Cascades of Multisite Phosphorylation Control Sic1 Destruction at the Onset of S Phase

Institute of Technology, University of Tartu, Tartu 50411, Estonia.
Nature (Impact Factor: 41.46). 12/2011; 480(7375):128-31. DOI: 10.1038/nature10560
Source: PubMed


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.

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    • "After the G1-S transition is complete and budding and replication have commenced, SBF and MBF are inactivated by Clb1,2-CDK (Figure 1). These assumptions of the model are consistent with experimental observations showing that Sic1 is phosphorylated by Cln-CDK and Clb-CDK, resulting in Sic1 ubiquitination by SCF (Skp, Cullin, F-box) and degradation by the 26S proteasome (Koivomagi et al., 2011). Moreover, CLN2 transcription is known to be turned off by Clb2 (Amon et al., 1993; Koch et al., 1996). "
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    • "The obligatory Cks1 subunit in both CDK complexes contains a pocket that preferentially binds phospho-TP sites (CDKs are generally highly selective for -SP-and -TP-sites); hence, once phosphorylated at any such site, the phosphoepi- tope–Cks1 interaction provides yet another docking interaction . After a sufficient amount of the Cln2-bound CDK1- Cks1 complex builds up during G 1 phase, it holds onto Sic1 via two contacts (active-site –phosphoacceptor-site binding and VLLPP-motif–Cln2-docking-pocket association); following the first phosphorylation, there are now three such interactions possible (active-site–phosphoacceptorsite binding, VLLPP-motif–Cln2-docking-pocket association , and phosphoepitope –Cks1 interaction), which explains initiation and establishment of processive phosphorylation (Kõivomägi et al. 2011). Similarly, as the amount of Clb5-bound CDK1-Cks1 builds up in late- G 1 /early-S phase, maintenance and completion of processive multisite phosphorylation by the Clb5-CDK1-Cks1 enzyme is very efficient, given that it has four times the probability of engaging an RxL motif than the Cln2- CDK1-Cks1 complex does a single VLLPP motif (Venta et al. 2012). "
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    • "Within a single protein, clusters of kinase target sites can act as a functional unit whereby the position of each site within the cluster might not be strongly constrained. This could be the case if the role of the modifications were to regulate the bulk electrostatics of a protein region (Strickfaden et al, 2007) or to achieve a non-linear regulatory outcome (K~ oivom€ agi et al, 2011). Phosphosites are often found in clusters within proteins (Schweiger & Linial, 2010; Christian et al, 2012) and the clustering of sites matching a kinase motif can serve as a predictor for kinase-target interactions further highlighting the functional importance of these clusters (Moses et al, 2007a). "
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