Fig. 5: Cell cycle-dependent phosphorylation dynamics.

A Workflow used to investigate phosphorylation dynamics during the cell cycle. CCD phosphorylation events detected in the Time Course and Mitotic Exit datasets were classified as protein-dependent and protein-independent based on their correlation with protein abundance. Furthermore, CCD phosphorylation events were annotated with structural features to define phosphorylation sites located at protein interfaces and phosphorylated residues that overlapped with known motifs. B Boxen plot comparing accessibility and disorder scores for CCD phosphorylation events. C Boxen plot showing the differences in conservation scores between CCD and non-CCD phosphorylation events, where higher −log10 conservation score values indicate more conserved residues. D CCD phosphorylation sites conservation across different species. The numbers on the right represent the phosphorylation events identified in each species, and the percentage of enrichment is reported. E Heatmap showing the oscillation of phosphorylation sites predicted to be CDK, Aurora, PLK, PIKK and other kinases substrates. Phosphorylation changes are coloured according to their average abundance (log₂ mean normalised values). Phosphorylation consensus motifs were obtained from Alexander et al.⁷¹. F Heatmaps showing the log₂ mean normalised protein (blue) and phosphorylation (red) abundance changes during the cell cycle detected by MS. CDKN1B and NCL are shown as representative examples of protein-dependent and protein-independent phosphorylation events. G Western blot validation of protein abundance changes shown in (F). H Structure of MKI67 bound to NIFK (PDB ID: 2AFF). MKI67 is shown in yellow and NIFK in pink. This interaction is mediated by the phosphorylation of NIFK on Thr234, shown in red sticks. NIFK protein (blue) and pThr234 (red) abundance changes during cell cycle progression detected by MS are shown in the heatmap (bottom). Protein and phosphorylation changes are coloured according to their abundance (log₂ mean normalised values). I Structure of KPNA2 bound to TP53BP1 (PDB ID: 6IU7). KPNA2 is shown in light blue and TP53BP1 in purple. This interaction is mediated by the phosphorylation of TP53BP1 on Ser1678, shown in red sticks. TP53BP1 protein (blue) and pSer1678 (red) changes during cell cycle progression detected by MS are shown in the heatmap (bottom). Protein (blue) and phosphorylation changes (red) are coloured according to their abundance (log₂ mean normalised values). J Examples of proteins containing motif consensus of phosphorylation sites adjacent to an NLS found in our dataset. Western blots shown in (G) are representative of at least three independent experiments with similar results. Source data are provided as a Source Data file.