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Teaser, summary, work performed and final results

Periodic Reporting for period 3 - Phosphoprocessors (Biological signal processing via multisite phosphorylation networks)

Teaser

Summary

Multisite phosphorylation of proteins is a powerful signal processing mechanism playing crucial roles in cell division and differentiation as well as in disease. Our goal in this application is to elucidate the molecular basis of this important mechanism. We recently demonstrated a novel phenomenon of multisite phosphorylation in cell cycle regulation. We showed that cyclin-dependent kinase (CDK)-dependent multisite phosphorylation of a crucial substrate is performed semiprocessively 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 study are: 1) We will 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. Next, we will restore the order by alternative wiring of the same switches; 2) To apply the proposed model in the context of different kinases and complex substrate arrangements, we will study the Cdk1-dependent multisite phosphorylation of kinetochore components, to understand the phospho-regulation of kinetochore formation, microtubule attachment and error correction; 3) We will apply multisite phosphorylation 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.

Work performed

We aimed to answer a longstanding biological question: How is the temporal order of cell cycle events achieved? To achieve this objective we have used two alternative approaches. First, we have applied live cell microscopy to study the CDK-phosphorylation induced degradation dynamics of key targets Sic1 and Far1. By re-wiring the multisite phosphorylation patterns, we have been able to shift the degradation timing as well as the rate of degradation of these targets. 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 timing of the GFP reporterâ€™s nuclear exit has been obtained.
In addition, under this objective we have continued to search for additional linear motifs that serve as specificity parametres controlling the CDK signal processing by the multisite phosphorylation networks. Such substrate docking interactions mediated by docking pockets on cyclins and short linear motifs in substrates play key role in defining the CDK activity thresholds and temporal order of substrate phosphorylation. In Saccharomyces cerevisiae several CDK targets involved in mating pathway, G1/S transcription, bud morphogenesis, and spindle pole body are specifically phosphorylated by G1 cyclin-Cdk1 complexes. The G1 cyclin Cln2 contains a docking site that interacts with a leucine- and proline-rich motif (LP motif) on substrates, providing substrate specificity for the Cln2-Cdk1 complexes. In studies we have found that a novel substrate docking interaction, different from the known hydrophobic LP interaction, governs the phosphorylation specificity of a group of key of G1-cyclin-Cdk1 targets. The novel docking motif was determined as a lysine- or arginine-rich sequence (K/R motif) that is found in substrates either as a linear K/R-x-K/R motif or as an Î±-helix with sequential basic amino acids on one side of the helix. We identified Cln mutants specifically defective in either hydrophobic LP interaction or basic motif docking and analyze the functions of the basic docking motif in vivo. In addition, we show that the C-terminal tail of Cln2 hinders LP docking, implicating a regulatory mechanism, where the phosphorylation status of the C-terminal tail alters substrate recognition by Cln-Cdk1 complexes.

A considerable progress has also been achieved with respect to dissecting the phosphorylation-dependent diversion (CDK inhibition) sequences. We have mapped critical sequences in Far1, Sic1 and Cdc6 that are responsible for phosphorylation-induced inhibition of the kinase. In combination with the cyclin mutants we are close to have a full map of interactions between the CDK complex and the inhibitors that is sufficient to solve a long-standing question of why some multisite phosphorylation networks of CDK are acting as substrates and some as inhibitors. Knowledge of these linear motifs and rules of their behaviour are of utmost importance for us to build a predictable and tunable toolbox of multisite phosphorylation tags.
In addition, we have discovered a set of new features with respect of orthogonality between 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. We have shown how phosphorylation signals with overlapping specificities leading to either a differentiation decision, or alternatively, to the cell cycle entry are filtered to make clean distinct choices. In S. cerevisiae, the MAPK kinase Fus3 of the mating pathway leads to CDK inhibition, while the CDK complex is counteracting its inhibitors to enter the cell cycle. In a boarderline situation with mixed signals, the MAPK and CDK, both directed to S/TP consensus sites in a CDK inhibitor

Final results

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. We will apply the principles of sequential signal processing via multisite phosphorylation to synthetic circuit design. For this, we will construct a number of synthetic signal response modules in which the outputs are phosphorylation-dependent degradation and localization signals and the input-output relationships are controlled by various easily tunable multisite phosphorylation networks. Advantages of the toolbox of multisite phosphorylation circuits: The lack of standardized synthetic circuits is a bottleneck in the field, and there is a pressing need to expand the synthetic biology 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. Secondly, the flexibility in encoding multisite phosphorylation provides nearly unlimited possibilities for differential connectivity. The result of this work will provide a useful toolbox for next-generation synthetic signaling networks that have real-world applications in medicine, biotechnology, bioremediation and bioenergy.