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Analysis of yeast protein kinases using protein chips

Nature Genetics volume 26, pages 283–289 (2000)Cite this article

Abstract

We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.

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Figure 1: Strategy to overproduce yeast protein kinases.
Figure 2: Protein chip fabrication and kinase assays.
Figure 3: The protein chip and kinase assays.
Figure 4: Quantitative analysis of protein kinase reactions.
Figure 5: Phylogenetic tree derived from the kinase core domain multiple sequence alignment, illustrating the correlation between functional specificity and amino sequences of the poly(Tyr-Glu) kinases.

References

  1. Fields, S., Kohara, Y. & Lockhart, D.J. Functional genomics. Proc. Natl Acad. Sci. USA 96, 8825–8826 ( 1999).

    Article  CAS  Google Scholar 

  2. Goffeau, A. et al. Life with 6000 genes. Science 274, 563–567 (1996).

    Article  Google Scholar 

  3. DeRisi, J.L., Iyer, V.R. & Brown, P.O. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686 (1997).

    Article  CAS  Google Scholar 

  4. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285 , 901–906 (1999).

    Article  CAS  Google Scholar 

  5. Heyman, J.A. et al. Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation. Genome Res. 9, 383–392 (1999).

    CAS  Google Scholar 

  6. Martzen, M.R. et al. A biochemical genomics approach for identifying genes by the activity of their products. Science 286, 1153–1155 (1999).

    Article  CAS  Google Scholar 

  7. Hunter, T. & Plowman, G.D. The protein kinases of budding yeast: six score and more. Trends Biol. Sci. 22, 18–22 (1997).

    Article  CAS  Google Scholar 

  8. Hudson, J.R. et al. The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form. Genome Res. 7, 1169–1173 (1997).

    Article  CAS  Google Scholar 

  9. Mitchell, D.A., Marshall, T.K. & Deschenes, R.J. Vector for the inducible overexpression of glutathione S-transferase fusion protein in yeast. Yeast 9, 715–723 (1993).

    Article  CAS  Google Scholar 

  10. Xia, Y. et al. Complex optical surfaces formed by replica molding against elastomeric masters. Science 273, 347– 349 (1996).

    Article  CAS  Google Scholar 

  11. Rogers, Y.-H. et al. Immobulization of oligonucleotides onto a glass support via disulfide bonds: a method for preparation of DNA microarrays. Anal. Biochem. 266, 23–30 (1999).

    Article  CAS  Google Scholar 

  12. Hunter, T. & Sefton, B.M. Protein phosphorylation . Methods Enzymol. 200, 35– 83 (1991).

    Google Scholar 

  13. Roemer, T.K. et al. Selection of axial growth sites in yeast requires Axl2p, a novel plasma membrane glycoprotein. Genes Dev. 10, 777–793 (1996).

    Article  CAS  Google Scholar 

  14. Weinert, T.A. & Hartwell, L.H. Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. Genetics 134, 63–80 ( 1993).

    CAS  Google Scholar 

  15. Jaquenoud, M., Gulli, M.P., Peter, K. & Peter, M. The Cdc42p effector Gic2p is targeted for ubiquitin-dependent degradation by the SCFGrr1 complex. EMBO J. 17, 5360 –5373 (1998).

    Article  CAS  Google Scholar 

  16. Menees, T.M., Ross-MacDonald, P.B. & Roeder, G.S. MEI4, a meiosis-specific yeast gene required for chromosome synapsis. Mol. Cell. Biol. 12, 1340– 1351 (1992).

    Article  CAS  Google Scholar 

  17. Bailis, J.M. & Roeder, G.S. Synaptonemal complex morphogenesis and sister-chromatid cohesion require Mek1-dependent phosphorylation of a meiotic chromosomal protein. Genes Dev. 12, 3551–3563 (1998).

    Article  CAS  Google Scholar 

  18. Stern, D.F., Zheng, P., Beidler, D.R. & Zerillo, C. Spk1, a new kinase from Saccharomyces cerevisiae phosphorylates proteins on serine, threonine, and tyrosine. Mol. Cell. Biol. 11, 987–1001 (1991).

    Article  CAS  Google Scholar 

  19. Kaouass, M. et al. The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae . Mol. Cell. Biol. 17, 2994– 3004 (1997).

    Article  CAS  Google Scholar 

  20. Barral, Y., Parra, M., Bidlingmaier, S. & Snyder, M. Nim1-related kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeast. Genes Dev. 13, 176–187 (1999).

    Article  CAS  Google Scholar 

  21. Madden, K., Sheu, Y.-J., Baetz, K., Andrews, B. & Snyder, M. SBF cell cycle regulator as a target of the yeast PKC-MAP kinase pathway. Science 275, 1781–1784 (1997).

    Article  CAS  Google Scholar 

  22. Sobel, S.G. & Snyder, M. A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae. J. Cell. Biol. 131, 1775–1788 (1995).

    Article  CAS  Google Scholar 

  23. Ferrigno, P., Posas, F., Koepp, D., Saito, H. & Silver, P.A. Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin β homologs NMD5 and XPO1. EMBO J. 17, 5606–5614 ( 1998).

    Article  CAS  Google Scholar 

  24. Ho, U., Mason, S., Kobayashi, R., Heokstra, M. & Andrew, B. Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 94, 581–586 (1997).

