CDK signaling via nonconventional CDK phosphorylation sites.

Since the discovery of cyclin-dependent kinases (CDKs), it has been perceived as a dogma that CDK signaling in the cell cycle is mediated via targeting the CDK consensus sites: the optimal and the minimal motifs S/T-P-x-K/R and S/T-P, respectively. However, more recent evidence suggests that often the CDK phosphorylation events of regulatory importance are mediated via nonconventional CDK sites that lack the required +1Pro of the consensus site motif. In these cases, the loss of specificity seems to be compensated via distant docking interactions facilitated by 1) phosphorylated priming sites binding to phospho-adaptor Cks1 and/or 2) cyclin-specific docking interactions via Short Linear Motifs (SLiMs) in substrates. This Perspective discusses the possible reasons why nonconventional CDK sites are used for CDK signaling. First, the nonconventional CDK sites can act as specificity filters to recognize and distinguish the CDK signal from many other proline-directed kinases in cells. Second, the nonconventional CDK sites in combination with the docking mechanisms provide a much wider range of phosphorylation rates, and thus, also a wider range of CDK thresholds during the accumulation and decline of CDK activity during the cell cycle. As a large number of Cks1-dependent nonconventional CDK sites have been discovered recently, past studies focusing on mutating only the consensus sites should likely be critically reexamined. It is also very likely that phosphorylation of nonconventional sites is crucial in many other kinase-signaling networks.

A synthetic biology approach reveals diverse and dynamic CDK response profiles via multisite phosphorylation of NLS-NES modules.

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.

Cdc6 is sequentially regulated by PP2A-Cdc55, Cdc14, and Sic1 for origin licensing in S. cerevisiae.

Cdc6, a subunit of the pre-replicative complex (pre-RC), contains multiple regulatory cyclin-dependent kinase (Cdk1) consensus sites, SP or TP motifs. In Saccharomyces cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, the Cdk1 phospho-adaptor in S phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates in mitosis and is tightly bound by Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing and degradation. It has been extensively studied how Cdc6 phosphorylation is regulated by the cyclin-Cdk1 complex. However, a detailed mechanism on how Cdc6 phosphorylation is reversed by phosphatases has not been elucidated. Here, we show that PP2ACdc55 dephosphorylates Cdc6 N-terminal sites to release Clb2. Cdc14 dephosphorylates the C-terminal phospho-degron, leading to Cdc6 stabilization in mitosis. In addition, Cdk1 inhibitor Sic1 releases Clb2·Cdk1·Cks1 from Cdc6 to load Mcm2-7 on the chromatin upon mitotic exit. Thus, pre-RC assembly and origin licensing are promoted by phosphatases through the attenuation of distinct Cdk1-dependent Cdc6 inhibitory mechanisms.

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription.

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.1-3 The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle,4-7 together with variations in substrate docking to sites on associated cyclin and Cks subunits.3,6,8-10 Many substrates are modified at multiple sites to provide more complex regulation.10-14 Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of budding yeast genes required for mitotic progression.11,12 As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback.13-16 Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that when high Cdk1 activity accumulates in a mitotic arrest, CLB2 expression is inhibited. Inhibition is accompanied by Ndd1 degradation, and we present evidence that degradation is triggered by multisite Ndd1 phosphorylation by high mitotic Cdk1-Clb2 activity. Complete Ndd1 phosphorylation by Clb2-Cdk1-Cks1 requires the phosphothreonine-binding site of Cks1, as well as a recently identified phosphate-binding pocket on the cyclin Clb2.17 We therefore propose that initial phosphorylation by Cdk1 primes Ndd1 for delayed secondary phosphorylation at suboptimal sites that promote degradation. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

The Eukaryotic Linear Motif resource: 2022 release.

