Global identification of protein kinase substrates by protein microarray analysis

Abstract

Herein, we describe a protocol for the global identification of in vitro substrates targeted by protein kinases using protein microarray technology. Large numbers of fusion proteins tagged at their carboxy-termini are purified in 96-well format and spotted in duplicate onto amino-silane-coated slides in a spatially addressable manner. These arrays are incubated in the presence of purified kinase and radiolabeled ATP, and then washed, dried and analyzed by autoradiography. The extent of phosphorylation of each spot is quantified and normalized, and proteins that are reproducibly phosphorylated in the presence of the active kinase relative to control slides are scored as positive substrates. This approach enables the rapid determination of kinase-substrate relationship on a proteome-wide scale, and although developed using yeast, has since been adapted to higher eukaryotic systems. Expression, purification and printing of the yeast proteome require about 3 weeks. Afterwards, each kinase assay takes approximately 3 h to perform.

Figure 1

Workflow of a protein microarray-based kinase assay to identify candidate substrates targeted by the yeast kinases. Shown are a representative probing of a yeast proteome microarray generated using an N-terminal GST overexpression ORF collection probed with anti-GST antibodies followed by Cy5-labeled anti-rabbit antibodies (entire array with all 40 blocks, left), a control kinase assay preformed in the absence of any kinase (single block of 256 spots, upper right), and a [γ-³³P]ATP Tpk1 kinase assay (single block of 256 spots, lower right). Dark spots in each of the kinase assays represent radiolabeled phosphorylated proteins. Positive controls for kinase assays are printed in each corner of the block and serve as reference points on the slide. Figure is adapted from Ptacek et al., Nature 438 (2005).