Multistep phosphorylation systems: tunable components of biological signaling circuits

Abstract

Multisite phosphorylation of proteins is a powerful signal processing mechanism that plays crucial roles in cell division and differentiation as well as in disease. We recently demonstrated a novel phenomenon in cell cycle regulation by showing that cyclin-dependent kinase-dependent multisite phosphorylation of a crucial substrate is performed sequentially 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 along disordered protein segments determine the signal-response function of a multisite phosphorylation switch. Here we discuss the general advantages and engineering principles of multisite phosphorylation networks as processors of kinase signals. We also address the idea of using the mechanistic logic of linear multisite phosphorylation networks to design circuits for synthetic biology applications.

FIGURE 1:

Multisite phosphorylation of Cdk1 targets. (A) A schematic model of a cyclin-Cdk1-Cks1 complex containing the catalytic Cdk1 subunit, the positive regulatory subunit (cyclin), and an accessory phosphate-binding subunit (Cks1). A disordered substrate is shown together with the docking motifs that interact with each of the subunits. These are as follows: the phosphorylated consensus site (S/T-P) interacts with the Cdk1 active site (red marks with -OH), a cyclin docking motif interacts with a hydrophobic pocket on the cyclin (blue boxes), and a phosphorylated threonine on the substrate interacts with Cks1 (red marks with –P). (B) Examples of multisite phosphorylation networks in selected Cdk1 targets of Saccharomyces cerevisiae. There are at least two types of cyclin-docking motifs in Cdk1 substrates. L-P rich motifs are specific docking sites for the G1-specific Cln2-Cdk1 complex (Bhaduri and Pryciak, 2011; Koivomagi et al., 2011b). RXL motifs are docking motifs specific for the S-phase complex Clb5-Cdk1. Phosphodegrons are sequence motifs composed of two phosphorylated sites positioned 2–3 amino acids apart and recognized by the ubiquitin ligase SCF-Cdc4. Networks are mostly located in disordered regions of the proteins. (C) Graph depicting the ultrasensitive signal-response curves created by multisite phosphorylation. For the model simulation, we created a system of ordinary differential equations describing the phosphorylation of a set of kinase substrates carrying different multisite phosphorylation networks. The differently colored lines indicate simulated phosphorylation output responses at different thresholds of the increasing input signal of the kinase (the diagonal dotted line). The number of phosphorylation sites was varied in each substrate to gain temporal resolution of the response curves (1, 4, 6, 9, and 12 phosphorylation sites in substrates represented by curves from left to right, respectively). In addition, the phosphorylation rate constants of selected steps were varied in some substrates to imitate the effect of different patterning of the sites on the phosphorylation rates on individual steps. Weak phosphatase activity was included to counteract the kinase activity. The dotted horizontal lines represent the kinase thresholds at which half of the substrate has reached the fully phosphorylated state.

FIGURE 2:

Sequential processing of kinase signals along linear multisite networks. (A) Schematic view of a specificity filter based on sequential phosphorylation of a substrate by cyclin-Cdk1-Cks1. The output signal is a diphosphodegron containing a non-CDK site. This system both prevents erroneous firing of the degradation switch by other proline-directed kinases and filters low-level Cdk1 signals to ensure that the commitment of the switch occurs only when adequate levels of Cdk1 activity are achieved. (B) A three-input AND gate created by sequential primed kinases that leads to the degradation of cohesin acetyltransferase Eco1 (Lyons et al., 2013). The three kinases recognize motifs primed by previous phosphorylation events (motifs are indicated by red letters on the scheme at right). The resulting ordered sequence of three phosphorylation events leads to the formation of a phosphorylated diphosphodegron.