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Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates processive phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains.

Progression through the cell cycle is controlled by a network of transcription factors that coordinate gene expression with cell-cycle events. One transcriptional activator in this network in budding yeast is the forkhead protein Hcm1, which controls the expression of genes that are transcribed during S-phase. Hcm1 activity is coordinated with the cell cycle via its regulation by cyclin-dependent kinase (Cdk1), which both activates Hcm1 and targets it for degradation, through phosphorylation of distinct sites. The mechanisms controlling the differential phosphorylation timing of the activating and destabilizing phosphosites are not clear. However, a recent study shows that the phosphatase calcineurin specifically removes activating phosphates from Hcm1 when cells are exposed to environmental stress, thus extinguishing its activity and slowing proliferation under unfavorable growth conditions. This regulatory mechanism, whereby a phosphatase actively alters the distribution of phosphosites on a cell cycle-regulatory transcription factor to elicit a change in cellular proliferation, adds an additional layer of complexity to the regulatory network controlling the cell cycle. Furthermore, this regulatory paradigm is likely to be a conserved mode of phosphoregulation that controls the cell cycle in diverse systems.