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  • br Conflicts of interest br Acknowledgments This research wa


    Conflicts of interest
    Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (NRF-2015R1A2A2A01004593).
    Introduction Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that participate in the biosynthetic protein folding process of heterotrimeric G protein α subunits [1,2]. Ric-8 proteins also act as guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange [[3], [4], [5], [6], [7], [8], [9]]. Ric-8A substrates include Gαi-, Gαq-, and Gα13-class subunits, whereas Gαs/Gαolf are substrates for Ric-8B [3,4,10,11]. We proposed that Ric-8 binds to nascent Gα subunits and renders a conformation that is capable of productively binding GTP for the first time [12]. Gα subunits that are produced in systems lacking Ric-8 protein(s) may be folded improperly and adopt a bottleneck conformation(s) that lacks guanine nucleotide. Mis-folded proteins are often degraded rapidly in cells, which is consistent with observations that newly produced Gα subunits are turned over nearly ten-times faster in Ric-8A-null sr 75 mg versus wild type cells [1]. This may account for the dramatic reductions (by ∼95%) of functional Gαi/q/13 or Gαs subunit abundances in Ric-8A or Ric-8B knock-out cells, respectively [1,2,[13], [14], [15]]. Purified Ric-8 proteins are also effective catalysts for the preparation of purified Gα proteins that are bound stoichiometrically to GTPγS for use in G protein effector enzyme studies [16]. Ric-8A is post-translationally modified by phosphorylation at various residues, which may affect its subcellular localization and ubiquitination [[17], [18], [19], [20]]. We recently demonstrated that phosphorylation of (Rat) Ric-8A residues S435 and T440 (Human Ric-8A S436 and T441) dramatically potentiated Gα subunit GEF and chaperoning activities [21]. Three additional C-terminal phosphosites, S522, S523, and S527 were identified and postulated to work in concert with S435-PO4 and T440-PO4 to affect Ric-8A activities and to promote high affinity Gα subunit binding. All five of these Ric-8A phosphosites are protein kinase CK2 consensus sites, four of which are functionally conserved in all Ric-8 homologs, including mammalian Ric-8B. The protein kinase CK2 phosphosites of Ric-8A and Ric-8B appear to be efficiently phosphorylated in normal cells and tissues, and when produced as recombinant proteins in Sf9 insect cells. These observations are consistent with the consideration that protein kinase CK2 is a constitutively active kinase [22]. Attempts to parse out the individual contributions of the five Ric-8A phosphosites by creating single and combinatorial alanine substitution mutant Ric-8A proteins produced in insect cells was met with mixed results. Some combinations of Ric-8A alanine substitution mutants were proscribed because they resulted in poor yields or completely dysfunctional recombinant proteins. This was not the case when the same mutant Ric-8A proteins were produced in E. coli, which lacks protein kinase CK2 and produces unmodified, recombinant Ric-8A. We interpret this to mean that there may be critical interactions among the different protein kinase CK2 phosphorylated residues that are important for Ric-8 structure and function. Additionally, the CK2 consensus sites of insect cell-produced Ric-8A or Ric-8B are only partially phosphorylated. This confounds the interpretation of Ric-8-regulated G protein guanine nucleotide exchange and protein folding measurements. Here we present a detailed method for the preparation of E. coli-produced Ric-8A proteins that are stoichiometrically-phosphorylated using recombinant protein kinase CK2 holoenzyme. The method entails a re-purification step to isolate phosphorylated Ric-8 proteins away from kinase. The phosphorylated Ric-8 proteins exhibited dramatically enhanced ability to stimulate Gα subunit guanine nucleotide exchange. This method will find utility for the production of homogenous preparations of phosphorylated Ric-8 proteins for structural studies, G protein enzymatic analyses, and as catalytic tools for the production of purified Gα-GTPγS.