Figure 5.

Analysis of Changes in Cdk1 Substrate Specificity during the Cell Cycle and of the Physiological Importance In Vivo of Different Substrate Docking Sites

(A and B) Cells constitutively expressing the indicated Sic1ΔC-3HA construct were released from α factor arrest, and at the indicated time points Phos-Tag SDS-PAGE and western blotting were used to assess phosphorylation state (see Figure S1). The constructs contained either the Clb2-specific P-X-T-P-X-K motif (A) or the Cln2-specific P-X-T-P-K-X motif (B) at positions T5, T33, T45, and T76. Other Cdk1 sites were mutated to alanines, and T2 was left unchanged. As controls, we also tested the same constructs with mutations of the LP and the RXL docking sites. Sic1 bands on the western blot were scanned using the GelDoc (GE), and each phosphorylated species was quantified. The relative steady-state phosphorylation at each time point was calculated as a ratio of pS/S_T, where S_T is the total signal of Sic1ΔC-3HA and pS is the total signal of phosphorylated bands multiplied by the number of phosphates each band contains. See also Figure S1. Analyses were performed in duplicates, and error bars indicate standard error of the mean.

(C and D) The importance of Clb5-specific RXL motifs (C) and the Cln2-specific LP motif (D) in the degradation of Sic1 was tested by overexpressing different Sic1 constructs under the GAL1 promoter. To assess the effect of the LP docking site, the strain used in (D) was sensitized by reducing Cln-Cdk1 activity in the cell by deleting Cln2.

(E) Western blotting analysis of Sic1 levels after the release of cells from a G1 arrest. Strains carrying CEN vectors with GAL-SIC1 or GAL-SIC1-1234rxl -vllpp were arrested in G1 with α factor for 2.5 hr. SIC1 expression was induced in the arrested cells by addition of galactose for 45 min. Both galactose and α factor were removed and the cells were released into glucose-containing medium. Sic1-3HA levels were analyzed at different time points using western blotting. In the lower panel the combined quantified Sic1 profiles from two independent experiments are presented. The error bars indicate standard error of the mean.

(F) To demonstrate how altered Cdk1 specificity toward Sic1 affects the timing of DNA replication, we performed flow cytometry of DNA content for cells taken at different time points of the α factor release experiment presented in (E).

(G) To explore further how altered Cdk1 specificity toward Sic1 affects the control of DNA replication, we performed flow cytometry of DNA content in asynchronous cells expressing the Sic1 mutant lacking cyclin docking motifs. Strains carrying CEN vectors with GAL-SIC1 or GAL-SIC1-1234rxl-vllpp were grown to log phase, and the expression of Sic1 was initiated by addition of galactose. Cells expressing Sic1 with mutations in the docking sites caused DNA rereplication (DNA > 2N). Cells expressing wild-type Sic1 did not exhibit any signs of rereplication.