Phosphorylation Analysis of C0-C2 Proteins

For the determination of phosphorylated serines, preliminary experiments tested phosphorylation at a wide range of PKA concentrations (0.02, 0.1, 0.5, 2.5, 13, 65, and 250 ng/μg C0–C2). Phosphorylation was monitored by in-gel staining of proteins with Pro-Q Diamond (Thermo Fisher) and staining total protein with SYPRO-Ruby (Thermo Fisher), according to the supplier's instructions. Maximal phosphorylation plateaued at 2.5 ng PKA/μg C0–C2. To ensure that all potential serines were phosphorylated, we used the sample treated with 65 ng/μg C0–C2. We excised the C0–C2 band as we described for LC-MS/MS (21 (link)) and utilized a probability-based approach for high-throughput protein phosphorylation analysis and site localization (28 (link)). The supporting information contains additional details of LC-MS/MS data processing and annotated spectra for all identified phosphopeptides (Table S1 and Fig. S2, A–E).
In separate preparations, C0–C2 was treated with GSK3β (G4296, Sigma) at 37 °C, for 2 h, in 10 mm imidazole, 145 mm KCl, 1 mm MgCl₂, 2 mm EGTA, 4 mm ATP, pH 7.0. Pro-Q Diamond staining of treated C0–C2 detected no phosphorylation at any concentration up to 100×. 1× treatment is the amount required to completely phosphorylate a GSK3β substrate, based on the specific activity of 175 pmol (of a standard substrate)/min/μg GSK3β. This was repeated with separate batches of GSK3β and C0–C2. As not all phosphorylation sites are equally detected by Pro-Q Diamond, we excised the C0–C2 band for LC-MS/MS analysis.