Research ArticleG Protein Signaling

Dual phosphorylation of Ric-8A enhances its ability to mediate G protein α subunit folding and to stimulate guanine nucleotide exchange

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Sci. Signal.  29 May 2018:
Vol. 11, Issue 532, eaap8113
DOI: 10.1126/scisignal.aap8113

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Phosphorylating a chaperone

Heterotrimeric G proteins, which consist of various combinations of α, β, and γ subunits, transduce a wide array of signals from G protein–coupled receptors (GPCRs). Proteins of the Ric-8 family are molecular chaperones that are required for the proper folding of Gα subunits and their assembly with βγ dimers to form functional G proteins. Papasergi-Scott et al. found that Ric-8A was itself regulated through the CK2-mediated phosphorylation of two serine and threonine residues that are conserved from worms to humans. Worms engineered to express a mutant Ric-8 protein deficient in these conserved sites exhibited defective locomotion and egg laying, functions that depend on G protein activation. Together, these data suggest that dual phosphorylation of Ric-8A is required for effective Gα subunit folding and activity.

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 partially rescued by chemical stimulation of Gq signaling. These results indicate that dual phosphorylation represents a critical form of conserved Ric-8 regulation and demonstrate that Ric-8 proteins are needed for effective Gα signaling. The position of the CK2-phosphorylated sites within a structural model of Ric-8A reveals that these sites contribute to a key acidic and negatively charged surface that may be important for its interactions with Gα subunits.

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