Abstract
Protein phosphorylation by protein kinases is the most important regulatory mechanism of cell function and signal transduction. In general, protein kinases exhibit specificities that are often primarily determined by the amino acrds around the phosphorylation sites (1). Identification of amino acids that contribute to substrate motifs are essential for the understanding of signal transduction pathways and for the development of specific peptide substrates and inhibitors. Many investigations with large numbers of individual peptides have been conducted in order to find high-affinity substrates as well as mhrbrtors (2). Peptide libraries offer the possibility to investigate the sequence dependence of the phosphorylation more thoroughly and systematically and may even allow the a priori delineation of peptide substrates of uncharacterized protein kinases.
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References
Kemp B E and Pearson R B (1990) Protein kmase recognition sequences motifs Trends Biochem. Sci 15, 342–346
Pearson R B and Kemp B E (1990) Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulation Methods Enzymol 200, 62–81
Wu J., Ma Q N., and Lam K S (1994) Identifyig substrate motifs of protein kinases by a random library approach Biochemistry 33, 14,825–14,833
Furka A., Sebestyen F., Asgedom M., and Dibo G (1991) General method for rapid synthesis of multicomponent peptide mixtures Int. J Pept Protem Res 37, 487–493.
Songyang Z., Blechner S., Hoagland N., Hoekstra M. F., Ptwmca-Worms H., and Cantley L C (1994) Use of an oriented peptrde library to determine the optimal substrates of protein kinases Curr. Biol 4, 973–981
Songyang Z. (1995) Catalytic specificity of protein tyrosme kmases IS critical for selective signalling Nature 373, 536–539
Frank R (1992) Spot-synthesis an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support Tetrahedron 48, 9217–9232
Frank R and Overwm H. (1996) SPOT-syntheses epitope analysts with arrays of synthetic peptrdes prepared on cellulose membranes III, Methods in Molecular Biology, vol 66: Epitope Mapping Protocols (Moms G. E., ed), Humana, Totowa, NJ, pp 149–169
Tegge W., Frank R., Hofmann F., and Dostmann W R G (1995) Determination of cychc nucleotide-dependent protein kmase substrate specificity by the use of peptlde libraries on cellulose paper Biochemistry 34, 10,569–10,577
Glass D B., Cheng H-C., Mende-Muller L., Reed J., and Walsh D A (1989) Primary structural determinants essential for potent inhibition of cAMP-dependent protein kmase by inhibitory peptides corresponding to the active portion of the heat-stable mhrbrtor protein J Biol Chem 264, 8802–8810
Butt E., Abel K., Krueger M., Palm D., Hoppe V., Hoppe J., and Walter U (1994) cAMP-and cGMP-dependent protein kmase phosphorylation sites of the focal adhesion vasodtlator-stimulated phosphoprotem (VASP) in vitro and in intact human platelets J Blol. Chem 269, 14,509–14,517
Glass D B (1990) Substrate specificity of the cyclic GMP-dependant protein kmase in, Peptides and Protem Phosphorylation (Kemp B E., ed), CRC, Boca Raton, FL, pp 209–238
Geysen H.M and Mason T J (1993) Screening chemically synthesized peptide librartes for biologically-relevant molecules Bioorg Med Chem. Lett 3, 397–404
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© 1998 Humana Press Inc., Totowa, NJ
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Tegge, W.J., Frank, R. (1998). Analysis of Protein Kinase Substrate Specificity by the Use of Peptide Libraries on Cellulose Paper (SPOT-Method). In: Cabilly, S. (eds) Combinatorial Peptide Library Protocols. Methods in Molecular Biology™, vol 87. Humana Press. https://doi.org/10.1385/0-89603-392-9:99
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DOI: https://doi.org/10.1385/0-89603-392-9:99
Publisher Name: Humana Press
Print ISBN: 978-0-89603-392-4
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