Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling

Despite the growing interest in using chemical genetics in plant research, small molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution mass spectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or posttranslational modifications, allows the discovery of drug targets or the study of protein–metabolite and protein–protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal and the phosphoproteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf, thus revealing cross-talk between brassinosteroid and auxin signaling.