RGS1 phospho-barcode: decoding how G signaling is modulated in plants through the phosphorylation of its components
Authors:   , Oliveira Célio Cabral 
Published: February 2023

Plants activate complex mechanisms of cellular signaling in response to environmental changes. Thus, signaling pathways controlled by the heterotrimeric G protein play an essential role. The G complex is composed of one guanine nucleotide-binding alpha subunit, one beta and one gamma. In spite of the fact that those proteins were conserved in eukaryotes during the evolution process, plants, algae, and protists, as well as organisms at the evolutionary base, possess self-activating alpha subunits with an accelerated GDP-to-GTP exchange activity at its catalytical domain. Thus, self-activating proteins differ from their orthologs at the activation level that is not controlled by G-protein coupled receptors (GPCRs). Instead, those are regulated by RGS (regulator of G-signaling) proteins with a seven-transmembrane (7TM) domain. Differently from the classical switching mechanism via the GTP/GDP state of Gα, the post- translational modifications on G-core components add a new layer of sensitization, discrimination of recognized stimulus, and propagation of stimulus signaling, which defines a four-state model of G-protein activation. Although several phosphorylation sites have already been identified, the underlying mechanisms and the specificity of each of them on signaling pathways are not fully elucidated. Therefore, to shed a light on this additional control layer in G-protein regulation, as well as on the mechanism of its regulator RGS, we used the model organism Arabidopsis thaliana and evaluated the effect of phosphorylation in the G subunits in the signal activation and distinction upon different conditions. In the first chapter, we have crossed in vivo and in vitro phosphorylation data of canonical and non-canonical subunits, and we highlight the phosphosite conservation among eukaryotes. We pointed out the kinase receptors involved and the potential effect of those modifications on signal plasticity. In the second chapter, through site-direct mutagenesis, we generated phosphomimetics and phosphonull mutants of AtRGS1 residues that presented higher conservation among species and were previously identified by their phosphorylation. Thus, we evaluated the central role of those residues in interacting with G complex proteins and with possible adaptors involved in its subcellular localization. These data, taken together with confocal microscopy results, immunodetection methods, and state-of-the-art protein structural modeling tools, reveal a phosphorylation mechanism that is possibly conserved in plants, in which multiple residues are phosphorylated in a cooperative manner to respond to different stimuli. Several lines of evidence suggest that the serine 278 residue is an essential modulator of RGS1 function and its signaling pathways. First, mutation on the serine residue to glutamic acid significantly decreases the interaction of RGS with G complex proteins and VPS26b (Vacuolar Protein Sorting 26). Second, the RGS internalization process upon flg22 stimulus requires the 278 residue phosphorylation, which is only partially required upon UDP-glucose stimulus. Third, phosphonull S278A mutation decreases the VPS26 proteins recruitment upon flg22 elicitation. Finally, the serine residue 278 is considerably conserved in a flexible protein region, and it regulates the phosphorylation levels of C-terminal tail residues, and one or more threonine residues that are detected for the first time in vivo. Moreover, molecular dynamics simulations evidenced the effect of this residue on domain positioning and flexibility of other regions. Accordingly, this work presents the relevance of post-translational modifications in the signal distinction triggered by G protein signaling, in addition to demonstrating the influence of those modifications on the structural alterations in the RGS proteins, which control their specific stimulus responses. Keywords: RGS. Phosphorylation. Flg22. G-protein. 7TM.