We aimed to answer a longstanding biological question: How is the temporal order of cell cycle events achieved? We used two alternative approaches. First, we have applied live-cell microscopy to study the cyclin-dependent kinase (CDK) phosphorylation-induced degradation dynamics of several key targets. By re-wiring the multisite phosphorylation patterns, we were able to shift the degradation timing as well as the rate of degradation of CDK targets and reporter constructs. The timing of these events was remarkably predictable by our models exhibiting few-minute precision along the whole span of the cell cycle. These experiments support our hypothesis outlined in the grant proposal suggesting that the multisite phosphorylation networks in the disordered regions of CDK targets act as time-tags for cell cycle switches.
As a second approach, we have used the GFP constructs fused to phospho-regulated NLS sequences. A promising set of new results on the timing of the GFP reporter’s nuclear exit was obtained. We continued to search for additional short linear motifs (SLiMs) that serve as specificity factors controlling the CDK signal processing. Docking of such motifs in CDK targets is mediated by pockets on cyclins and short linear motifs in substrates play a key role in defining the CDK activity thresholds and temporal order of substrate phosphorylation. We discovered several new cyclin-specific SLiMs and completed the set of docking specificities for the four major cyclins: LP, RxL, PxxPxF, and LxF motifs for G1-, S-, G2-, and M-phase CDKs, respectively. These linear motifs helped us to build a synbio toolbox of multisite phosphorylation tags. We have also discovered a set of new features to orthogonality between protein kinase MAPK and CDK signaling. These orthogonality mechanisms are important for designing the synthetic circuits that do not cross-react with other kinase pathways in the cell. Based on this knowledge we constructed the first set of logic gates with two orthogonal kinase inputs based on MAPK and CDK in yeast pheromone pathway circuit. These synthetic logic gates and the general principle that leads to new and more complex circuits can find practical applications in cell-based biosensors, microbial cell factories, in therapeutic solutions and production of protein drugs, in the development of artificial tissues and prosthetic networks, and many other potential applications of synthetic biology.
