Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

Abstract

Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.

Figure 1

The phosphoadaptor subunit Cks1 provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and ³²P-ATP. The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site (mut; see Supplementary Methods). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of ³²P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.

Figure 2

Phosphorylated priming sites provide docking interactions for efficient phosphorylation of suboptimal sites in phosphodegrons. (a) Schematic view of phosphorylation sites, docking motifs (Clb5- and Cln2-specific), phosphodegrons (ovals ) in Sic1. (b) Phosphorylation specificity of Clb5- and Cln2-Cdk1 towards different Cdk sites was studied using Sic1ΔC constructs containing a single fixed Cdk site. For Clb5-Cdk1, the dependence of the site specificity profile on RXL docking sites was assessed using Sic1ΔC constructs containg a single Cdk site and a single fixed RXL motif. (c) The impact of different priming phosphorylation sites on cooperative phosphorylation of the degron cluster S69/S76/S80. Phospho-site mutants of Sic1ΔC carrying the intact S69/S76/S80 cluster and the indicated sites left unmutated were used in a kinase assay with Cln2-Cdk1 and Clb5-Cdk1 using Phos-Tag SDS-PAGE. (d) Full-length Sic1 versions containing the combination of sites described in (c) were overexpressed under the galactose promoter to assay the ability of cells to degrade Sic1. (e) Comparison of the in vitro phosphorylation profiles of Sic1ΔC versions containing only the phosphorylation sites T33/T45 or T33/T45 with mutation T48A. (f) The nonconsensus Cdk1 site T48 is important for viability of cells overexpressing Sic1. The same assay as (d) was used. In panels d and f, the labels indicate unmutated amino acids, and all other consensus Cdk sites are mutated; mutations in the nonconsensus Cdk sites are highlighted in red. (g) The phosphorylation and degradation dynamics of Sic1 were followed after the release of cells from α-factor in a system constitutively expressing mutated versions of noninhibitory Sic1ΔC-3HA. The asterisk indicates a G1-specific phosphorylation by an unknown kinase.

Figure 3

Differential roles of Cln2 and Clb5 in Sic1 multiphosphorylation and degradation. (a) Pairwise mapping of the docking connections underlying Sic1 multiphosphorylation, using purified Sic1ΔC mutants containing just two of the Cdk phosphorylation sites per mutant. Representative examples of autoradiographs of phosphorylation assays, showing different docking specificities between Cln2-Cdk1 and Clb5-Cdk1. (b) The specificity profiles for different pair-wise docking connections. The error bars indicate standard errors of the means of at least two independent experiments. (See Supplementary Information and Supplementary Table 1). (c) Schematic view of fast and slow docking-dependent phosphorylation steps for Cln2- and Clb5-Cdk1. (d) Sic1 mutants with improved Cdk recognition determinants in suboptimal phosphodegrons rescue the inviability of cells overexpressing Sic1-1234rxl. The Cln2-dependent docking site becomes essential under these conditions. (e) Cells carrying SIC1wt-TAP or versions with docking site mutations at the endogeneous SIC1 locus were released from an α-factor arrest and the degradation pattern of Sic1-TAP protein was followed by western blotting, using standard SDS-PAGE. (f) A wild-type strain (SIC1wt-TAP) and a strain also expressing the nondegradable inhibitory domain of Sic1 (SIC1ΔN) under the GAL1 promoter were arrested in α-factor, followed by the addition of galactose. After 45 minutes, the cells were washed into galactose media lacking α-factor and Sic1-TAP levels were followed by western blotting (see also Supplementary Fig. 7). (g) Sic1 mutants with improved Cln-Cdk recognition determinants in suboptimal phosphodegrons trigger rapid degradation of Sic1 in the presence of SIC1- ΔN. Experiment was performed as in panel (f).