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 alpha subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr(166) in the Arabidopsis G alpha 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 Tyr(166)-dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr(166) forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr(166) phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound G alpha substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr(166) 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."