Dual phosphorylation of Ric-8A enhances its ability to mediate G protein α subunit folding and to stimulate guanine nucleotide exchange.
Abstract
Resistance to inhibitors of cholinesterase-8A (Ric-8A) and Ric-8B are essential biosynthetic chaperones for heterotrimeric G protein α subunits. We provide evidence for the direct regulation of Ric-8A cellular activity by dual phosphorylation. Using proteomics, Western blotting, and mutational analyses, we determined that Ric-8A was constitutively phosphorylated at five serines and threonines by the protein kinase CK2. Phosphorylation of Ser435 and Thr440 in rat Ric-8A (corresponding to Ser436 and Thr441 in human Ric-8A) was required for high-affinity binding to Gα subunits, efficient stimulation of Gα subunit guanine nucleotide exchange, and mediation of Gα subunit folding. The CK2 consensus sites that contain Ser435 and Thr440 are conserved in Ric-8 homologs from worms to mammals. We found that the homologous residues in mouse Ric-8B, Ser468 and Ser473, were also phosphorylated. Mutation of the genomic copy of ric-8 in Caenorhabditis elegans to encode alanine in the homologous sites resulted in characteristic ric-8 reduction-of-function phenotypes that are associated with defective Gq and Gs signaling, including reduced locomotion and defective egg laying. The C. elegans ric-8 phosphorylation site mutant phenotypes were parti...Continue Reading
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