Multisite Phosphorylation Underlies Ultrasensitive Response of CDK Inhibitor Sic1 In Yeast Cell Cycle Regulation

Sic1 is a cyclin dependent kinase (CDK) inhibitor in the budding yeast Saccharomyces cerevisiae. This protein inhibits the Clb5,6-Cdc28 kinase required for DNA replication in the S phase and is believed to contribute to precise timing of the G1 to S transition by undergoing switch-like multisite phosphorylation-mediated degradation. During the late G1 phase, as the activity of Cln2-Cdc28 kinase complex increases, Sic1 becomes phosphorylated at up to nine sites. The phosphorylated Sic1 is recruited by the Cdc4 protein for proteolysis which eventually leads to Clb5,6-Cdc28 kinase release and DNA replication. Computational studies suggest that multisite phosphorylation could lead to highly non-linear degradation of Sic1 and thus a switch-like entry into the S phase. Here, we demonstrate experimentally that the in vitro binding of phosphorylated Sic1 to Cdc4 in response to the increasing activity of Cln2-Cdc28 kinase is indeed ultrasensitive (Hill coefficient n_H ~ 3.2) and the ultrasensitivity decreases as the number of phosphorylable sites decreases to five (n_H ~1.0). In our steady-state binding assays, mutant Sic1 proteins with less than five phosphorylable sites do not bind to Cdc4 and the Cdc4-bound fraction of Sic1 increases as the number of phosphorylable sites increases. In parallel, mass spectrometry analysis showes that the wild-type Sic1 is phosphorylated mostly on less than five sites in the initial lag phase of the binding curve. Interestingly, the distribution shifts dramatically toward six- and higher orders of phosphorylated states as the concentration of Cln2-Cdc28 increases.  This study highlights the potential role of multisite protein phosphorylation in converting a graded input (Cln2-Cdc28 activity) to an ultrasensitive output (binding to Cdc4) and thereby generating a switch-like cellular behavior such as the G1/S transition.