Almost twenty years after its initial release, the Eukaryotic Linear Motif (ELM) resource remains an invaluable source of information for the study of motif-mediated protein-protein interactions. ELM provides a comprehensive, regularly updated and well-organised repository of manually curated, experimentally validated short linear motifs (SLiMs). An increasing number of SLiM-mediated interactions are discovered each year and keeping the resource up-to-date continues to be a great challenge. In the current update, 30 novel motif classes have been added and five existing classes have undergone major revisions. The update includes 411 new motif instances mostly focused on cell-cycle regulation, control of the actin cytoskeleton, membrane remodelling and vesicle trafficking pathways, liquid-liquid phase separation and integrin signalling. Many of the newly annotated motif-mediated interactions are targets of pathogenic motif mimicry by viral, bacterial or eukaryotic pathogens, providing invaluable insights into the molecular mechanisms underlying infectious diseases. The current ELM release includes 317 motif classes incorporating 3934 individual motif instances manually curated from 3867 scientific publications. ELM is available at: http://elm.eu.org.

Docking to a Basic Helix Promotes Specific Phosphorylation by G1-Cdk1.

Cyclins are the activators of cyclin-dependent kinase (CDK) complex, but they also act as docking scaffolds for different short linear motifs (SLiMs) in CDK substrates and inhibitors. According to the unified model of CDK function, the cell cycle is coordinated by CDK both via general CDK activity thresholds and cyclin-specific substrate docking. Recently, it was found that the G1-cyclins of S. cerevisiae have a specific function in promoting polarization and growth of the buds, making the G1 cyclins essential for cell survival. Thus, while a uniform CDK specificity of a single cyclin can be sufficient to drive the cell cycle in some cells, such as in fission yeast, cyclin specificity can be essential in other organisms. However, the known G1-CDK specific LP docking motif, was not responsible for this essential function, indicating that G1-CDKs use yet other unknown docking mechanisms. Here we report a discovery of a G1 cyclin-specific (Cln1,2) lysine-arginine-rich helical docking motif (the K/R motif) in G1-CDK targets involved in the mating pathway (Ste7), transcription (Xbp1), bud morphogenesis (Bud2) and spindle pole body (Spc29, Spc42, Spc110, Sli15) function of S. cerevisiae. We also show that the docking efficiency of K/R motif can be regulated by basophilic kinases such as protein kinase A. Our results further widen the list of cyclin specificity mechanisms and may explain the recently demonstrated unique essential function of G1 cyclins in budding yeast.

Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle.

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. It is still poorly understood how efficient, centromere-dependent kinetochore assembly is accomplished from hundreds of individual protein building blocks in a cell cycle-dependent manner. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U, which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-phase and targets the protein for degradation. Binding of the Mtw1Mis12 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic in SCF mutants, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits facilitates efficient centromere-dependent kinetochore assembly. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.

Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction.

Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi-site phosphorylation using the yeast trehalase Nth1 and its activator, the 14-3-3 protein Bmh1, as a model. Nth1 is known to be phosphorylated by the metabolic kinase PKA on four serine residues and by the cell cycle kinase CDK on one residue. However, how these five phospho-sites adjust Nth1 activity remains unclear. Using a novel reporter construct, we investigated the contribution of the individual sites for the regulation of the trehalase and its 14-3-3 interactor. In contrast to the constitutively phosphorylated S20 and S83, the weaker sites S21 and S60 are only phosphorylated by increased PKA activity. For binding Bmh1, S83 functions as the high-affinity "gatekeeper" site, but successful binding of the Bmh1 dimer and thus Nth1 activation requires S60 as a secondary site. Under nutrient-poor conditions with low PKA activity, S60 is not efficiently phosphorylated and the cell cycle dependent phosphorylation of S66 by Cdk1 contributes to Nth1 activity, likely by providing an alternative Bmh1 binding site. Additionally, the PKA sites S20 and S21 modulate the dephosphorylation of Nth1 on downstream Bmh1 sites. In summary, our results expand our molecular understanding of Nth1 regulation and provide a new aspect of the interaction of 14-3-3 proteins with their targets.

A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling.

