Analysis of yeast protein kinases using protein chips

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.

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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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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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