Coevolving MAPK and PID phosphosites indicate an ancient environmental control of PIN auxin transporters in land plants.

Plant growth flexibly adapts to environmental conditions, implying cross-talk between environmental signalling and developmental regulation. Here, we show that the PIN auxin efflux carrier family possesses three highly conserved putative mitogen-activated protein kinase (MAPK) sites adjacent to the phosphorylation sites of the well-characterised AGC kinase PINOID, which regulates the polar localisation of PINs and directional auxin transport, thereby underpinning organ growth. The conserved sites of PIN1 are phosphorylated in vitro by two environmentally activated MAPKs, MPK4 and MPK6. In contrast to AGC kinases, MAPK-mediated phosphorylation of PIN1 at adjacent sites leads to a partial loss of the plasma membrane localisation of PIN1. MAPK-mediated modulation of PIN trafficking may participate in environmental adjustment of plant growth.

Kinase-Associated Phosphoisoform Assay: a novel candidate-based method to detect specific kinase-substrate phosphorylation interactions in vivo.

BACKGROUND: Protein kinases are important components of signalling pathways, and kinomes have remarkably expanded in plants. Yet, our knowledge of kinase substrates in plants is scarce, partly because tools to analyse protein phosphorylation dynamically are limited. Here we describe Kinase-Associated Phosphoisoform Assay, a flexible experimental method for directed experiments to study specific kinase-substrate interactions in vivo. The concept is based on the differential phosphoisoform distribution of candidate substrates transiently expressed with or without co-expression of activated kinases. Phosphorylation status of epitope-tagged proteins is subsequently detected by high-resolution capillary isoelectric focusing coupled with nanofluidic immunoassay, which is capable of detecting subtle changes in isoform distribution. RESULTS: The concept is validated by showing phosphorylation of the known mitogen-activated protein kinase (MAPK) substrate, ACS6, by MPK6. Next, we demonstrate that two transcription factors, WUS and AP2, both of which are shown to be master regulators of plant development by extensive genetic studies, exist in multiple isoforms in plant cells and are phosphorylated by activated MAPKs. CONCLUSION: As plant development flexibly responds to environmental conditions, phosphorylation of developmental regulators by environmentally-activated kinases may participate in linking external cues to developmental regulation. As a counterpart of advances in unbiased screening methods to identify potential protein kinase substrates, such as phosphoproteomics and computational predictions, our results expand the candidate-based experimental toolkit for kinase research and provide an alternative in vivo approach to existing in vitro methodologies.