Skip to main content
SpringerLink
Log in
Book cover

Kinase Screening and Profiling pp 183–202Cite as

Determination of the Substrate Specificity of Protein Kinases with Peptide Micro- and Macroarrays

  • Protocol

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1360))

Abstract

Elucidation of the key determinants for the phosphorylation site specificities of protein kinases facilitates identification of their physiological substrates, and serves to better define their critical roles in the signaling networks that underlie a multitude of cellular activities. Albeit with some apparent limitations, such as the lack of contextual information for secondary substrate-binding sites, the synthetic peptide-based approach has been adopted widely for the kinase specificity profiling studies, especially when they are used in an array format, which permits the screening of large numbers of potential peptide substrates in parallel. In this chapter, we present detailed protocols for determining protein kinase substrate specificity using an approach that involves both peptide microarrays and macroarrays. In particular, SPOT synthesis on macroarrays can be used to follow up on in silico predictions of protein kinase substrate specificity with predictive algorithms.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Manning G, Whyte DB, Martinez R et al. (2002) The protein kinase complement of the human genome. Science 298:1912–1934

    Article  CAS  PubMed  Google Scholar 

  2. Songyang Z, Cantley LC (1995) Recognition and specificity in protein tyrosine kinase-mediated signalling. Trends Biochem Sci 20:470–475

    Article  CAS  PubMed  Google Scholar 

  3. Songyang Z, Lu KP, Kwon YT et al. (1996) A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1. Mol Cell Biol 16:6486–6493

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Faux MC, Scott JD (1996) More on target with protein phosphorylation: conferring specificity by location. Trends Biochem Sci 21:312–315

    CAS  PubMed  Google Scholar 

  5. Kreegipuu A, Blom N, Brunak S (1998) Statistical analysis of protein kinase specificity determinants. FEBS Lett 430:45–50

    Article  CAS  PubMed  Google Scholar 

  6. Songyang Z (1999) Recognition and regulation of primary-sequence motifs by signaling modular domains. Prog Biophys Mol Biol 71:359–372

    Article  CAS  PubMed  Google Scholar 

  7. Kemp BE, Benjamini E, Krebs EG (1976) Synthetic hexapeptide substrates and inhibitors of 3′:5′-cyclic AMP-dependent protein kinase. Proc Natl Acad Sci U S A 73:1038–1042

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Kemp BE, Graves DJ, Benjamini E et al. (1977) Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J Biol Chem 252:4888–4894

    CAS  PubMed  Google Scholar 

  9. Casnellie JE, Krebs EG (1984) The use of synthetic peptides for defining the specificity of tyrosine protein kinases. Adv Enzyme Regul 22:501–515

    Article  CAS  PubMed  Google Scholar 

  10. Safaei J, Manuch J, Gupta A, Stacho L, Pelech S (2011) Prediction of 492 human protein kinase substrate specificities. Proteome Sci 9(Suppl 1):S6

    Article  PubMed Central  PubMed  Google Scholar 

  11. Tegge W, Frank R, Hofmann F et al. (1995) Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper. Biochemistry 34:10569–10577

    Article  CAS  PubMed  Google Scholar 

  12. MacBeath G, Schreiber SL (2000) Printing proteins as microarrays for high-throughput function determination. Science 289:1760–1763

    CAS  PubMed  Google Scholar 

  13. Houseman BT, Huh JH, Kron SJ et al. (2002) Peptide chips for the quantitative evaluation of protein kinase activity. Nat Biotechnol 20:270–274

    Article  CAS  PubMed  Google Scholar 

  14. Reimer U, Reineke U, Schneider-Mergener J (2002) Peptide arrays: from macro to micro. Curr Opin Biotechnol 13:315–320

    Article  CAS  PubMed  Google Scholar 

  15. Frank R (2002) The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports—principles and applications. J Immunol Methods 267:13–26

    Article  CAS  PubMed  Google Scholar 

  16. Kramer A, Schneider-Mergener J (1998) Synthesis and application of peptide libraries bound to continuous cellulose membranes. Methods Mol Biol 87:25–39

    CAS  PubMed  Google Scholar 

  17. Reineke U, Volkmer-Engert R, Schneider-Mergener J (2001) Applications of peptide arrays prepared by the SPOT-technology. Curr Opin Biotechnol 12:59–64

    Article  CAS  PubMed  Google Scholar 

  18. Blackwell HE (2006) Hitting the SPOT: small-molecule macroarrays advance combinatorial synthesis. Curr Opin Chem Biol 10:203–212

    Article  CAS  PubMed  Google Scholar 

  19. Hilpert K, Winkler DFH, Hancock REW (2007) Peptide arrays on cellulose support: SPOT synthesis—a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nat Protoc 2:1333–1349

    Article  CAS  PubMed  Google Scholar 

  20. Winkler DFH, Campbell WD (2008) The Spot technique: Synthesis and screening of peptide macroarrays on cellulose membranes. Methods Mol Biol 494:47–70

    Google Scholar 

  21. Schutkowski M, Reineke U, Reimer U (2005) Peptide arrays for kinase profiling. Chembiochem 6:513–521

    Article  CAS  PubMed  Google Scholar 

  22. Diks SH, Kok K, O’Toole T et al. (2004) Kinome profiling for studying lipopolysaccharide signal transduction in human peripheral blood mononuclear cells. J Biol Chem 279:49206–49213

