Tyrosine phosphorylation switching of a G protein

Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that post-translational modification of the G subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr¹⁶⁶ in the Arabidopsis G subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr¹⁶⁶-dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr¹⁶⁶ forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr¹⁶⁶ phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound G substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr¹⁶⁶ in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism “substrate phosphoswitching.”.