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

2020; Springer Nature; Volume: 40; Issue: 2 Linguagem: Inglês

10.15252/embj.2020105839

ISSN

1460-2075

Autores

Ilona Faustova, Luka Bulatovic, Frida Matiyevskaya, Ervin Valk, Mihkel Örd, Mart Loog,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

Article19 November 2020Open Access Source DataTransparent process A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling Ilona Faustova Ilona Faustova Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Luka Bulatovic Luka Bulatovic Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Frida Matiyevskaya Frida Matiyevskaya Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Ervin Valk Ervin Valk Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Mihkel Örd Mihkel Örd Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Mart Loog Corresponding Author Mart Loog [email protected] orcid.org/0000-0003-1330-8453 Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Ilona Faustova Ilona Faustova Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Luka Bulatovic Luka Bulatovic Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Frida Matiyevskaya Frida Matiyevskaya Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Ervin Valk Ervin Valk Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Mihkel Örd Mihkel Örd Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Mart Loog Corresponding Author Mart Loog [email protected] orcid.org/0000-0003-1330-8453 Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Author Information Ilona Faustova1, Luka Bulatovic1, Frida Matiyevskaya1, Ervin Valk1, Mihkel Örd1 and Mart Loog *,1 1Institute of Technology, University of Tartu, Tartu, Estonia *Corresponding author. Tel: +372 517 5698; E-mail: [email protected] The EMBO Journal (2021)40:e105839https://doi.org/10.15252/embj.2020105839 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis Short linear motifs (SLiMs) mediate protein interactions e.g. between cyclin-dependent kinase (CDK) substrates and cyclins. A new cyclin docking motif uncovered in yeast CDK substrates targets them specifically for phosphorylation by S-phase CDK. A cyclin docking motif with consensus NLxxxL contributes to phosphorylation-dependent degradation of the yeast CDK inhibitor Far1 at the G1/S stage. The NLxxxL motif is present in multiple yeast CDK substrates and exclusively mediates phosphorylation by Clb5/6-Cdk1 (S-CDK) complexes. The NLxxxL motif is homologous to the canonical RxL cyclin-docking motif, but more potent in promoting phosphorylation. In addition to directing the timing of phosphorylation, cyclin-docking specificity can affect the reversal of Cdk1-dependent phosphorylation during mitotic exit. Introduction Cyclin-dependent protein kinases (CDKs) are central regulators executing hundreds of phosphorylation events that trigger, order, check, and finalize the complex process of eukaryotic cell division cycle (Morgan, 2007; Enserink & Kolodner, 2010). CDKs are unique among hundreds of other representatives of the eukaryotic protein kinase superfamily, as they use complex, sequential multi-docking phosphorylation mechanisms; these involve interactions of a phosphoadaptor subunit Cks1 with primed phospho-sites, as well as recognition of short linear motifs (SLiMs) in target proteins by cyclin docking pockets (Örd & Loog, 2019). The initial view of cyclins, in which they function as periodically synthesized and degraded activators of CDK kinase subunits, each acting at their specific cell cycle stage, has been considerably broadened recently. First, it was found that different cyclins modulate the intrinsic active site specificity toward the phosphorylation motifs (Loog & Morgan, 2005; Kõivomägi et al, 2011a; Topacio et al, 2019). Second, for a long time it was known that a subset of CDK targets are tethered to CDK complexes via RxL docking motifs (a SLiM with the consensus sequence R/K-x-L-φ or R/K-x-L-x–φ) that bind to the hydrophobic patch (hp) in human cyclin A or E (Russo et al, 1996; Schulman et al, 1998; Takeda et al, 2001) or in S-phase cyclin Clb5 in budding yeast (Wilmes et al, 2004; Loog & Morgan, 2005). However, recent studies have uncovered several other cyclin-specific SLiMs that interact either with the hp or other areas on the cyclins (Bhaduri & Pryciak, 2011; Kõivomägi et al, 2011a; Bhaduri et al, 2015; Topacio et al, 2019; Örd et al, 2019a; Allan et al, 2020; Jackman et al, 2020). These studies widened the scope of the third general function of cyclins, namely in addition to being an activator and activity modulating factor for CDK subunit, cyclins also act as protein scaffolds that present various pocket configurations to dock different SLiMs in target proteins (Tatum & Endicott, 2020). A full set of highly specific and selective motifs have been defined for each of the four major cyclins in budding yeast: the LP, RxL, PxF, and LxF motifs for G1-, S-, G2-, and M-phase cyclins, respectively (Bhaduri & Pryciak, 2011; Kõivomägi et al, 2011a; Örd et al, 2019a; Örd et al, 2020). Further, recent studies have also revealed that human cyclins utilize a wider variety of docking motifs, as cyclin D and cyclin B1 were found to bind different helical docking motifs (Topacio et al, 2019; Allan et al, 2020; Jackman et al, 2020). Altogether, these findings define cyclins as versatile proteins with a plethora of docking options for SLiMs to achieve specific cell cycle signaling via CDK phosphorylation. Such a complexity of substrate docking motifs highlights the uniqueness of CDK among protein kinases. Its grand task of temporally ordering hundreds, if not thousands, of individual phosphorylation events at precise activity thresholds during the cell cycle (Stern & Nurse, 1996; Coudreuse & Nurse, 2010; Swaffer et al, 2016) is quite different from the tasks performed by many other kinases and signaling enzymes, whose mode of action can be more simply described as binary switching between active and inactive state. In addition to the differential docking mechanisms provided by cyclins, a key to the combinatorial complexity of CDK substrate phosphorylation is conferred by the phosphoadaptor subunit Cks1 (Kõivomägi et al, 2011b; McGrath et al, 2013), which provides a functional feature to the CDK complex that is unique among protein kinases. The majority of CDK targets contain multiple phosphorylation sites in intrinsically disordered regions (Holt et al, 2009), and the cyclin-CDK-Cks1 complex functions as a scaffold with three fixed points of substrate contact (Fig 1A), which allows the process of multisite phosphorylation to proceed in an ordered manner (Kõivomägi et al, 2013; Örd et al, 2019b). The phosphorylated TP motifs bind to the Cks1 phospho-pocket and facilitate the phosphorylation of secondary sites located C-terminally from the pTP priming sites (Kõivomägi et al, 2013; McGrath et al, 2013). This mechanism enables three types of substrate sequences—pTP priming sites bound by Cks1, phospho-acceptor sites recognized by the CDK active site, and diverse docking motifs recognized by cyclin—to be combined in "barcoded" linear patterns along the disordered targets. These patterns can be read by the CDK complexes to achieve a different input–output function of the net phosphorylation rate for a target (Örd et al, 2019b). In other words, the barcoded patterns act as timing tags that assign a target to a specific CDK activity threshold and hence a specific time point during the cell cycle. These findings created a unified model bringing together the CDK threshold model and cyclin specificity model: cyclin specificity and multisite phosphorylation patterns help to encode different execution orders and the CDK thresholds into the target proteins. Figure 1. NLxxxL motif promotes S-CDK-mediated phosphorylation and degradation of N-Far1 A. Scheme showing the major cyclin-substrate interactions of Cdk1 complex. B. Scheme showing the CDK phosphorylation sites and the potential Clb5 docking region in the disordered N terminus of Far1 (residues 1–150). C. Co-immunoprecipitation of Cdc4 using either unphosphorylated or Clb5-Cdk1-phosphorylated Far1(85–150) as bait. The experiment was repeated twice, a representative example is shown. D. Time-lapse fluorescence images showing the expression and degradation of wild-type and S87A S91A mutant Far1(1–150)-GFP and nuclear-cytoplasmic shuttling of Whi5-mCherry. Nuclear export of Whi5-mCherry takes place at 0 min (Start point) and was used to synchronize the cells for quantitative analysis. E, F. Mean ± SEM fluorescence intensities of the indicated