Letter | Published:

Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling

Nature volume 546, pages 307311 (08 June 2017) | Download Citation

Abstract

As key executers of biological functions, the activity and abundance of proteins are subjected to extensive regulation. Deciphering the genetic architecture underlying this regulation is critical for understanding cellular signalling events and responses to environmental cues. Using random mutagenesis in haploid human cells, we apply a sensitive approach to directly couple genomic mutations to protein measurements in individual cells. Here we use this to examine a suite of cellular processes, such as transcriptional induction, regulation of protein abundance and splicing, signalling cascades (mitogen-activated protein kinase (MAPK), G-protein-coupled receptor (GPCR), protein kinase B (AKT), interferon, and Wingless and Int-related protein (WNT) pathways) and epigenetic modifications (histone crotonylation and methylation). This scalable, sequencing-based procedure elucidates the genetic landscapes that control protein states, identifying genes that cause very narrow phenotypic effects and genes that lead to broad phenotypic consequences. The resulting genetic wiring map identifies the E3-ligase substrate adaptor KCTD5 (ref. 1) as a negative regulator of the AKT pathway, a key signalling cascade frequently deregulated in cancer. KCTD5-deficient cells show elevated levels of phospho-AKT at S473 that could not be attributed to effects on canonical pathway components. To reveal the genetic requirements for this phenotype, we iteratively analysed the regulatory network linked to AKT activity in the knockout background. This genetic modifier screen exposes suppressors of the KCTD5 phenotype and mechanistically demonstrates that KCTD5 acts as an off-switch for GPCR signalling by triggering proteolysis of Gβγ heterodimers dissociated from the Gα subunit. Although biological networks have previously been constructed on the basis of gene expression2,3, protein–protein associations4,5,6, or genetic interaction profiles7,8, we foresee that the approach described here will enable the generation of a comprehensive genetic wiring map for human cells on the basis of quantitative protein states.

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References

  1. 1.

    et al. KCTD5, a putative substrate adaptor for cullin3 ubiquitin ligases. FEBS J. 275, 3900–3910 (2008)

  2. 2.

    , , & Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA 95, 14863–14868 (1998)

  3. 3.

    et al. Functional discovery via a compendium of expression profiles. Cell 102, 109–126 (2000)

  4. 4.

    et al. The BioPlex network: a systematic exploration of the human interactome resource. Cell 162, 425–440 (2015)

  5. 5.

    et al. A census of human soluble protein complexes. Cell 150, 1068–1081 (2012)

  6. 6.

    et al. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 163, 712–723 (2015)

  7. 7.

    . et al. A global genetic interaction network maps a wiring diagram of cellular function. Science 353, aaf1420 (2016)

  8. 8.

    et al. Global mapping of the yeast genetic interaction network. Science 303, 808–813 (2004)

  9. 9.

    Genome-wide mapping of cellular traits using yeast. Yeast 31, 197–205 (2014)

  10. 10.

    et al. Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-seq. Cell 167, 1883–1896.e15 (2016)

  11. 11.

    et al. Perturb-seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic resource. Cell 167, 1853–1866.e17 (2016)

  12. 12.

    et al. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162, 675–686 (2015)

  13. 13.

    et al. Gene essentiality and synthetic lethality in haploid human cells. Science 350, 1092–1096 (2015)

  14. 14.

    , , , & Genetics of single-cell protein abundance variation in large yeast populations. Nature 506, 494–497 (2014)

  15. 15.

    et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006)

  16. 16.

    et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009)

  17. 17.

    et al. Ebola virus entry requires the cholesterol transporter Niemann–Pick C1. Nature 477, 340–343 (2011)

  18. 18.

    Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat. Rev. Immunol. 5, 375–386 (2005)

  19. 19.

    & Polycomb complexes and silencing mechanisms. Curr. Opin. Cell Biol. 16, 239–246 (2004)

  20. 20.

    et al. Intracellular crotonyl-CoA stimulates transcription through p300-catalyzed histone crotonylation. Mol. Cell 58, 203–215 (2015)

  21. 21.

    et al. Diagnosis of lysosomal storage disorders: evaluation of lysosome-associated membrane protein LAMP-1 as a diagnostic marker. Clin. Chem. 43, 1325–1335 (1997)

  22. 22.

    , , & Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101 (2005)

  23. 23.

