Abstract
Protein tyrosine kinases phosphorylate their substrates within the context of specific consensus sequences surrounding the site of modification. We describe a peptide microarray approach to rapidly determine tyrosine kinase phosphorylation site motifs. This method uses a peptide library that systematically substitutes each of the amino acid residues at multiple positions surrounding a central tyrosine residue. Peptide substrates are synthesized as biotin conjugates for immobilization on avidin-coated slides. Following incubation of the slide with protein kinase and radiolabeled ATP, the relative extent of phosphorylation of each of the peptides is quantified by phosphor imaging. This method allows small quantities of kinase to be analyzed rapidly in parallel, facilitating analysis of large numbers of kinases.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298:1912–1934
Brognard J, Hunter T (2011) Protein kinase signaling networks in cancer. Curr Opin Genet Dev 21:4–11
Zhang J, Yang PL, Gray NS (2009) Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 9:28–39
Songyang Z, Carraway KL III, Eck MJ, Harrison SC, Feldman RA, Mohammadi M et al (1995) Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature 373:536–539
Allen MD, DiPilato LM, Rahdar M, Ren YR, Chong C, Liu JO et al (2006) Reading dynamic kinase activity in living cells for high-throughput screening. ACS Chem Biol 1:371–376
Zaman GJ, Garritsen A, de Boer T, van Boeckel CA (2003) Fluorescence assays for high-throughput screening of protein kinases. Comb Chem High Throughput Screen 6:313–320
Yaffe MB, Leparc GG, Lai J, Obata T, Volinia S, Cantley LC (2001) A motif-based profile scanning approach for genome-wide prediction of signaling pathways. Nat Biotechnol 19:348–353
Linding R, Juhl Jensen L, Pasculescu A, Olhovsky M, Colwill K, Bork P et al (2008) NetworKIN: a resource for exploring cellular phosphorylation networks. Nucleic Acids Res 36:D695–D699
Kettenbach AN, Wang T, Faherty BK, Madden DR, Knapp S, Bailey-Kellogg C et al (2012) Rapid determination of multiple linear kinase substrate motifs by mass spectrometry. Chem Biol 19:608–618
Hutti JE, Jarrell ET, Chang JD, Abbott DW, Storz P, Toker A et al (2004) A rapid method for determining protein kinase phosphorylation specificity. Nat Methods 1:27–29
Tegge WJ, Frank R (1998) Analysis of protein kinase substrate specificity by the use of peptide libraries on cellulose paper (SPOT-method). Methods Mol Biol 87:99–106
Schutkowski M, Reimer U, Panse S, Dong L, Lizcano JM, Alessi DR et al (2004) High content peptide microarrays for deciphering kinase specificity and biology. Angew Chem Int Ed 43:2761–2674
Uttamchandani M, Chan EW, Chen GY, Yao SQ (2003) Combinatorial peptide microarrays for the rapid determination of kinase specificity. Bioorg Med Chem Lett 13:2997–3000
Deng Y, Alicea-Velazquez NL, Bannwarth L, Lehtonen SI, Boggon TJ, Cheng HC et al (2014) Global analysis of human nonreceptor tyrosine kinase specificity using high-density Peptide microarrays. J Proteome Res 13:4339–4346
Marttila AT, Hytonen VP, Laitinen OH, Bayer EA, Wilchek M, Kulomaa MS (2003) Mutation of the important Tyr-33 residue of chicken avidin: functional and structural consequences. Biochem J 369:249–254
Deng Y, Zhu XY, Kienlen T, Guo A (2006) Transport at the air/water interface is the reason for rings in protein microarrays. J Am Chem Soc 128:2768–2769
Deng Y, Couch BA, Koleske AJ, Turk BE (2012) A peptide photoaffinity probe specific for the active conformation of the Abl tyrosine kinase. Chembiochem 13:2510–2512
Adams JA (2001) Kinetic and catalytic mechanisms of protein kinases. Chem Rev 101:2271–2290
Edelhoch H (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948–1954
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Deng, Y., Turk, B.E. (2016). Analysis of Protein Tyrosine Kinase Specificity Using Positional Scanning Peptide Microarrays. In: Cretich, M., Chiari, M. (eds) Peptide Microarrays. Methods in Molecular Biology, vol 1352. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3037-1_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3037-1_3
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3036-4
Online ISBN: 978-1-4939-3037-1
eBook Packages: Springer Protocols