    Article  CAS  Google Scholar 

  25. Wurgler-Murphy, S.M., Maeda, T., Witten, E.A. & Saito, H. Regulation of the Saccharomyces cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine phosphatases. Mol. Cell. Biol. 17, 1289–1297 (1997).

    Article  CAS  Google Scholar 

  26. Santos, T. & Hollingsworth, N.M. Red1p, a MEK1-dependent phosphoprotein that physically interacts with Hop1p during meiosis in yeast . J. Biol. Chem. 274, 1783– 1790 (1999).

    Article  Google Scholar 

  27. Holly, S.P. & Blumer, K.J. PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae. J. Cell Biol. 147, 845– 856 (1999).

    Article  CAS  Google Scholar 

  28. Richman, T.J., Sawyer, M.M. & Johnson, D.I. The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast. J. Biol. Chem. 274, 16861 –16870 (1999).

    Article  CAS  Google Scholar 

  29. Malathi, K., Xiao, Y. & Mitchell, A.P. Catalytic roles of yeast GSK3β/shaggy homolog Rim11p in meiotic activation. Genetics 153, 1145–1152 (1999).

    CAS  Google Scholar 

  30. Owen, D.J., Noble, M.E., Garman, E.F., Papageorgiou, A.C. & Johnson, L.N. Two structures of the catalytic domain of phosphorylase kinase: an active protein kinase complexed with substrate analogue and product. Structure 3, 467–474 (1995).

    Article  CAS  Google Scholar 

  31. Jackman, R.J., Duffy, D.C., Cherniavskaya, O. & Whitesides, G.M. Using elastomeric membranes as dry resists and for dry lift-off. Langmuir 15, 2973– 2984 (1999).

    Article  CAS  Google Scholar 

  32. Mylin, L.M., Hofmann, K.J., Schultz, L.D. & Hopper, J.E. Regulated GAL4 expression cassette providing controllable and high-level output from high-copy galactose promoters in yeast. Methods Enzymol. 185, 297–308 (1990).

    Article  CAS  Google Scholar 

  33. Higgins, D.G., Thompson, J.D. & Gibson, T.J. Using CLUSTAL for multiple sequence alignments . Methods Enzymol. 266, 383– 402 (1996).

    Article  CAS  Google Scholar 

  34. Gonnet, G.H., Cohen, M.A. & Benner, S.A. Exhaustive matching of the entire protein sequence database. Science 256, 1443– 1445 (1992).

    Article  CAS  Google Scholar 

  35. Bairoch, A. & Apweiler, R. The SWISS-PROT protein sequence data bank and its supplement TrEMBL. Nucleic Acids Res. 27, 49–54 ( 1999).

    Article  CAS  Google Scholar 

  36. Barker, W.C. et al. The PIR-International Protein Sequence Database. Nucleic Acids Res. 27, 39–43 (1999).

    Article  CAS  Google Scholar 

  37. Benson, D.A. et al. GenBank. Nucleic Acids Res. 27, 12–17 (1999).

    Article  CAS  Google Scholar 

  38. Lipman, D.J. & Pearson, W.R. Rapid and sensitive protein similarity searches. Science 277, 1435–1441 (1985).

    Article  Google Scholar 

  39. Pearson, W.R. & Lipman, D.J. Improved tools for biological sequence comparison. Proc. Natl Acad. Sci. USA 85, 2444–2448 (1988).

    Article  CAS  Google Scholar 

  40. Dayhoff, M.O., Schwartz, R.M. & Orcutt, B.C. A model of evolutionary change in proteins . in Atlas of Protein Sequence and Structure (ed. Dayhoff, M.O.) 345–352 (National Biomedical Research Foundation, Washington DC, 1978).

    Google Scholar 

  41. Hanks, S.K. & Hunter, T. Protein Kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9, 576– 596 (1995).

    Article  CAS  Google Scholar 

  42. Felsenstein, J. PHYLIP-Phylogeny Inference Package (Version 3.2). Cladistics 5, 164–166 (1989).

    Google Scholar 

  43. Fitch, W.M. & Margoliash, E. Construction of phylogenetic trees. Science 155, 279– 284 (1967).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Schwartz, D. Stern, J. Bailus, G. Michaud, M. Jaquenoud and M. Peter for substrates; G. Michaud for devising methods for preparing GST:fusions; G. Michaud, B. Manning, C. Horak and S. Bidlingmaier for critical comments on the manuscript; E. Skoufas for the list of protein kinases; and F.J. Sigworth for the use of his laboratory facilities to cast silicone elastomer microwells. This research was supported by grants from the National Institutes of Health, Defense Research Project Agency and the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation.

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Authors and Affiliations

  1. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA

    Heng Zhu, Paul Bertone, Antonio Casamayor, David Smith & Michael Snyder

  2. Department of Electrical Engineering, Yale University, New Haven, Connecticut, USA

    James F. Klemic, Swan Chang & Mark A. Reed

  3. Department of Applied Physics, Yale University, New Haven, Connecticut, USA

    James F. Klemic & Mark A. Reed

  4. Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut, USA

    Kathryn G. Klemic

  5. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Mark Gerstein & Michael Snyder

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  1. Heng Zhu
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Correspondence to Michael Snyder.

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Zhu, H., Klemic, J., Chang, S. et al. Analysis of yeast protein kinases using protein chips. Nat Genet 26, 283–289 (2000). https://doi.org/10.1038/81576

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