Global Analysis of Human Nonreceptor Tyrosine Kinase Specificity Using High-Density Peptide Microarrays

Protein
kinases phosphorylate substrates in the context of specific
phosphorylation site sequence motifs. The knowledge of the specific
sequences that are recognized by kinases is useful for mapping sites
of phosphorylation in protein substrates and facilitates the generation
of model substrates to monitor kinase activity. Here, we have adapted
a positional scanning peptide library method to a microarray format
that is suitable for the rapid determination of phosphorylation site
motifs for tyrosine kinases. Peptide mixtures were immobilized on
glass slides through a layer of a tyrosine-free Y33F mutant avidin
to facilitate the analysis of phosphorylation by radiolabel assay.
A microarray analysis provided qualitatively similar results in comparison
with the solution phase peptide library “macroarray”
method. However, much smaller quantities of kinases were required
to phosphorylate peptides on the microarrays, which thus enabled a
proteome scale analysis of kinase specificity. We illustrated this
capability by microarray profiling more than 80% of the human nonreceptor
tyrosine kinases (NRTKs). Microarray results were used to generate
a universal NRTK substrate set of 11 consensus peptides for in vitro
kinase assays. Several substrates were highly specific for their cognate
kinases, which should facilitate their incorporation into kinase-selective
biosensors.