    Article  CAS  PubMed  Google Scholar 

  23. Schutkowski M, Reimer U, Panse S et al. (2004) High-content peptide microarrays for deciphering kinase specificity and biology. Angew Chem Int Ed Engl 43:2671–2674

    Article  CAS  PubMed  Google Scholar 

  24. Uttamchandani M, Chan EW, Chen GY et al. (2003) Combinatorial peptide microarrays for the rapid determination of kinase specificity. Bioorg Med Chem Lett 13:2997–3000

    Article  CAS  PubMed  Google Scholar 

  25. Wildemann D, Erdmann F, Alvarez BH et al. (2006) Nanomolar inhibitors of the peptidyl prolyl cis/trans isomerase Pin1 from combinatorial peptide libraries. J Med Chem 49:2147–2150

    Google Scholar 

  26. Atherton E, Sheppard RC (1989) 7.2. Activated esters of Fmoc-amino acids. In: Solid phase peptide synthesis—a practical approach. IRL press at Oxford University Press, Oxford, UK, pp 76–78

    Google Scholar 

  27. Fields GB, Noble RL (1990) Solid phase synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Pept Protein Res 35:161–214

    Article  CAS  PubMed  Google Scholar 

  28. Zander N, Gausepohl H (2002) Chemistry of Fmoc peptide synthesis on membranes. In: Koch J, Mahler M (eds) Peptide Arrays on Membrane Support. Springer, Berlin, Heidelberg, pp 23–39

    Chapter  Google Scholar 

  29. Hilpert K, Winkler DFH, Hancock REW (2007) Cellulose-bound peptide arrays: Preparation and applications. Biotechnol Genet Eng Rev 24:31–106

    Article  CAS  Google Scholar 

  30. Frank R (1992) Spot-synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48:9217–9232

    Article  CAS  Google Scholar 

  31. Volkmer R, Kretzschmar I, Tapia V (2012) Mapping receptor–ligand interactions with synthetic peptide arrays: Exploring the structure and function of membrane receptors. Eur J Cell Biol 91:349–356

    Article  CAS  PubMed  Google Scholar 

  32. Kamradt T, Volkmer-Engert R (2004) Cross-reactivity of T lymphocytes in infection and autoimmunity. Mol Divers 8:271–280

    Article  CAS  PubMed  Google Scholar 

  33. Krchnak V, Wehland J, Plessmann U et al. (1988) Noninvasive continuous monitoring of solid phase peptide synthesis by acid-base indicator. Collect Czech Chem Comm 53:2542–2548

    Article  CAS  Google Scholar 

  34. Molina F, Laune D, Gougat C et al. (1996) Improved performances of spot multiple peptide synthesis. Pept Res 9:151–155

    CAS  PubMed  Google Scholar 

  35. Portwich M, Keller S, Strauss HM et al. (2007) A network of coiled-coil associations derived from synthetic GCN4 leucine-zipper arrays. Angew Chem Int Ed 46:1654–1657, Supporting Information

    Article  CAS  Google Scholar 

  36. Kramer A, Reineke U, Dong L et al. (1999) Spot-synthesis: observations and optimizations. J Pept Res 54:319–327

    Article  CAS  PubMed  Google Scholar 

  37. Martin K, Steinberg TH, Cooley LA et al. (2003) Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye. Proteomics 3:1244–1255

    Article  CAS  PubMed  Google Scholar 

  38. Petter C, Scholz C, Wessner H et al. (2008) Phage display screening for peptidic chitinase inhibitors. J Mol Recognit 21:401–409

    Article  CAS  PubMed  Google Scholar 

  39. Lizcano JM, Deak M, Morrice N et al. (2002) Molecular basis for the substrate specificity of NIMA-related kinase-6 (NEK6). Evidence that Nek6 does not phosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- and glucocorticoid-induced protein kinase in vivo. J Biol Chem 277:27839–27849

    Google Scholar 

  40. Panse S, Dong L, Burian A et al. (2004) Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays. Mol Divers 8:291–299

    Article  CAS  PubMed  Google Scholar 

  41. Licha K, Bhargava S, Rheinlander C et al. (2000) Highly parallel nano-synthesis of cleavable peptide-dye conjugates on cellulose membranes. Tetrahedron Lett 41:1711–1715

    Article  CAS  Google Scholar 

  42. Ast T, Heine N, Germeroth L et al. (1999) Efficient assembly of peptomers on continuous surfaces. Tetrahedron Lett 40:4317–4318

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, British Columbia, Canada, V6P 6T3

    Shenshen Lai, Dirk F. H. Winkler, Hong Zhang & Steven Pelech

  2. Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada

    Steven Pelech

Authors
  1. Shenshen Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dirk F. H. Winkler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Steven Pelech
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Steven Pelech .

Editor information

Editors and Affiliations

  1. Promega Corporation, Madison, Wisconsin, USA

    Hicham Zegzouti

  2. Promega Corporation, Madison, Wisconsin, USA

    Said A. Goueli

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Lai, S., Winkler, D.F.H., Zhang, H., Pelech, S. (2016). Determination of the Substrate Specificity of Protein Kinases with Peptide Micro- and Macroarrays. In: Zegzouti, H., Goueli, S. (eds) Kinase Screening and Profiling. Methods in Molecular Biology, vol 1360. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3073-9_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3073-9_14

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3072-2

  • Online ISBN: 978-1-4939-3073-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation