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Immunoaffinity profiling of tyrosine phosphorylation in cancer cells

Abstract

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

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Figure 1: Characteristics of the immunoaffinity profiling strategy.
Figure 2: Immunoaffinity profiling of NIH 3T3 cells expressing oncogenic Src Y527F.
Figure 3: Distribution of amino acid residues surrounding phosphotyrosine among the phosphopeptides identified from three distinct cell types.

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References

  1. Blume-Jensen, P. & Hunter, T. Oncogenic kinase signalling. Nature 411, 355–365 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Sawyers, C.L. Rational therapeutic intervention in cancer: kinases as drug targets. Curr. Opin. Genet. Dev. 12, 111–115 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Mann, M. et al. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol. 20, 261–268 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Ficarro, S.B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Ficarro, S. et al. Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation. J. Biol. Chem. 278, 11579–11589 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Pandey, A. et al. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl. Acad. Sci. USA 97, 179–184 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pandey, A. et al. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways. J. Biol. Chem. 275, 38633–38639 (2000).

    Article  CAS  PubMed  Google Scholar 

  8. Pandey, A. et al. Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases. Oncogene 21, 8029–8036 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Salomon, A.R. et al. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc. Natl. Acad. Sci. USA 100, 443–448 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Brill, L.M. et al. Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry. Anal. Chem. 76, 2763–2772 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Marcus, K., Immler, D., Sternberger, J. & Meyer, H.E. Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins. Electrophoresis 21, 2622–2636 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Quadroni, M. & James, P. Phosphopeptide analysis. EXS 88, 199–213 (2000).

    CAS  PubMed  Google Scholar 

  13. Schneider, U., Schwenk, H.U. & Bornkamm, G. Characterization of EBV-genome negative “null” and “T” cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int. J. Cancer 19, 621–626 (1977).

    Article  CAS  PubMed  Google Scholar 

  14. Srivastava, A.K. & St-Louis, J. Smooth muscle contractility and protein tyrosine phosphorylation. Mol. Cell. Biochem. 176, 47–51 (1997).

    Article  CAS  PubMed  Google Scholar 

  15. Eng, J.K., McCormack, A.L. & Yates, J.R., III . An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. Kane, L.P., Lin, J. & Weiss, A. Signal transduction by the TCR for antigen. Curr. Opin. Immunol. 12, 242–249 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Calalb, M.B., Polte, T.R. & Hanks, S.K. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol. Cell. Biol. 15, 954–963 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Belsches, A.P., Haskell, M.D. & Parsons, S.J. Role of c-Src tyrosine kinase in EGF-induced mitogenesis. Front. Biosci. 2, d501–d518 (1997).

    Article  CAS  PubMed  Google Scholar 

  19. Bache, K.G., Raiborg, C., Mehlum, A., Madshus, I.H. & Stenmark, H. Phosphorylation of Hrs downstream of the epidermal growth factor receptor. Eur. J. Biochem. 269, 3881–3887 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Schaller, M.D. et al. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol. Cell. Biol. 14, 1680–1688 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bouton, A.H., Riggins, R.B. & Bruce-Staskal, P.J. Functions of the adapter protein Cas: signal convergence and the determination of cellular responses. Oncogene 20, 6448–6458 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Curtis, D.J. et al. Adaptor protein SKAP55R is associated with myeloid differentiation and growth arrest. Exp. Hematol. 28, 1250–1259 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Wang, Y.H., Li, F., Schwartz, J.H., Flint, P.J. & Borkan, S.C. c-Src and HSP72 interact in ATP-depleted renal epithelial cells. Am. J. Physiol. Cell Physiol. 281, C1667–C1675 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Frame, M.C., Fincham, V.J., Carragher, N.O. & Wyke, J.A. v-Src′s hold over actin and cell adhesions. Nat. Rev. Mol. Cell Biol. 3, 233–245 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Pulford, K. et al. Detection of anaplastic lymphoma kinase (ALK) and nucleolar protein nucleophosmin (NPM)-ALK proteins in normal and neoplastic cells with the monoclonal antibody ALK1. Blood 89, 1394–1404 (1997).

