR 5237, Montpellier Cedex 5, France

    Benjamin Lacroix

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kommer, D.C., Stamatiou, K., Vagnarelli, P. (2024). Cell Cycle–Specific Protein Phosphatase 1 (PP1) Substrates Identification Using Genetically Modified Cell Lines. In: Castro, A., Lacroix, B. (eds) Cell Cycle Control. Methods in Molecular Biology, vol 2740. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3557-5_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3557-5_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3556-8

  • Online ISBN: 978-1-0716-3557-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation