Phosphorylation Studies Using Plant Protein Microarrays

Identifying protein kinase substrates is one major focus of protein kinase research and supports the elucidation of signal transduction pathways and their complex regulation. In this chapter we describe a protein microarray-based in vitro method, which permits a systematic screening of immobilized proteins for their phosphorylation by specific protein kinases. This high-throughput method allows the identification of potential kinase substrates. The method starts with plant protein microarrays containing hundreds of purified recombinant His-tagged proteins. The microarrays have to be incubated with the soluble and active kinase in the presence of radioactive [γ³³ -P]ATP. Only small volumes of active kinase are needed for one microarray experiment. Radioactive signals are then detectable by phosphor imager or X-ray film. The identified potential substrates have to be verified by other in vitro or in vivo methods, as both the kinase and the substrate may not interact with each other under in vivo conditions. This screening method could be generally applicable for direct identification of candidate substrates of various protein kinases.