Far1(1–150)-GFP mutants over the cell cycle. The number of cells analyzed for each construct is noted in Table EV2. G. Time from Start to degradation of 50% of the indicated Far1-GFP sensor in single cells. The bars show median ± 95% confidence intervals. The numbers above the plot show median values. ****P-value < 0.0001, *** 0.05 for pairwise comparisons with wild type using Mann–Whitney U-test. The number of cells analyzed for each construct is noted in Table EV2. H. In vitro kinase assay showing the effect of N130A, L131A, L135A, and L136A mutations on Far1(1–150)-GFP phosphorylation by Clb5-Cdk1. WT/mut shows the decrease in phosphorylation rate of the mutant compared to wild type. The experiment was repeated twice, a representative example is shown. Source data are available online for this figure. Source Data for Figure 1 [embj2020105839-sup-0006-SDataFig1.xlsx] Download figure Download PowerPoint Despite these recent advances, it has remained unclear how the two major elements of multisite substrate docking, the Cks1-binding priming phosphorylation sites, and the cyclin-binding SLiM (Fig 1A), are cooperating with or compensating for each other's effects to define the CDK thresholds and timings of the cell cycle events. It is also not understood what is the possible sequence diversity, functional interrelationships and the balance between specificity and affinity of different SLiMs binding the hp of B-type cyclins. Here we report a Saccharomyces cerevisiae SLiM docking motif, with a consensus sequence NLxxxL, that promotes phosphorylation exclusively by S-CDK (Clb5 cyclin bound to the yeast CDK Cdc28). This motif was found in several S-phase CDK targets and, intriguingly, in multiple cases it overlapped with conventional RxL motifs. The NLxxxL-mediated Clb5 docking enabled fast phosphorylation at the G1/S transition as well as early dephosphorylation in anaphase. The NLxxxL motif, when part of a CDK substrate, stands out among the known cyclin docking motifs with its considerably lower KM value. Finally, we also discovered an unexpected difference in the Cks1-dependent phosphorylation behavior depending on whether the substrate utilized a weaker RxL motif versus a more efficient NLxxxL motif. Results NLxxxL motif promotes S-CDK-mediated phosphorylation of a di-phosphodegron in Far1 An S-CDK-specific putative linear docking motif was initially mapped via truncations of the N-terminal disordered region of Far1 within the segment of 130–140 of Far1 (Valk & Loog, manuscript in preparation). In the current study, the motif was further mapped using site-directed mutagenesis and quantitative time-lapse fluorescence microscopy. For this, we employed an approach using CDK threshold sensors, a set of variable GFP-tagged substrate constructs that are degraded in response to phosphorylation at defined levels of CDK activity (Örd et al, 2019b). We based the sensors on a fragment comprised of the first 150 N-terminal amino acids from Far1 (Far1(1–150)) (Fig 1B). This fragment contained Cdk1 phosphorylation site S87, which is necessary for cell cycle-dependent degradation of Far1 (Gartner et al, 1998). Far1 is ubiquitinated by SCFCdc4 (Blondel et al, 2000), and S87–S91 matches the consensus for a Cdc4 di-phosphodegron (Hao et al, 2007; Fig EV1A). To confirm that the Clb5-Cdk1-mediated phosphorylation of S87 and S91 is necessary for Far1-Cdc4 interaction, we performed Cdc4 pull-down experiments with truncated Far1(85–150). These experiments showed that Clb5-dependent phosphorylation of Far1(85–150) and the presence of both S87 and S91 was necessary for interaction with Cdc4 (Fig 1C). Click here to expand this figure. Figure EV1. Mapping of the S87/S91 di-phosphodegron and the linker region Alignment of S. cerevisiae Far1(80–100) with homologs from other yeast species. Far1 segment 85–91 matches the consensus for Cdc4 di-phosphodegron. Sequence of Far1 positions 85–140 showing the linker sequence between the S87/S91 degron and the NLxxxL motif. To study the linker region, positions 103–109 and 112–119 were replaced with glycine-serine linkers in two constructs that were analyzed in time-lapse microscopy in panel "C". Mean ± SEM fluorescence intensities of the indicated Far1(1–150)-GFP constructs after Start. For sample size, please see Table