High-throughput in vitro identification of direct MAPK/Erk substrates

Abstract

Phosphorylation mediated by cellular protein kinases is an effective mechanism employed by an organism to regulate central processes such as cell-cycle progression, metabolic pathways, cytoskeletal function, cell migration and differentiation. Thus, for example, various signaling pathways utilize sequential phosphorylation events to relay external cues from the cell surface to the nucleus, where eventually gene expression profiles are altered and, consequently, changes in cell fates and function are induced. Accordingly, recognizing the direct targets of key effector kinases is of utmost importance for understanding the cellular responses to pathway activity. Here we describe a high-throughput genome-wide proteomics approach aimed at uncovering novel nuclear targets for the single Drosophila MAPK/Erk. Briefly, pools of cDNA are transcribed and translated in vitro in the presence of [35S]Methionine, generating a library of radiolabeled protein pools which are subsequently subjected to biochemical kinase assays using recombinant, active Erk2. Phosphorylated proteins representing potential MAPK/Erk substrates are then detected due to their shifted mobility on SDS-PAGE gels. This protocol can be easily adjusted and applied toward identifying targets of other kinases for which in vitro phosphorylation assays are available.