RESUMO
The complexity of multisite phosphorylation mechanisms in regulating nuclear localization signals (NLSs) and nuclear export signals (NESs) is not understood, and its potential has not been used in synthetic biology. The nucleocytoplasmic shuttling of many proteins is regulated by cyclin-dependent kinases (CDKs) that rely on multisite phosphorylation patterns and short linear motifs (SLiMs) to dynamically control proteins in the cell cycle. We studied the role of motif patterns in nucleocytoplasmic shuttling using sensors based on the CDK targets Dna2, Psy4, and Mcm2/3 of Saccharomyces cerevisiae. We designed multisite phosphorylation modules by rearranging phosphorylation sites, cyclin-specific SLiMs, phospho-priming, phosphatase specificity, and NLS/NES phospho-regulation and obtained very different substrate localization dynamics. These included ultrasensitive responses with and without a delay, graded responses, and different homeostatic plateaus. Thus, CDK can do much more than trigger sequential switches during the cell cycle as it can drive complex patterns of protein localization and activity by using multisite phosphorylation networks.
RESUMO
The hydrophobic patch (hp), a docking pocket on cyclins of CDKs (cyclin-dependent kinases), has been thought to accommodate a single short linear motif (SLiM), the "RxL or Cy" docking motif. Here we show that hp can bind different motifs with high specificity. We identify a PxxPxF motif that is necessary for G2-cyclin Clb3 function in S. cerevisiae, and that mediates Clb3-Cdk1 phosphorylation of Ypr174c (proposed name: Cdc5 SPB anchor-Csa1) to regulate the localization of Polo kinase Cdc5. Similar motifs exist in other Clb3-Cdk1 targets. Our work completes the set of docking specificities for the four major cyclins: LP, RxL, PxxPxF, and LxF motifs for G1-, S-, G2-, and M-phase cyclins, respectively. Further, we show that variations in motifs can change their specificity for human cyclins. This diversity could provide complexity for the encoding of CDK thresholds to achieve ordered cell-cycle phosphorylation.
