Fig. 3: The pulsed activation of Akt1 reveals sequential signaling events and functional clusters with variable longitudinal endurance.

a The rationale of the clustering analysis performed, which is based on the results of periodic and pulsed light activations. b The fuzzy c-means clustering (FCM) analysis on these P-sites and four groups (fC1-fC4) clustered. The yellow-green-purple-red color palette corresponds to the low to high membership value for each P-site, a similarity score of vectors to each cluster reported by FCM analysis. c The sequence features and functions of P-sites among fC1-fC4 extracted by motifeR. The number of P-sites for each motif sequence and the fold enrichment compared to the background of the entire proteome background were shown. d The numbers of P-sites of fC1-fC4 were shown in histograms, with the secondary axis (and the orange line) showing different percentages of Akt1 strict motifs in fC1-fC4. e P-site-level annotation and enrichment analysis against kinase~P-site relationships and regulatory processes in fC1-fC4. P values are reported by Fisher’s exact test (see “Methods”). f The signaling network was reconstructed using PHONEMeS, and the Akt1 subnetworks were extracted by selecting 3-step downstream nodes of Akt1. The node fill color corresponds to the protein activity inferred by PHONEMeS, with yellow and gray marking up- and downregulated activity, respectively. The border color corresponds to the cluster (fC1-fC4) in which a kinase was predominantly overrepresented based on the lowest P-value. The node size corresponds to the number of outgoing edges. Source data are provided as a Source data file.