    Article  CAS  PubMed  Google Scholar 

  26. Morris, S.W. et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 263, 1281–1284 (1994).

    Article  CAS  PubMed  Google Scholar 

  27. Fujimoto, J. et al. Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5). Proc. Natl. Acad. Sci. USA 93, 4181–4186 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. MacCoss, M.J. et al. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc. Natl. Acad. Sci. USA 99, 7900–7905 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bai, R.Y., Dieter, P., Peschel, C., Morris, S.W. & Duyster, J. Nucleophosmin-anaplastic lymphoma kinase of large-cell anaplastic lymphoma is a constitutively active tyrosine kinase that utilizes phospholipase C-γ to mediate its mitogenicity. Mol. Cell. Biol. 18, 6951–6961 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. van der Geer, P., Wiley, S., Gish, G.D. & Pawson, T. The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein-protein interactions. Curr. Biol. 6, 1435–1444 (1996).

    Article  CAS  PubMed  Google Scholar 

  31. Zamo, A. et al. Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death. Oncogene 21, 1038–1047 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Zhang, Q. et al. Multilevel dysregulation of STAT3 activation in anaplastic lymphoma kinase-positive T/null-cell lymphoma. J. Immunol. 168, 466–474 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Khoury, J.D. et al. Differential expression and clinical significance of tyrosine-phosphorylated STAT3 in ALK(+) and ALK(−) anaplastic large cell lymphoma. Clin. Cancer Res. 9, 3692–3699 (2003).

    CAS  PubMed  Google Scholar 

  34. Darnell, J.E. Jr., Kerr, I.M. & Stark, G.R. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264, 1415–1421 (1994).

    Article  CAS  PubMed  Google Scholar 

  35. Szebeni, A., Hingorani, K., Negi, S. & Olson, M.O. Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23. J. Biol. Chem. 278, 9107–9115 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Bischof, D., Pulford, K., Mason, D.Y. & Morris, S.W. Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis. Mol. Cell. Biol. 17, 2312–2325 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Carr, S. et al. The need for guidelines in publication of peptide and protein identification data: working group on publication guidelines for peptide and protein identification data. Mol. Cell. Proteomics 3, 531–533 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Nesvizhskii, A.I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Hornbeck, P.V., Chabra, I., Kornhauser, J.M., Skrzypek, E. & Zhang, B. PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics 4, 1551–1561 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Gerber, S.A., Rush, J., Stemman, O., Kirschner, M.W. & Gygi, S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100, 6940–6945 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are grateful to Jeffrey Knott, Jason Reynolds and Matthew Prokop for providing synthetic peptides, Sandra Schieferl for the 3T3-Src cell line and David Mason for advice on the use of ALCL cell lines. We thank Ross M. Tomaino, Scott A. Gerber and Steven P. Gygi for helping us collect tandem mass spectra in the early stages of this project, and Daniel Schwartz, Sean A. Beausoleil, Rob Duarte and Steven P. Gygi for producing heat maps for the phosphotyrosine peptides we identified. This work was supported by a Small Business Innovation Research phase I grant 1R43CA101106 of the Innovative Molecular Analysis Technologies program from the National Cancer Institute (J.R.).

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Correspondence to Michael J Comb.

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Supplementary information

Supplementary Fig. 1

The reference library-searching strategy. (PDF 269 kb)

Supplementary Table 1

Phosphotyrosine peptides from signaling proteins found in pervanadate-treated Jurkat cells (PDF 52 kb)

Supplementary Table 2

Phosphotyrosine peptides from Jurkat-pervanadate-trypsin/P-Tyr-100 (PDF 70 kb)

Supplementary Table 3

Phosphotyrosine peptides found in Jurkat-pervanadate-trypsin 15–25% acetonitrile fraction/P-Tyr-100 during reproducibility studies (PDF 55 kb)

Supplementary Table 4

Phosphotyrosine peptides from signaling proteins found in 3T3-Src (PDF 54 kb)

Supplementary Table 5

Phosphotyrosine peptides from 3T3-Src-trypsin/P-Tyr-100 (PDF 70 kb)

Supplementary Table 6

Comparison of non-redundant phosphotyrosine peptides from anaplastic large cell lymphoma cell lines, Karpas 299 or SU-DHL-1-trypsin/P-Tyr-100 (PDF 65 kb)

Supplementary Table 7

Phosphotyrosine peptides from anaplastic large cell lymphoma cell lines, Karpas 299 or SU-DHL-1-trypsin/P-Tyr-100 (PDF 106 kb)

Supplementary Table 8

Phosphotyrosine peptides from SU-DHL-1-trypsin or chymotrypsin or endoproteinase GluC or elastase/P-Tyr-100 (PDF 78 kb)

Supplementary Table 9

Number of above-threshold peptide assignments from forward and reverse databases (PDF 33 kb)

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Rush, J., Moritz, A., Lee, K. et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23, 94–101 (2005). https://doi.org/10.1038/nbt1046

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