Phosphosite Scanning reveals a complex phosphorylation code underlying CDK-dependent activation of Hcm1

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.