Journal: Science signaling

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

doi: 10.1126/scisignal.aap8113

Figure Lengend Snippet: (A) GPS 3.0 was used to evaluate sites within Ric-8A with potential for CK2-mediated phosphorylation (47). The prediction was run at the high-stringency setting using a maximal false-positive rate threshold cutoff of 2%. Higher relative scores represent greater potential for phosphorylation. (B) Phosphosite identification in rat Ric-8A purified from insect cells and from E. coli-produced Ric-8A treated with CK2 was made by MS analysis of peptides from tryptic and chymotryptic digests. Red boxed residues were phosphorylated sites in insect cell–produced Ric-8A, yellow boxed residues were phosphorylated sites in E. coli–produced Ric-8A treated with CK2, and blue boxed residues were phosphorylated sites obtained for both proteins. All phosphosites were identified by Mascot software and confirmed by analysis of CID spectra. Peptide coverage of the three C-terminal phosphosites (Ser522, Ser523, and Ser527) was not attained for insect cell–produced Ric-8A. The 10-residue acidic sequence that contains the two consecutive CK2 sites of Ser435 and Thr440 is underlined. (C) The 10-residue acidic sequence is highly conserved across vertebrate Ric-8 proteins. The phosphosites corresponding to rat Ric-8A Ser435 and Thr440 are invariantly serine or threonine residues in all homologs examined. Dictyostelium Ric-8 is a shortened protein that lacks the acidic sequence stretch altogether. (D) Insect cell–produced rat wild-type (WT), S435A, and T440A Ric-8A proteins were treated with or without CK2 and then analyzed by Western blotting with an antibody specific for the CK2 consensus site (pS/pT)-D-X-E, which recognizes the phosphorylated Thr440 site (p-Thr440), the 6383 Ric-8A antiserum that recognizes the phosphorylated Ser435 site (p-Ser435), and the 1184 antiserum that recognizes Ric-8A. (E) Insect cell–produced mouse Ric-8B was treated with and without alkaline phosphatase and analyzed by Western blotting with the Ric-8A phosphosite-specific antibodies that also detect mouse Ric-8B p-Ser468 and p-Ser472, respectively. Ric-8B antiserum 2413 was used to detect the Ric-8B alkaline phosphatase–dependent gel shift after protein resolution by Phos-tag PAGE. (F) Purified Ric-8A from insect cells was treated with or without alkaline phosphatase, whereas E. coli–produced Ric-8A was treated with or without CK2. The proteins were analyzed by Western blotting with the p-Thr440, p-Ser435, and 1184 antibodies. (G) Insect–produced WT Ric-8A and the indicated Ric-8A alanine substitution mutant proteins produced in E. coli were treated with or without CK2 and analyzed by Western blotting with the p-Thr440 and 1184 Ric-8A antibodies. The proteins were also resolved by Phos-tag PAGE and analyzed by Western blotting with the 1184 Ric-8A antibody, as indicated. (H) WT, T440A, and S435A/T440A Ric-8A-ΔCT purified from E. coli were treated with or without CK2, resolved by SDS-PAGE and Phos-tag PAGE, and analyzed by Western blotting with the p-Thr440, p-Ser435, and 1184 Ric-8A antibodies, as indicated. Data in (D) to (H) are representative of more than three independent experiments.

Article Snippet: Monoclonal rabbit phospho-CK2 motif (pS/pT)-D-X-E antibody (Cell Signaling Technology) was found to specifically recognize phosphorylation at site Thr 440 of recombinant rat Ric-8A ( pT -D-T-E) but failed to recognize human Ric-8A ( pT -D-T-D).

Techniques: Purification, Produced, Software, Sequencing, Western Blot, Electrophoretic Mobility Shift Assay, Mutagenesis, SDS Page