Tyrosine phosphorylation switching of a G protein

Abstract

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 posttranslational modification of the Gα subunit in the G-protein complex regulates the RGSdependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr-166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occurs. 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 Gα 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." __________________________________ http://www.jbc.org/cgi

The Nucleotide‐Dependent Interactome of Rice Heterotrimeric G‐Protein α ‐Subunit

“…Phosphorylation plays an important role in regulating the G‐protein activation state . We observed five cytosolic kinases/dikinases as well as two serine/threonine‐protein phosphatase in our interactome (Table S1, Supporting Information).…”

“…AtRGS1 accelerates the intrinsic GTPase activity of the Arabidopsis Gα subunit (AtGPA1) to promote the inactive (OFF) state by stabilizing the transition state, consequently facilitating re‐binding with the βγ dimer to form the heterotrimeric complex and to maintain the G‐protein complex in the inactive state . In addition, AtRGS1 binds tyrosine phosphorylated AtGPA1 in its GDP‐bound state by an unprecedented mechanism dubbed “substrate phosphoswitching” . Ligand‐dependent endocytosis of AtRGS1 protein physically uncouples itself and its GAP activity from the Gα protein, consequently de‐repressing the G cycle to allow self‐activation .…”

Receptor‐Like Kinase Phosphorylation of Arabidopsis Heterotrimeric G‐Protein Gα ‐Subunit AtGPA1

“…Although SAPH‐ire (Structural Analysis of PTM Hot spots) predicted multiple key modified alignment positions in the Gα family, including N‐terminal α‐helix (αN), P‐loop, and αE helix, the presence of those predicted phosphosites needs to be verified experimentally. AtGPA1 is a substrate of the Ser/Thr and Tyr dual‐specificity RLK, BAK1 . Arabidopsis has approximately 400 RLKS .…”

“…Arabidopsis has approximately 400 RLKS . A set of 70 LRR RLKs was screened for AtGPA1 phosphorylation and 18 RLKs were identified as AtGPA1 phosphokinases . Recently, Xue et al .…”

“…Twelve RLKs previously shown to phosphorylate AtGPA1 were chosen for this study ( Table ) to identify AtGPA1 phosphorylation sites via LC–MS/MS (See Supporting Information Methods for details). The cytoplasmic domains of RLKs were used in the kinase assays.…”