    , , , & Phosducin-like protein acts as a molecular chaperone for G protein βγ dimer assembly. EMBO J. 24, 1965–1975 (2005)

  24. 24.

    et al. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein βγ subunits. Cell 77, 83–93 (1994)

  25. 25.

    et al. Gβγ stimulates phosphoinositide 3-kinase-γ by direct interaction with two domains of the catalytic p110 subunit. J. Biol. Chem. 273, 7024–7029 (1998)

  26. 26.

    et al. Regulation of the G-protein regulatory-Gαi signaling complex by nonreceptor guanine nucleotide exchange factors. J. Biol. Chem. 288, 3003–3015 (2013)

  27. 27.

    et al. Mutations in G protein β subunits promote transformation and kinase inhibitor resistance. Nat. Med. 21, 71–75 (2015)

  28. 28.

    et al. Native GABAB receptors are heteromultimers with a family of auxiliary subunits. Nature 465, 231–235 (2010)

  29. 29.

    et al. Cullin 3 recognition is not a universal property among KCTD proteins. PLoS ONE 10, e0126808 (2015)

  30. 30.

    et al. Auxiliary GABAB receptor subunits uncouple G protein βγ subunits from effector channels to induce desensitization. Neuron 82, 1032–1044 (2014)

  31. 31.

    et al. A generic strategy for CRISPR-Cas9-mediated gene tagging. Nat. Commun. 6, 10237 (2015)

  32. 32.

    et al. Deciphering the glycosylome of dystroglycanopathies using haploid screens for Lassa virus entry. Science 340, 479–483 (2013)

  33. 33.

    et al. Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties. Nucleic Acids Res. 33, 135–142 (2005)

  34. 34.

    , , & Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

  35. 35.

    & BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010)

  36. 36.

    et al. Reactome: a knowledgebase of biological pathways. Nucleic Acids Res. 33, D428–D432 (2005)

  37. 37.

    , , & ConsensusPathDB–a database for integrating human functional interaction networks. Nucleic Acids Res. 37, D623–D628 (2009)

  38. 38.

    , & TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009)

  39. 39.

    , & HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015)

  40. 40.

    et al. Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases. J. Struct. Biol. 175, 113–119 (2011)

  41. 41.

    et al. TGFβ signaling directs serrated adenomas to the mesenchymal colorectal cancer subtype. EMBO Mol. Med. 8, 745–760 (2016)

  42. 42.

    , & Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat. Biotechnol. 28, 868–873 (2010)

  43. 43.

    , , & Large-scale identification of ubiquitination sites by mass spectrometry. Nat. Protocols 8, 1950–1960 (2013)

  44. 44.

    et al. A novel Fanconi anaemia subtype associated with a dominant-negative mutation in RAD51. Nat. Commun. 6, 8829 (2015)

  45. 45.

    et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell Proteomics 13, 2513–2526 (2014)

  46. 46.

    et al. BioGRID: a general repository for interaction datasets. Nucleic Acids Res. 34, 535–539 (2006)

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Acknowledgements

We thank J. Goedhart, L. Wessels, B. van Steensel, S. Nijman, and members of the Brummelkamp, Perrakis, and Sixma laboratories for discussions. We thank R. Spaapen for providing CUL3 knockout cells, P. Celie and M. Stadnik for assistance with the recombinant protein expression, as well as E. Fessler and J. P. Medema for generation of WNT3A/R-spondin-conditioned medium. This work was supported by the Dutch Cancer Society (NKI 2015-7609), the Cancer Genomics Center, an Ammodo KNAW Award 2015 for Biomedical Sciences to T.R.B., by the Netherlands Organization for Scientific Research (NWO) as part of the National Roadmap Large-scale Research Facilities of the Netherlands, Proteins@Work (project number 184.032.201) to O.B.B. and A.F.M.A., and by a Vidi grant (723.012.102) to A.F.M.A.

Author information

Author notes

    • Lucas T. Jae

    Present address: Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, 81377 Munich, Germany.

    • Markus Brockmann
    •  & Vincent A. Blomen

    These authors contributed equally to this work.

Affiliations

  1. Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

    • Markus Brockmann
    • , Vincent A. Blomen
    • , Joppe Nieuwenhuis
    • , Elmer Stickel
    • , Matthijs Raaben
    • , Onno B. Bleijerveld
    • , A. F. Maarten Altelaar
    • , Lucas T. Jae
    •  & Thijn R. Brummelkamp
  2. Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands.

    • A. F. Maarten Altelaar
  3. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

    • Thijn R. Brummelkamp
  4. Cancergenomics.nl, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

    • Thijn R. Brummelkamp

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Contributions

M.B., V.A.B., J.N., M.R. L.T.J., and T.R.B. were responsible for the overall design of the study. V.A.B. and E.S. performed the bioinformatics. E.S. developed the Phenosaurus platform. O.B.B. and A.F.M.A. designed, performed, and analysed the proteomics experiments. M.B., V.A.B., L.T.J., and T.R.B. wrote the manuscript; all authors commented on it.

Competing interests

T.R.B. is co-founder and shareholder of Haplogen GmbH and Scenic Biotech BV, and M.B., V.B; J.N., M.R., L.T.J., and T.R.B. are listed as inventors on a patent application related to the technology.

Corresponding authors

Correspondence to Lucas T. Jae or Thijn R. Brummelkamp.

Reviewer Information Nature thanks J. Moffat and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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https://doi.org/10.1038/nature22376

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