EV2. Plot showing the 50% degradation timing of indicated Far1(1–150)-GFP mutants in individual cells. The bars show median ± 95% CI. The numbers above the plot show median values for each construct. ns denotes P-value > 0.05 for pairwise comparisons with wild type using Mann–Whitney U-test. The number of cells analyzed is shown in Table EV2. Source data are available online for this figure. Download figure Download PowerPoint To estimate the effects of systematic alanine mutations within the docking motif on CDK-dependent phosphorylation of the degron, we measured the dynamics of sensor degradation. Ubiquitination-driven degradation of sensors with di-phospho-degrons is mediated by E3 ligase SCF-Cdc4 and proteasome, that are constitutively active throughout the cell cycle (Zhou & Howley, 1998), allowing to measure the dynamics of sensor phosphorylation alone. Using a previously described live-cell fluorescent microscopy protocol (Örd et al, 2019b), we followed the timing of phosphorylation-dependent degradation of GFP-tagged sensors (Fig 1D and E). For time point zero, we used the cell cycle Start, defined as the nuclear export of 50% of Whi5-mCherry, the transcriptional inhibitor of early cell cycle genes (Doncic et al, 2011). We found that a construct based on the wild type Far1(1–150) was degraded rapidly, declining to 50% of its maximal levels by ~ 14 min from Start (Fig 1E). This is in good agreement with our previous observations of the timing of G1/S transition, marked by Sic1 degradation and accumulation of free Clb5-Cdk1 complex (Venta et al, 2020). In contrast, a double-alanine mutation of the di-phosphodegron stabilized the sensor for the length of the cell cycle, which for budding yeast in rich media is about 90 min (Fig 1D and E). We introduced alanine mutations within the identified docking region and tested a set of them in the context of the sensors (Fig 1F and G). Single mutants L131A and L135A showed a considerable delay in sensor degradation, the mutation N130A caused an intermediate effect with a ~ 22-min half-time, while L136A mutation, and a triple mutation of the motif TTS in the middle of the segment behaved like a wild type. We also introduced simultaneous alanine mutants into four amino acid segments flanking the 10 amino acid region initially mapped by truncations. The 4xAla mutations of the N-terminal flanking sequence IKAT and the C-terminal RESI individually did not cause a delay in degradation, and however, a minor 3-min delay was detected with simultaneous mutation of both flanking regions (Fig 1F and G). As the sequence of the segment did not bear any obvious resemblance to the conventional RxLxF motif, we wondered if a region between the docking motif and the degron phosphorylation sites would provide crucial additional contacts with the CDK complex. To test this, we replaced two segments in this linker region with Gly-Ser stretches (Fig EV1B). However, no changes in degradation dynamics were observed (Fig EV1C and D). These results indicate that phosphorylation of the degron is controlled by the NLxxxL motif, and this motif fulfills the general criteria of a SLiM, defined as a recognition sequence of up to ten amino acids within a disordered segment (Davey et al, 2012). To compare the in vivo data with the biochemical measurements of CDK specificity in vitro, we performed assays to determine the initial velocity of phosphorylation using purified CDK complexes and the sensor proteins, by 32P autoradiography of SDS–PAGE. The phosphorylation by Clb5-Cdk1 was disrupted by mutations at the three key positions (N130A, L131A, L135A) and to a degree that correlated well with their degradation orders observed in vivo (Fig 1H). This indicates that NLxxxL is a docking motif for Clb5-Cdk1, which is necessary for timely phosphorylation of the Far1 N terminus. NLxxxL motif is specific for S-phase cyclins Clb5 and Clb6 To analyze the cyclin specificity of the identified docking motif, we performed in vitro phosphorylation assays with eight cyclin-Cdk1 complexes (Figs 2A and EV2A–C). For the four major complexes, we also tested their corresponding cyclin docking pocket mutants (Fig 2A). As controls, we used: (i) histone H1, a substrate that lacks cyclin docking motifs and relies only on a consensus phosphorylation