Figure 5.

NetworKIN analysis of phosphorylated substrate peptides changing with treatment predicts kinases involved with GPCR signaling and cytoskeletal regulation. A) Kinase activation analysis performed on regulated (BH adjusted p-value of <0.1, moderated t-statistic) phosphopeptides separated into increasing and decreasing sets and matched to kinases in NetworKIN. The resultant rank-ordered predictions for each kinase were filtered to include only kinases observed in MC3T3-E1 cells and then evaluated for changes in activation status by a one-sided Kolmogorov-Smirnov test with a 0.05 FDR filter. B) Linear phosphorylation motif analysis of the MC3T3-E1 phosphoproteome. To gain more site-specific information, significantly overrepresented phosphorylation motifs were determined by Motif-X and matched to kinases with NetworKIN. NetworKIN output was filtered by score minimum of 0.22, maximum score difference of 4, and maximum of ten predictions per site. For the purposes of this analysis, lysine and arginine were considered degenerate. Predictions were ranked by frequency of presence in total number of sites bearing the indicated motif analyzed by NetworKIN. Due to NetworKIN’s human protein-limited analysis abilities, the peptides were converted from mouse to human site numbers in PhosphositePlus. This conversion process provided varying levels of efficiency (ranging from 50–100%) per motif set and introduced moderate bias in the analysis. For example, ‘SP down’ motif sites were converted at 50% efficiency (n= 67/134), yet RxxS down motif sites were converted at 100% efficiency (42/42). This biases the output as both motifs were also present in the increasing sets.