SymRK-dependent phosphorylation of Gα protein and its role in signaling during soybean (Glycine max) nodulation.

Heterotrimeric G proteins, comprised of Gα, Gβ and Gγ subunits, influence signaling in most eukaryotes. In metazoans, G proteins are activated by G protein-coupled receptor (GPCR)-mediated GDP to GTP exchange on Gα; however, the role(s) of GPCRs in regulating plant G-protein signaling remains equivocal. Mounting evidence suggests the involvement of receptor-like kinases (RLKs) in regulating plant G-protein signaling, but their mechanistic details remain scarce. We have previously shown that during Glycine max (soybean) nodulation, the nod factor receptor 1 (NFR1) interacts with G-protein components and indirectly affects signaling. We explored the direct regulation of G-protein signaling by RLKs using protein-protein interactions, receptor-mediated in vitro phosphorylations and the effects of such phosphorylations on soybean nodule formation. Results presented in this study demonstrate a direct, phosphorylation-based regulation of Gα by symbiosis receptor kinase (SymRK). SymRKs interact with and phosphorylate Gα at multiple residues in vitro, including two in its active site, which abolishes GTP binding. Additionally, phospho-mimetic Gα fails to interact with Gβγ, potentially allowing for constitutive signaling by the freed Gβγ. These results uncover an unusual mechanism of G-protein cycle regulation in plants where the receptor-mediated phosphorylation of Gα not only affects its activity but also influences the availability of its signaling partners, thereby exerting a two-pronged check on signaling.