site; and (ii) a non-inhibitory truncated version of Cdk1 inhibitor Sic1 (Sic1ΔC), containing a conventional cyclin docking motif, RxL. The results show that the discovered docking motif was specific for both S-phase complexes, Clb5- and Clb6-Cdk1. The other closely related G2- and M-CDK complexes showed very weak phosphorylation rate. The G1-CDK complexes Cln1- and Cln2-Cdk1 also showed considerable phosphorylation specificity, and however, this was neither dependent on the discovered motif in Far1, nor on the known hydrophobic substrate-binding pocket on Cln2 (Bhaduri et al, 2015). Figure 2. NLxxxL is a specific SLiM for docking by S-phase Clb5/6-Cdk1 Autoradiograph of a cyclin specificity analysis of the indicated substrates with wild-type or docking site mutant cyclin-Cdk1 complexes. Far1(1–150 mut) is L131A L135A L136A mutant, Sic1ΔC is the non-inhibitory mutant of Sic1 (deletion of 216–284). The experiment was repeated twice, a representative example is shown. Mean ± SEM fluorescence intensities of wild-type and NLxxxL motif mutant Far1(1–150)-GFP in either wild-type or clb5(hpm) clb6(hpm) strain over the cell cycle. The number of cells analyzed is noted in Table EV2. Source data are available online for this figure. Source Data for Figure 2 [embj2020105839-sup-0007-SDataFig2.xlsx] Download figure Download PowerPoint The Clb5 specificity and Clb5 interaction with the Far1 N terminus were dependent on the conserved hp docking pocket in the cyclin and the NLxxxL motif in Far1 (Figs 2A and EV2D). Also, in vitro phosphorylation experiments in the presence of competitor peptide with NLxxxL motif inhibited the rate of Clb5 phosphorylation of both wild-type Far1(1–150 WT) and Far1(1–150 RxL), where NLxxxL motif is replaced with an RxL motif, further indicating that the NLxxxL motif binds to the hp (Fig EV2E and F, for Far1(1–150 RxL) see also Fig 3 later). Finally, we observed that NLxxxL motif is required for Clb5-dependent phosphorylation of both Cdc4 di-phosphodegron sites S87 and S91 (Fig EV2G). Figure 3. NLxxxL motif is more effective than RxL motif in directing Clb5-Cdk1 activity Schemes of Far1(1–150)-GFP constructs, either wild-type or mutant where NLxxxL motif is replaced with RxL motif (VKRTLF), that are used in time-lapse microscopy experiments in panel (C). Sequence alignment showing the introduction of wild-type or mutated RxL motif to Far1. Plot showing the mean ± SEM fluorescence intensities of Far1(1–150)-GFP constructs with the indicated docking site. In rxl mutant, the NLxxxL motif is replaced with a mutated non-functional RxL motif (VKATAF). The number of cells analyzed is noted in Table EV2. Kinetic characterization of Far1(1–150 WT)-GFP and Far1(1–150 RxL)-GFP phosphorylation by Clb5-Cdk1. The data are from two experimental replicates. Mean ± SEM fluorescence intensities of wild-type or RxL-dependent Far1(1–150)-GFP in wild-type or the indicated cyclin hpm mutant strains. The number of cells analyzed is noted in Table EV2. Pheromone sensitivity halo assay with PGAL1-3HA-CLB5 sic1Δ strains carrying either wild-type FAR1, far1Δ, far1(L135A), or far1(RxL). Different concentrations of α-factor were pipetted on the paper disks. On glucose plates, α-factor triggers cell cycle arrest. CLB5 overexpression from PGAL1 in sic1Δ strain causes lethality presumably by inhibition of replication licensing, which is rescued by the presence of α-factor, which leads to Far1-dependent inhibition of excess Clb5-Cdk1 activity. The experiment was repeated twice, a representative example is shown. Source data are available online for this figure. Source Data for Figure 3 [embj2020105839-sup-0008-SDataFig3.