Figure 1

AKT SILAC kinase screen. (a) In-vitro kinase reaction steps between either no kinase (control, CTL), recombinant active kinase (active kinase, AK) or heat-treated kinase (denatured kinase, DK) with recombinant protein substrates. Up to four reaction conditions were tested (30 min and 60 min incubations, each in the presence of 0.5 mM ATP or 1 mM ATP). The resulting reaction mixtures were separated by SDS-PAGE and analyzed by western blot or further processed with in-gel digestion followed by liquid chromatography tandem mass spectrometry (LC–MS/MS). (b) The reaction between AKT1/YB1 was validated by immunoblot of known phospho-sites on active AKT1 (T-308), and the AKT1-mediated YB1 phospho-site (S-102). Pan-AKT was used to show the absence, presence and denaturing effect on active AKT-1, while Total YB1 was used to show the presence of YB1 substrate in each reaction. (c) Phospho-peptides identified by LC–MS/MS were analyzed by label-free quantitation. The abundance of phospho-peptides resulting from the AKT1/YB1 reaction (0.5 mM ATP, 60 min) are represented as a heatmap (mean of two independent reactions). Square brackets indicate peptide locations within YB1 on which each phosphorylation was found. Hashtags denote a co-modification within that peptide, e.g. carbamidomethylation, deamidation, and/or oxidation. (d) Graphical summary explaining the SILAC based whole cell lysate (in vitro) kinase assay. (e) Volcano plot of all identified phospho-peptides identified from the AKT2 SILAC kinase screen. A minimal Log₂ Fold change cut-off < 0.5 (red shading) was taken as potential increased phosphorylation events. (f) STRING cluster analysis⁴⁹ of proteins corresponding to increased phospho-peptides identified in E. (g) KEGG pathway analysis⁵⁰ of proteins identified in F. (h) Phospho-peptides with a Log₂ Fold change > 1.0 were aligned using WebLogo v3.7.4⁵². Residue colors indicate chemical properties of amino acids.