Periodic Reporting for period 4 - Phosphoprocessors (Biological signal processing via multisite phosphorylation networks)
Período documentado: 2019-11-01 hasta 2020-10-31
Multisite phosphorylation of proteins is a signal processing mechanism playing crucial roles in cell division and differentiation as well as in disease. This process is catalyzed by enzymes called protein kinases, whose role is to find the right target protein and switch its function by covalently adding a coded pattern of phosphates to its specific positions. Our goal in this project was to elucidate the molecular basis of this important mechanism. Previously, we had demonstrated a novel phenomenon of multisite phosphorylation in cell cycle regulation. We showed that the master regulator of the cell cycle, the cyclin-dependent kinase (CDK) phosphorylates one of its crucial multisite targets semi-processively in the N-to-C terminal direction along the disordered protein. The process is controlled by key parameters including the distance between phosphorylation sites, the distribution of serines and threonines in sites, and the position of docking motifs. According to our model, linear patterns of phosphorylation networks along the disordered protein segments determine the net phosphorylation rate of the protein. This concept provides a new interpretation of CDK signal processing, and it can explain how the temporal order of cell cycle events is achieved. The goals of this were: 1) To seek proof of the model by rewiring the patterns of budding yeast Cdk1 multisite networks according to the rules we have identified, so to change the order of cell cycle events; 2) To apply the proposed model in the context of different kinases and complex substrate arrangements, we studied the CDK-dependent phosphorylation of yeast centrosome and kinetochore components; 3) To apply the obtained knowledge on multisite phosphorylation code to design circuits for synthetic biology. A toolbox of synthetic parts based on multisite phosphorylation would revolutionize the field because of the fast time scales and wide combinatorial possibilities.
Why is it important for society? This work will provide a useful toolbox for next-generation synthetic signaling networks. It was the first attempt to explore the potential and possibilities to use multisite phosphorylation and its combinatorial encoding as a way to design synthetic signaling circuits for synthetic biology applications. Such future applications will range from medicine to bio-based chemical production.
Why is it important for society? This work will provide a useful toolbox for next-generation synthetic signaling networks. It was the first attempt to explore the potential and possibilities to use multisite phosphorylation and its combinatorial encoding as a way to design synthetic signaling circuits for synthetic biology applications. Such future applications will range from medicine to bio-based chemical production.
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.
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.
Multisite phosphorylation of proteins by protein kinases is a powerful signal processing mechanism whose diverse possibilities are not well understood. In this process, multiple phosphates are added in a random or defined order to the serine, threonine, or tyrosine residues in kinase substrates. When a crucial set of key sites becomes phosphorylated up to a certain degree, the downstream signaling switch will be triggered. In this project, we applied the principles of sequential signal processing via multisite phosphorylation to synthetic circuit design. For this, we constructed several synthetic signal response modules with the output effects being phosphorylation-dependent degradation and localization, and whose input-output relationships are controlled by various tunable parameters of multisite phosphorylation networks.
The lack of standardized synthetic circuits is a bottleneck in the field of synthetic biology, and there is a pressing need to expand the toolkit of available parts and modules. The majority of synthetic circuits demonstrated to date are based on transcriptional control with inducible promoters. The primary drawback of these systems is the slow signal response time scales. The phosphorylation networks proposed here would revolutionize the field by being the first synthetic circuit toolbox that could provide fast signal-response time scales and flexibility in the encoding of multisite phosphorylation that provides nearly unlimited possibilities for differential connectivity.
The lack of standardized synthetic circuits is a bottleneck in the field of synthetic biology, and there is a pressing need to expand the toolkit of available parts and modules. The majority of synthetic circuits demonstrated to date are based on transcriptional control with inducible promoters. The primary drawback of these systems is the slow signal response time scales. The phosphorylation networks proposed here would revolutionize the field by being the first synthetic circuit toolbox that could provide fast signal-response time scales and flexibility in the encoding of multisite phosphorylation that provides nearly unlimited possibilities for differential connectivity.