Cyclin-dependent kinases (CDKs), the master regulators of cell division, are activated by different cyclins at different cell cycle stages. In addition to being activators of CDKs, cyclins recognize various linear motifs to target CDK activity to specific proteins. We uncovered a cyclin docking motif, NLxxxL, that contributes to phosphorylation-dependent degradation of the CDK inhibitor Far1 at the G1/S stage in the yeast Saccharomyces cerevisiae. This motif is recognized exclusively by S-phase CDK (S-CDK) Clb5/6-Cdc28 and is considerably more potent than the conventional RxL docking motif. The NLxxxL and RxL motifs were found to overlap in some target proteins, suggesting that cyclin docking motifs can evolve to switch from one to another for fine-tuning of cell cycle events. Using time-lapse fluorescence microscopy, we show how different docking connections temporally control phosphorylation-driven target degradation. This also revealed a differential function of the phosphoadaptor protein Cks1, as Cks1 docking potentiated degron phosphorylation of RxL-containing but not of NLxxxL-containing substrates. The NLxxxL motif was found to govern S-cyclin-specificity in multiple yeast CDK targets including Fin1, Lif1, and Slx4, suggesting its wider importance.

The sequence at Spike S1/S2 site enables cleavage by furin and phospho-regulation in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV.

The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680SPRRAR↓SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous cleavage motif was present at S1/S2 of the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR↓SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furin's ability to cleave the site. Although phospho-regulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.

Comprehensive Analysis of G1 Cyclin Docking Motif Sequences that Control CDK Regulatory Potency In Vivo.

Many protein-modifying enzymes recognize their substrates via docking motifs, but the range of functionally permissible motif sequences is often poorly defined. During eukaryotic cell division, cyclin-specific docking motifs help cyclin-dependent kinases (CDKs) phosphorylate different substrates at different stages, thus enforcing a temporally ordered series of events. In budding yeast, CDK substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs were unknown. Here, we comprehensively analyze LP motif requirements in vivo by combining a competitive growth assay with deep mutational scanning. We quantified the effect of all single-residue replacements in five different LP motifs by using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results uncover substantial tolerance for deviations from the consensus sequence, plus requirements at some positions that are contingent on the favorability of other motif residues. They also reveal the basis for variations in functional potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the strength of other candidate motif sequences. Finally, we find that variation in docking motif potency can advance or delay the time at which CDK substrate phosphorylation occurs, and thereby control the temporal ordering of cell cycle regulation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in docking strength can tune the degree and timing of regulatory modifications.

Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Phos-tag SDS-PAGE.

Phos-tagTM SDS-PAGE is a method that enables electrophoretic separation of proteins based on their phosphorylation status. With Phos-tagTM SDS-PAGE, it is possible to discriminate between different phosphoforms of proteins based on their phosphorylation level and the number of phosphorylated sites, and to determine the stoichiometry of different phosphorylation products. Phos-tagTM SDS-PAGE is useful for analyzing disordered proteins with multiple phosphorylation sites and can be used for any of the downstream applications used in combination with conventional SDS-PAGE, for example, Western blotting and mass-spectrometry. To obtain the best results with Phos-tagTM SDS-PAGE, however, it is often necessary to optimize the gel composition. Depending on the molecular weight and number of phosphoryl groups added to the protein, different gel composition or running conditions should be used. Here, we provide protocols for Mn2+- and Zn2+-Phos-tagTM SDS-PAGE and give examples of how disordered proteins with different characteristics behave in gels with various Phos-tag concentrations.

Proline-Rich Motifs Control G2-CDK Target Phosphorylation and Priming an Anchoring Protein for Polo Kinase Localization.

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.

A processive phosphorylation circuit with multiple kinase inputs and mutually diversional routes controls G1/S decision.

Studies on multisite phosphorylation networks of cyclin-dependent kinase (CDK) targets have opened a new level of signaling complexity by revealing signal processing routes encoded into disordered proteins. A model target, the CDK inhibitor Sic1, contains linear phosphorylation motifs, docking sites, and phosphodegrons to empower an N-to-C terminally directed phosphorylation process. Here, we uncover a signal processing mechanism involving multi-step competition between mutually diversional phosphorylation routes within the S-CDK-Sic1 inhibitory complex. Intracomplex phosphorylation plays a direct role in controlling Sic1 degradation, and provides a mechanism to sequentially integrate both the G1- and S-CDK activities while keeping S-CDK inhibited towards other targets. The competing phosphorylation routes prevent premature Sic1 degradation and demonstrate how integration of MAPK from the pheromone pathway allows one to tune the competition of alternative phosphorylation paths. The mutually diversional phosphorylation circuits may be a general way for processing multiple kinase signals to coordinate cellular decisions in eukaryotes.

Multisite phosphorylation code of CDK.

The quantitative model of cyclin-dependent kinase (CDK) function states that cyclins temporally order cell cycle events at different CDK activity levels, or thresholds. The model lacks a mechanistic explanation, as it is not understood how different thresholds are encoded into substrates. We show that a multisite phosphorylation code governs the phosphorylation of CDK targets and that phosphorylation clusters act as timing tags that trigger specific events at different CDK thresholds. Using phospho-degradable CDK threshold sensors with rationally encoded phosphorylation patterns, we were able to predictably program thresholds over the entire range of the Saccharomyces cerevisiae cell cycle. We defined three levels of CDK multisite phosphorylation encoding: (i) serine-threonine swapping in phosphorylation sites, (ii) patterning of phosphorylation sites, and (iii) cyclin-specific docking combined with modulation of CDK activity. Thus, CDK can signal via hundreds of differentially encoded targets at precise times to provide a temporally ordered phosphorylation pattern required for cell division.

Cyclin-Specific Docking Mechanisms Reveal the Complexity of M-CDK Function in the Cell Cycle.

Cyclin-dependent kinases (CDKs) coordinate hundreds of molecular events during the cell cycle. Multiple cyclins are involved, but the global role of cyclin-specific phosphorylation has remained unsolved. We uncovered a cyclin docking motif, LxF, that mediates binding of replication factor Cdc6 to mitotic cyclin. This interaction leads to phospho-adaptor Cks1-mediated inhibition of M-CDK to facilitate Cdc6 accumulation and sequestration in mitosis. The LxF motif and Cks1 also mediate the mutual inhibition between M-CDK and the tyrosine kinase Swe1. Additionally, the LxF motif is critical for targeting M-CDK to phosphorylate several mitotic regulators; for example, Spo12 is targeted via LxF to release the phosphatase Cdc14. The results complete the full set of G1, S, and M-CDK docking mechanisms and outline the unified role of cyclin specificity and CDK activity thresholds. Cooperation of cyclin and Cks1 docking creates a variety of CDK thresholds and switching orders, including combinations of last in, first out (LIFO) and first in, first out (FIFO) ordering.

How the cell cycle clock ticks.

Eukaryotic cell division has been studied thoroughly and is understood in great mechanistic detail. Paradoxically, however, we lack an understanding of its core control process, in which the master regulator of the cell cycle, cyclin-dependent kinase (CDK), temporally coordinates an array of complex molecular events. The core elements of the CDK control system are conserved in eukaryotic cells, which contain multiple cyclin-CDK forms that have poorly defined and partially overlapping responsibilities in the cell cycle. However, a single CDK can drive all events of cell division in both mammalian and yeast cells, and in fission yeast a single mitotic cyclin can drive the cell cycle without major problems. But how can the same CDK induce different events when activated at different times during the cell cycle? This question, which has bewildered cell cycle researchers for decades, now has a sufficiently clear mechanistic answer. This Perspective aims to provide a synthesis of recent data to facilitate a better understanding of this central cellular control system.

Multistep phosphorylation systems: tunable components of biological signaling circuits.

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.

Multisite phosphorylation networks as signal processors for Cdk1.

The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, distribution of serines and threonines as phosphoacceptors and positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phosphoadaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within networks of phosphorylation sites in different targets provides a wide range of possibilities for differential amplification of Cdk1 signals, thus providing a mechanism to generate a wide range of thresholds in the cell cycle.