Kinase peptide specificity: Improved determination and relevance to protein phosphorylation
Open Access
- 8 September 2004
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 101 (38), 13744-13749
- https://doi.org/10.1073/pnas.0401881101
Abstract
Specificity of phosphorylation is critical to signal transduction. Recent emphasis on colocalization of substrate and kinase has eclipsed emphasis on peptide specificity, i.e., kinase preference for particular amino acids surrounding the phosphorylation site. We describe an approach to determining peptide specificity by using positional scanning of biotinylated oriented peptide libraries and insights emerging from those determinations. We accurately determine preference (or disfavor) for residues at a given substrate position (such as P+2) by comparison of in vitro phosphorylation of peptide libraries differing by a single residue at that position. By analysis of all positions near the phosphorylation site, position-specific scoring matrices are generated and used both to understand the basis of specificity and to predict phosphorylation. PKC-δ and -ζ predictions have been validated rigorously by comparisons with measured phosphorylation. The results demonstrate specificity and sensitivity (80–90%) much better than the previous predictive method. These predictions can be accessed at http://mpr.nci.nih.gov. The accuracy of the specificity determination allows identification of an important difference in peptide specificity between these closely related kinases; Ile/Leu at the P–1 position is disfavored by PKC-ζ but not PKC-δ. Our findings and visual representation of peptide specificity highlight the importance of disfavored residues. Finally, analysis of 124 experimentally determined PKC sites from the literature demonstrates a very strong role of peptide specificity in many of those sites. Thus, position-specific scoring matrices generated by this method provide a foundation for quantitative analyses of kinase specificity and improved predictions of previously determined physiologically relevant phosphorylation sites.Keywords
This publication has 34 references indexed in Scilit:
- Conservation of Electrostatic Properties within Enzyme Families and SuperfamiliesBiochemistry, 2003
- AGC protein kinase phosphorylation and protein kinase CBiochemical Society Transactions, 2001
- AGC protein kinase phosphorylation and protein kinase CBiochemical Society Transactions, 2001
- Regulation of ARNO nucleotide exchange by a PH domain electrostatic switchCurrent Biology, 1999
- Examination of an active‐site electrostatic node in the cAMP‐dependent protein kinase catalytic subunitProtein Science, 1996
- Use of an oriented peptide library to determine the optimal substrates of protein kinasesCurrent Biology, 1994
- Studies on the primary sequence requirements for PKC‐α, ‐β1 and ‐γ peptide substratesFEBS Letters, 1990
- Sequence logos: a new way to display consensus sequencesNucleic Acids Research, 1990
- Unique substrate specificity and regulatory properties of PKC‐ε: a rationale for diversityFEBS Letters, 1989
- Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membraneNature, 1984