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Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function

Nature Biotechnology volume 22, pages 993–1000 (2004)Cite this article

Abstract

Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.

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Figure 1: Synthesis of caged phosphopeptides that target 14-3-3.
Figure 2: UV photolysis of the phosphoserine cage dramatically enhances peptide binding to 14-3-3 proteins.
Figure 3: Uncaged but not caged phosphopeptides compete with endogenous cellular proteins for binding to 14-3-3.
Figure 4: Intracellular delivery of caged phosphopeptides.
Figure 5: Coordinated, synchronous loss of 14-3-3 function in cells results in aberrant cell cycle progression and G1 release.
Figure 6: Coordinated, synchronous loss of 14-3-3 function results in a dysfunctional, DNA damage–induced S-phase cell cycle checkpoint.

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References

  1. Yaffe, M.B. & Elia, A.E. Phosphoserine/threonine-binding domains. Curr. Opin. Cell Biol. 13, 131–138 (2001).

    Article  CAS  Google Scholar 

  2. Lu, P.J., Zhou, X.Z., Shen, M. & Lu, K.P. Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science 283, 1325–1328 (1999).

    Article  CAS  Google Scholar 

  3. Nash, P. et al. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414, 514–521 (2001).

    Article  CAS  Google Scholar 

  4. Elia, A.E., Cantley, L.C. & Yaffe, M.B. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 299, 1228–1231 (2003).

    Article  CAS  Google Scholar 

  5. Manke, I.A., Lowery, D.M., Nguyen, A. & Yaffe, M.B. BRCT repeats as phosphopeptide-binding modules involved in protein targeting. Science 302, 636–639 (2003).

    Article  CAS  Google Scholar 

  6. Yaffe, M.B. & Smerdon, S.J. PhosphoSerine/threonine binding domains: you can't pSERious? Structure 9, R33–R38 (2001).

    Article  CAS  Google Scholar 

  7. Aitken, A. 14-3-3 proteins on the MAP. Trends Biochem. Sci. 20, 95–97 (1995).

    Article  CAS  Google Scholar 

  8. Fu, H., Subramanian, R.R. & Masters, S.C. 14-3-3 proteins: structure, function, and regulation. Annu. Rev. Pharmacol. Toxicol. 40, 617–647 (2000).

    Article  CAS  Google Scholar 

  9. Tzivion, G. & Avruch, J. 14-3-3 proteins: active cofactors in cellular regulation by serine/threonine phosphorylation. J. Biol. Chem. 277, 3061–3064 (2002).

    Article  CAS  Google Scholar 

  10. Jones, D.H., Ley, S. & Aitken, A. Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins. FEBS Lett. 368, 55–58 (1995).

    Article  CAS  Google Scholar 

  11. Tzivion, G., Luo, Z. & Avruch, J. A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature 394, 88–92 (1998).

    Article  CAS  Google Scholar 

  12. Yaffe, M.B. et al. The structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91, 961–971 (1997).

    Article  CAS  Google Scholar 

  13. Muslin, A.J., Tanner, J.W., Allen, P.M. & Shaw, A.S. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889–897 (1996).

    Article  CAS  Google Scholar 

  14. Rittinger, K. et al. Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Mol. Cell 4, 153–166 (1999).

    Article  CAS  Google Scholar 

  15. Subramanian, R.R., Masters, S.C., Zhang, H. & Fu, H. Functional conservation of 14-3-3 isoforms in inhibiting bad-induced apoptosis. Exp. Cell Res. 271, 142–151 (2001).

    Article  CAS  Google Scholar 

  16. Zhang, L., Wang, H., Liu, D., Liddington, R. & Fu, H. Raf-1 kinase and exoenzyme S interact with 14-3-3zeta through a common site involving lysine 49. J. Biol. Chem. 272, 13717–13724 (1997).

    Article  CAS  Google Scholar 

  17. Wang, B. et al. Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display. Biochemistry 38, 12499–12504 (1999).

    Article  CAS  Google Scholar 

  18. Henriksson, M.L. et al. A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo. Eur. J. Biochem. 269, 4921–4929 (2002).

