SILAKin: A novel high throughput SILAC and mass spectrometry-based assay to identify the substratome of kinases secreted by pathogens

Abstract

ABSTRACTProtein phosphorylation is one of the most important reversible post-translational modifications. It affects every cellular process including differentiation, metabolism and cell cycle. Eukaryotic protein kinases (ePK) catalyse the transfer of a phosphate from ATP onto proteins, which regulates fast changes in protein activity, structure or subcellular localisation. The systematic identification of substrates is thus crucial to characterise the functions of kinases and determine the pathways they regulate, and even more so when studying the impact of pathogens-excreted kinases on the host cell signal transduction. Several strategies and approaches have been used to identify substrates, but all show important limitations thus calling for the development of new efficient and more convenient approaches for kinase substrate identification.Herein, we present SILAkin, a novel and easy method to identify substrates that is applicable to most kinases. It combines phosphatase treatment, pulse heating, in vitro kinase assay (IVKA) and SILAC (Stable Isotope Labeling with Amino acids in Cell culture)-based quantitative mass spectrometry (MS). We developed SILAkin using the Leishmania casein kinase 1 (L-CK1.2) as experimental model. Leishmania, an intracellular parasite causing Leishmaniasis, releases L-CK1.2 in its host cell. Applying this novel assay allowed us to gain unprecedented insight into host-pathogen interactions through the identification of host substrates phosphorylated by pathogen-excreted kinases. We identified 225 substrates, including 85% previously unknown that represent novel mammalian CK1 targets, and defined a novel CK1 phosphorylation motif. The substratome was validated experimentally by L-CK1.2 and human CK1δ, demonstrating the efficiency of SILAkin to identify new substrates and revealing novel regulatory pathways. Finally, SILAkin was instrumental in highlighting host pathways potentially regulated by L-CK1.2 in Leishmania-infected host cells, described by the GO terms ‘viral & symbiotic interaction’, ‘apoptosis’, ‘actin cytoskeleton organisation’, and ‘RNA processing and splicing’. SILAkin thus can generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogen adapted for intracellular survival.