A global analysis of Cdk1 substrate phosphorylation sites in budding yeast reveals insights into the evolution of cell-cycle control. Researchers identified 547 phosphorylation sites on 308 Cdk1 substrates using chemical inhibition and mass spectrometry. They found that most phosphorylation sites are not conserved across species, but instead shift in rapidly evolving disordered regions. This suggests that phosphorylation often regulates protein function through simple, non-specific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in these regions may facilitate the evolution of kinase signaling circuits. Cdk1 substrates were analyzed in three cell populations: asynchronous, mitotic-arrested, and late-mitotic. The study identified 10,656 unique phosphorylation sites, with 8,710 assigned precise positions with high confidence. Phosphorylation of Cdk1 consensus sites was observed on 67% of previously identified substrates. Gene ontology analysis showed enrichment for cell cycle-related functions, as well as other processes like translation and nuclear transport. Phosphorylation can drive conformational changes in proteins or disrupt interactions with other proteins. The study found that most Cdk1 phosphorylation sites are in loops and disordered regions, consistent with previous findings. Cdk1-dependent phosphorylation sites tend to cluster in the primary amino acid sequence, suggesting multiple phosphorylations modulate the same protein surface. The evolution of Cdk1 phosphorylation sites was analyzed across 32 fungal species. While some sites are conserved, others show different evolutionary patterns. Hierarchical clustering showed few sites with strong conservation of precise position, while many show conservation of consensus sites. This suggests context-independent regulation. The study also found a significant overlap between Cdk1 substrates and binding partners of 14-3-3 proteins, indicating that multisite phosphorylation can create generic interactions with phosphate-binding domains. The evolution of Cdk1 signaling shares features with transcriptional regulation, with both maintaining biochemical specificity over long evolutionary timescales. The flexibility in phosphorylation site positioning allows for combinatorial control by multiple kinases, as seen in Ime2, a Cdk1 relative that phosphorylates Cdk1 substrates at distinct sites. Overall, the study highlights the importance of phosphorylation in cell-cycle control and its role in the evolution of kinase signaling circuits.A global analysis of Cdk1 substrate phosphorylation sites in budding yeast reveals insights into the evolution of cell-cycle control. Researchers identified 547 phosphorylation sites on 308 Cdk1 substrates using chemical inhibition and mass spectrometry. They found that most phosphorylation sites are not conserved across species, but instead shift in rapidly evolving disordered regions. This suggests that phosphorylation often regulates protein function through simple, non-specific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in these regions may facilitate the evolution of kinase signaling circuits. Cdk1 substrates were analyzed in three cell populations: asynchronous, mitotic-arrested, and late-mitotic. The study identified 10,656 unique phosphorylation sites, with 8,710 assigned precise positions with high confidence. Phosphorylation of Cdk1 consensus sites was observed on 67% of previously identified substrates. Gene ontology analysis showed enrichment for cell cycle-related functions, as well as other processes like translation and nuclear transport. Phosphorylation can drive conformational changes in proteins or disrupt interactions with other proteins. The study found that most Cdk1 phosphorylation sites are in loops and disordered regions, consistent with previous findings. Cdk1-dependent phosphorylation sites tend to cluster in the primary amino acid sequence, suggesting multiple phosphorylations modulate the same protein surface. The evolution of Cdk1 phosphorylation sites was analyzed across 32 fungal species. While some sites are conserved, others show different evolutionary patterns. Hierarchical clustering showed few sites with strong conservation of precise position, while many show conservation of consensus sites. This suggests context-independent regulation. The study also found a significant overlap between Cdk1 substrates and binding partners of 14-3-3 proteins, indicating that multisite phosphorylation can create generic interactions with phosphate-binding domains. The evolution of Cdk1 signaling shares features with transcriptional regulation, with both maintaining biochemical specificity over long evolutionary timescales. The flexibility in phosphorylation site positioning allows for combinatorial control by multiple kinases, as seen in Ime2, a Cdk1 relative that phosphorylates Cdk1 substrates at distinct sites. Overall, the study highlights the importance of phosphorylation in cell-cycle control and its role in the evolution of kinase signaling circuits.