    Article  CAS  Google Scholar 

  19. Corrie, J.E.T. & Trentham, D.R. Caged Nucleotides and Neurotransmitters. in Biological Applications of Photochemical Switches (ed. Morrison, H.) 243–305 (Wiley & Sons, New York, NY, 1993).

    Google Scholar 

  20. de Gruijl, F.R. Photocarcinogenesis: UVA vs UVB. Methods Enzymol. 319, 359–366 (2000).

    Article  CAS  Google Scholar 

  21. Kleiman, N.J., Wang, R.R. & Spector, A. Ultraviolet light induced DNA damage and repair in bovine lens epithelial cells. Curr. Eye Res. 9, 1185–1193 (1990).

    Article  CAS  Google Scholar 

  22. Park, C.-H. & Givens, R.S. New photoactivated protecting groups. 6. p-hydroxy-phenacyl: a phototrigger for chemical and biologcial probes. J. Am. Chem. Soc. 119, 2453–2463 (1997).

    Article  CAS  Google Scholar 

  23. Ellis-Davies, G.C. Development and application of caged calcium. Methods Enzymol. 360, 226–238 (2003).

    Article  CAS  Google Scholar 

  24. Wootton, J.F. et al. Kinetics of cytosolic Ca2+ concentration after photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate from a caged derivative in guinea pig hepatocytes. Biophys. J. 68, 2601–2607 (1995).

    Article  CAS  Google Scholar 

  25. Patton, W.F. et al. Mercury-arc photolysis: a method for examining second messenger regulation of endothelial cell monolayer integrity. Anal. Biochem. 196, 31–38 (1991).

    Article  CAS  Google Scholar 

  26. Walker, J.W. et al. Signaling pathways underlying eosinophil cell motility revealed by using caged peptides. Proc. Natl. Acad. Sci. USA 95, 1568–1573 (1998).

    Article  CAS  Google Scholar 

  27. Vazquez, M.E., Nitz, M., Stehn, J., Yaffe, M. & Imperiali, B. Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations. J. Am. Chem. Soc. 125, 10150–10151 (2003).

    Article  CAS  Google Scholar 

  28. Ghosh, M. et al. Cofilin promotes actin polymerization and defines the direction of cell motility. Science 304, 743–746 (2004).

    Article  CAS  Google Scholar 

  29. Tong, Y. et al. Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel. J. Gen. Physiol. 117, 103–118 (2001).

    Article  CAS  Google Scholar 

  30. Rothman, D.M., Vazquez, M.E., Vogel, E.M. & Imperiali, B. General method for the synthesis of caged phosphopeptides: tools for the exploration of signal transduction pathways. Org. Lett. 4, 2865–2868 (2002).

    Article  CAS  Google Scholar 

  31. Rothman, D.M., Vazquez, M.E., Vogel, E.M. & Imperiali, B. Caged phospho-amino acid building blocks for solid phase peptide synthesis. J. Org. Chem. 68, 6795–6798 (2003).

    Article  CAS  Google Scholar 

  32. Ellis-Davies, G.C.R. & Kaplan, J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc. Natl. Acad. Sci. USA 91, 187–191 (1994).

    Article  CAS  Google Scholar 

  33. Derossi, D., Chassaing, G. & Prochiantz, A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 8, 84–87 (1998).

    Article  CAS  Google Scholar 

  34. Dunican, D.J. & Doherty, P. Designing cell-permeant phosphopeptides to modulate intracellular signaling pathways. Biopolymers 60, 45–60 (2001).

    Article  CAS  Google Scholar 

  35. Chen, L. et al. Molecular transporters for peptides: delivery of a cardioprotective epsilonPKC agonist peptide into cells and intact ischemic heart using a transport system, R(7). Chem. Biol. 8, 1123–1129 (2001).

