Regulation of Ric‐8A activity by phosphorylation (843.4)

Ric‐8A is required for the biosynthetic folding of Gαi/q/13. Ric‐8A is also a guanine nucleotide exchange factor (GEF) that stimulates in vitro Gα subunit GTP binding. Although Ric‐8A is crucial for Gα folding, and therefore, proper G protein heterotrimer function, mechanisms by which Ric‐8A itself is regulated have yet to be elucidated. We experimentally identified multiple stoichiometrically phosphorylated sites of recombinant Ric‐8A produced in S f 9 cells and demonstrate that endogenous mammalian Ric‐8A is a phosphorylated protein. Recombinant Ric‐8A was enzymatically dephosphorylated, and the potency of Ric‐8A stimulation of Gα nucleotide exchange was measured. Dephosphorylated Ric‐8A was substantially less potent than phosphorylated Ric‐8A in stimulation of Gαq and Gα13 nucleotide exchange, whereas Gαi activity was largely unaffected by Ric‐8A phosphorylation status. These results were corroborated by comparing the potency of phosphorylated Ric‐8A produced in Sf 9 cells to non‐phosphorylated Ric‐8A produced in E. coli . To determine the functional significance of Ric‐8A phosphorylation, identified phospho‐sites and additional sites predicted to be phosphorylated using the Scansite engine were mutated alone or in combination to alanine or phospho‐mimetic aspartic acids. Mutant Ric‐8A proteins were purified and will be tested using GEF and Gα subunit folding assays in cell‐free lysates. The significance of Ric‐8A phosphorylation will be further evaluated in cells. Ric‐8A ‐/‐ mouse embryonic stem cells have greatly reduced Gα levels that are rescued by stable Ric‐8A expression. Using this complementation assay we identified Ric‐8A phospho‐mutants that do not rescue the Ric‐8A ‐/‐ Gα abundance defects. Grant Funding Source : NIH RGM0882428