Fig 1.
A) Flowchart of clustering procedure. Substrates for a kinase (for example Akt) are extracted from the KSR knowledgebase and can either be exclusive (blue) or not (pink). In the first step tight clustering is performed on exclusive substrates and core substrates (purple) identified. In the second step tight clustering is performed using all substrates and the characteristic temporal activity of a kinase is identified. B) Heatmap of scaled log fold change of the characteristic temporal activity of 9 kinases over time. High log fold change is represented in red, low log fold change is shown in blue C) Table showing the time points included in the accuracy analysis and the accuracy of using a database or KSR-LIVE for Akt and mTOR.
Fig 2.
Scaled log fold change over time of kinase (shown in blue) and the corresponding CTA (shown in red, mean ± SD) for multiple kinases.
Fig 3.
Analysis of Emdal et al. data using KSR-LIVE.
A) Log fold change of MAPK1, MTOR and CDK1 CTAs (shown in red, mean ± SD). B) Log fold change of kinase phosphorylation (blue) and the corresponding CTA (shown in red, mean ± SD) for multiple kinases.
Fig 4.
Validation of IRS1 S265 as an AKT substrate.
A) Comparison of AKT and RPS6KB1 consensus motif and IRS1 S265 site. B) CTA of AKT (green) and RPS6KB1 (purple) and time profile of IRS1 S265 (blue). (CTA is depicted with mean ± SD) C) Scatter plot of RPS6KB1 prediction scores (y-axis) against RPS6KB1 prediction score—AKT prediction score (x-axis). AKT training substrates are shown in red and RPS6KB1 training substrates are shown in blue. IRS1 S265 is shown in green. D) Insulin signaling via AKT and RPS6KB1. See main text for details. E) 3T3-L1 adipocytes were stimulated with insulin alone or in the presence of inhibitors of AKT (MK, GDC) or mTORC1 (Rapa), after which AKT and RPS6KB1 signaling were assessed by Western blotting. Blots shown are representative of 3 separate experiments. F) Quantification of IRS1 S265 phosphorylation from (E), depicted as mean ± SEM.