Fig. 1.

Miniaturized peptide array approach enables high-throughput analysis of kinase consensus phosphorylation motifs. (A) Scheme for kinase peptide screening. Capillary pin-based liquid transfer devices were used to add components to reactions (2 μl per well) and spot 0.2 μl aliquots onto the streptavidin-coated membrane following incubation. The 1536-well format allows four kinases to be analyzed simultaneously. (B) Representative peptide screening results for Atg1, Gin4, Mps1, and Prk1. (C) Phosphorylation of consensus peptide substrates by Atg1, Gin4, Mps1, and Prk1. The sequence of each peptide is as follows: ATGtide, YANWLAASIYLDGKKK; GINtide, YALRRSRSMWNLGKKK; MPStide, YADHDDDTMHFRGKKK; and PRKtide, YALKPQYTGPRGKKK. Peptide phosphorylation was assayed at 10 μM concentration by radiolabel kinase assay. Incorporation of radiolabeled phosphate into peptides was determined by phosphocellulose filter binding assay. Maximal rates for each kinase in these assays were: Atg1, 250 nM/min, Gin4, 510 nM/min, Mps1, 130 nM/min, Prk1, 330 nM/min. (D) Rates of Atg1 phosphorylation of ATGtide variants with individual point substitutions. Peptide phosphorylation was assayed as for panel C.