The MAPK pathway bisects heterotrimeric G protein signaling through control of G protein subunit biosynthetic protein folding
Authors: Papasergi‐Scott Makaía M , Patel Bharti R. , Gabbay Meital , Tall Gregory 
Published: November 2023

G‐protein coupled receptor (GPCR) signaling is broadly utilized across physiologic systems and the aberrant signaling of G‐protein pathways contributes to pathologies including cardiovascular, neurodegenerative, and endocrine diseases. The correct folding of heterotrimeric G protein α subunits is requisite for GPCR signal transduction. Our lab identified Ric‐8A and Ric‐8B as necessary biosynthetic folding chaperones for G protein α i/q/13 and Gα s/olf subunit classes, respectively. In Ric‐8 gene deletion or Ric‐8 protein depletion models Gα subunits are folded improperly, fail to become membrane associated, and are degraded more rapidly. Although Ric‐8 function as an essential G protein chaperone is reasonably well understood, the mechanisms regulating Ric‐8 activity have not been addressed. We found that mammalian Ric‐8A is constitutively phosphorylated at multiple sites. Recombinant Ric‐8A purified from E.coli or enzymatically dephosphorylated Ric‐8A purified from insect cells had markedly less activity than phosphorylated Ric‐8A in stimulation of in vitro Gα guanine nucleotide exchange. Using a combination of mass spectrometry‐based phospho‐site identification, analysis of phospho‐deficient and phospho‐mimetic Ric‐8A mutants, and Ric‐8A−/− genetic complementation assays we identified two Ric‐8A phosphorylation sites that are required for efficient Gα subunit folding capacity. The casein‐kinase 2 (CK2) consensus Ric‐8A phospho‐sites, S435 and T440, reside in an acidic region and are invariantly conserved. When E.coli‐produced Ric‐8A was phosphorylated with CK2, full Ric‐8A activity was restored. To further investigate regulation of Ric‐8A activity in vivo we utilized immortalized mouse Ric‐8Aflox/flox melanocytes that had been transformed and made to stably express constitutively active HRas‐G12V. We made the profound observation that Ric‐8A in HRAS‐G12V‐transformed melanocytes was dephosphorylated and that HRAS‐G12V expressing cells had marked reductions in steady state Gα protein levels, with no change in Gα transcript levels. The dephosphorylation event downstream of Ras signaling was recapitulated in NIH3T3 cells stably expressing HRAS‐G12V and in KRAS‐G13D positive human colorectal cancer cell lines. Moreover, stimulation of HEK293 or Mouse Embryonic Fibroblasts with epidermal growth factor led to rapid Ric‐8A dephosphorylation. These data support our hypothesis that Ric‐8A activity is constitutively “on” due to constitutive CK2‐phosphorylation, but can be down regulated via dephosphorylation. Specifically, MAPK pathway oncogenes (e.g. RAS‐G to V/D) or exposure to mitogenic growth factors may activate a downstream phosphatase that dephosphorylates Ric‐8A. This may be a means by which the MAPK pathway acts to reduce total cellular Gαi/q/13 levels to enhance cell proliferation given that multiple Ric‐8A knock‐out cell models have enhanced growth rates.Support or Funding InformationNIH RO1GM088242 to G.G.T., PhRMA Foundation Pre‐doctoral Fellowship to M.M.P.S., NIH T32‐GM068411 to M.M.P.S., HHMI “Med‐into‐Grad” Fellowship to M.M.P.S.