    Article  CAS  Google Scholar 

  36. Lalande, M. A reversible arrest point in the late G1 phase of the mammalian cell cycle. Exp. Cell Res. 186, 332–339 (1990).

    Article  CAS  Google Scholar 

  37. Peng, C.Y. et al. Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science 277, 1501–1505 (1997).

    Article  CAS  Google Scholar 

  38. Jiang, K. et al. Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser-345. J. Biol. Chem. 278, 25207–25217 (2003).

    Article  CAS  Google Scholar 

  39. Wang, Y. et al. Binding of 14-3-3beta to the carboxyl terminus of Wee1 increases Wee1 stability, kinase activity, and G2-M cell population. Cell Growth Differ. 11, 211–219 (2000).

    CAS  PubMed  Google Scholar 

  40. Rothblum-Oviatt, C.J., Ryan, C.E. & Piwnica-Worms, H. 14-3-3 binding regulates catalytic activity of human Wee1 kinase. Cell Growth Differ. 12, 581–589 (2001).

    CAS  PubMed  Google Scholar 

  41. Laronga, C., Yang, H.Y., Neal, C. & Lee, M.H. Association of the cyclin-dependent kinases and 14-3-3 sigma negatively regulates cell cycle progression. J. Biol. Chem. 275, 23106–23112 (2000).

    Article  CAS  Google Scholar 

  42. Bartek, J. & Lukas, J. Pathways governing G1/S transition and their response to DNA damage. FEBS Lett. 490, 117–122 (2001).

    Article  CAS  Google Scholar 

  43. Busino, L. et al. Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage. Nature 426, 87–91 (2003).

    Article  CAS  Google Scholar 

  44. Ravanat, J.L., Douki, T. & Cadet, J. Direct and indirect effects of UV radiation on DNA and its components. J. Photochem. Photobiol. B 63, 88–102 (2001).

    Article  CAS  Google Scholar 

  45. Alberola-Ila, J., Hogquist, K.A., Swan, K.A., Bevan, M.J. & Perlmutter, R.M. Positive and negative selection invoke distinct signaling pathways. J. Exp. Med. 184, 9–18 (1996).

    Article  CAS  Google Scholar 

  46. Esteban, L.M. et al. Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development. Mol. Cell. Biol. 21, 1444–1452 (2001).

    Article  CAS  Google Scholar 

  47. Dalal, S.N., Schweitzer, C.M., Gan, J. & DeCaprio, J.A. Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site. Mol. Cell. Biol. 19, 4465–4479 (1999).

    Article  CAS  Google Scholar 

  48. Ford, J.C. et al. 14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast. Science 265, 533–535 (1994).

    Article  CAS  Google Scholar 

  49. Chan, T.A., Hermeking, H., Lengauer, C., Kinzler, K.W. & Vogelstein, B. 14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage. Nature 401, 616–620 (1999).

    Article  CAS  Google Scholar 

  50. Chen, M.S., Ryan, C.E. & Piwnica-Worms, H. Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding. Mol. Cell. Biol. 23, 7488–7497 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by NIH grant GM60594 and a Burroughs-Wellcome Career Development Award to M.B.Y., and the Cell Migration Consortium grant GM64346 to B.I.

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Author notes

  1. Anhco Nguyen and Deborah M Rothman: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Anhco Nguyen, Justine Stehn & Michael B Yaffe

  2. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Anhco Nguyen, Justine Stehn, Barbara Imperiali & Michael B Yaffe

  3. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Deborah M Rothman & Barbara Imperiali

  4. Division of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Michael B Yaffe

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  1. Anhco Nguyen
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Correspondence to Michael B Yaffe.

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Supplementary information

Supplementary Table 1

Percentage of Cells in S-phase. (PDF 5 kb)

Supplementary Methods

Characterization of individual peptides and additional synthesis details. (PDF 12 kb)

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Nguyen, A., Rothman, D., Stehn, J. et al. Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function. Nat Biotechnol 22, 993–1000 (2004). https://doi.org/10.1038/nbt997

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