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.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Fields, S., Kohara, Y. & Lockhart, D.J. Functional genomics. Proc. Natl Acad. Sci. USA 96, 8825–8826 ( 1999).
Goffeau, A. et al. Life with 6000 genes. Science 274, 563–567 (1996).
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).
Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285 , 901–906 (1999).
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).
Martzen, M.R. et al. A biochemical genomics approach for identifying genes by the activity of their products. Science 286, 1153–1155 (1999).
Hunter, T. & Plowman, G.D. The protein kinases of budding yeast: six score and more. Trends Biol. Sci. 22, 18–22 (1997).
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).
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).
Xia, Y. et al. Complex optical surfaces formed by replica molding against elastomeric masters. Science 273, 347– 349 (1996).
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).
Hunter, T. & Sefton, B.M. Protein phosphorylation . Methods Enzymol. 200, 35– 83 (1991).
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).
Weinert, T.A. & Hartwell, L.H. Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. Genetics 134, 63–80 ( 1993).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Malathi, K., Xiao, Y. & Mitchell, A.P. Catalytic roles of yeast GSK3β/shaggy homolog Rim11p in meiotic activation. Genetics 153, 1145–1152 (1999).
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).
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).
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).
Higgins, D.G., Thompson, J.D. & Gibson, T.J. Using CLUSTAL for multiple sequence alignments . Methods Enzymol. 266, 383– 402 (1996).
Gonnet, G.H., Cohen, M.A. & Benner, S.A. Exhaustive matching of the entire protein sequence database. Science 256, 1443– 1445 (1992).
Bairoch, A. & Apweiler, R. The SWISS-PROT protein sequence data bank and its supplement TrEMBL. Nucleic Acids Res. 27, 49–54 ( 1999).
Barker, W.C. et al. The PIR-International Protein Sequence Database. Nucleic Acids Res. 27, 39–43 (1999).
Benson, D.A. et al. GenBank. Nucleic Acids Res. 27, 12–17 (1999).
Lipman, D.J. & Pearson, W.R. Rapid and sensitive protein similarity searches. Science 277, 1435–1441 (1985).
Pearson, W.R. & Lipman, D.J. Improved tools for biological sequence comparison. Proc. Natl Acad. Sci. USA 85, 2444–2448 (1988).
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).
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).
Felsenstein, J. PHYLIP-Phylogeny Inference Package (Version 3.2). Cladistics 5, 164–166 (1989).
Fitch, W.M. & Margoliash, E. Construction of phylogenetic trees. Science 155, 279– 284 (1967).
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.
About this article
Cite this article
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
Australian Journal of Chemistry (2020)
From Beef to Bees: High-Throughput Kinome Analysis to Understand Host Responses of Livestock Species to Infectious Diseases and Industry-Associated Stress
Frontiers in Immunology (2020)
Development of a Bacterial Macroarray for the Rapid Screening of Targeted Antibody-Secreted Hybridomas
SLAS DISCOVERY: Advancing the Science of Drug Discovery (2019)
Polymers for Advanced Technologies (2019)