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Cyclin hp-dependency and S-CDK specificity of the NLxxxL motif Coomassie-stained SDS–PAGE gels from kinase assays presented in Fig 2A. Phosphorylation of histone H1, Far1(1–150), and Far1(1–150 mut) by Cln1-, Clb6-, Clb4-, Clb1-, and Clb5-Cdk1 in vitro. In Far1(1–150 mut), the NLxxxL motif is mutated. The relative phosphorylation rate of wild-type Far1(1–150) compared to NLxxxL motif mutant Far1(1–150) by the indicated cyclin-Cdk1 complex. The data are mean from at least two experiments, and the error bars show standard deviation. Individual experiment data points are also shown. Co-immunoprecipitation of either wild-type or hpm Clb5 using either wild-type or NLxxxL motif mutant Far1(85–150) as bait. The experiment was repeated twice, a representative example is shown. Alignment of the competitor peptides used in Fig EV2F. The peptides were fused via ELGGGGG linker to GB1 domain. In vitro phosphorylation experiment showing the phosphorylation of Far1(1–150) with either NLxxxL (WT) or RxL motif (RxL) by Clb5-Cdk1 in the presence of 210 µM competitor peptides with the indicated motifs. The experiment was repeated twice, a representative example is shown. Sequences of the competitor peptides are shown in Fig EV2E. The indicated Far1(85–150) variant was phosphorylated with Clb5-Cdk1, and the multisite phosphorylation was studied using Phos-tag SDS–PAGE autoradiography. The arrow points to the product with phosphorylation at both S87 and S91. Source data are available online for this figure. Download figure Download PowerPoint To confirm that the effects of NLxxxL motif mutation observed in microscopy (Fig 1F) were indeed due to the hp-mediated docking of the substrate to Clb5 and Clb6, and not due to changes in counteracting phosphatase specificity in the case of the mutated NLxxxL motif, we performed a microscopy experiment with strains where S-phase cyclins were replaced with versions containing mutated docking pockets, the Clb5(hpm) and Clb6(hpm) (Fig 2B). As the degradation curves for wild-type and NLxxxL mutant sensor in clb5(hpm) clb6(hpm) strain were nearly identical, we can conclude that phosphatase specificity toward NLxxxL mutant was not changed. This also provides in vivo evidence for the Clb5/6-specificity of the NLxxxL motif. We have previously shown that conventional RxLxF cyclin docking motifs usually enhance the specificity of targets via binding into the cyclin hp pockets of different cyclins, showing a trend of compensation for the gradually weakening specificity toward the histone H1-based consensus phosphorylation peptide (kcat/KM: Clb2>Clb3>Clb5), such that the docking effect is strong for Clb5, intermediate for Clb3, and mild for Clb2 (Kõivomägi et al, 2011a). More recently, we have shown that the hp pockets of different Clbs recognize distinct non-RxL motifs, such as LxF for Clb2 and PxxPxF for Clb3 (Örd et al, 2020). In this respect, the S-CDK-specific motif described here falls into the latter category, being specific for only a particular cyclin despite using the hydrophobic docking pocket conserved in multiple Clb cyclins. Substitution of the NLxxxL motif with an RxL results in partial loss of function Having found that NLxxxL is an hp-binding motif like RxL, we asked if the two motifs have any functional differences other than the S-CDK specificity of NLxxxL. For this, we replaced the NLxxxL motif in the Far1(1-150)-GFP sensor with an RxL motif from Sic1 (Fig 3A and B, and Appendix Fig S1A). This substitution caused a minor 3-min delay in degradation, whereas a sensor with a mutated RxL motif showed a 30-min delay (Fig 3C, Appendix Fig S1B), comparable to mutations in the NLxxxL determinants (Fig 1G). To directly analyze the phosphorylation kinetics of Far1(1–150) with the different docking motifs, we performed Michaelis–Menten steady-state kinetic analysis with Clb5-Cdk1. The KM value for Far1(1–150) with the NLxxxL motif was around 1.5 µM, and substitution of NLxxxL with RxL increased KM to 10 µM (Fig 3D). This results in around 13-fold higher specificity (ratio of kcat/KM values) of the substrate with NLxxxL compared to the one with RxL. Interestingly, however, this large difference in specificity manifests only in a minor difference in degradation timing (Fig 3C). This result implies that early accumulation of CDK activity around G1/S has very high fold change, as the activity of Clb5-Cdk1 seems to increase by roughly 10-fold in the 3-min period around G1/S. This suggests that extensive changes in specificity are needed to precisely order phosphorylation events at the onset of S-phase due to the drastic increase in Cdk1 activity mediated by the activation of S-Cdk1 (Örd et al, 2019b). Further, mutation of Clb5 hp caused a considerable delay in Far1(1–150 RxL)-GFP degradation (Fig 3E, Appendix Fig S1C). Far1(1-150 WT)-GFP with NLxxxL motif, however, was not delayed by clb5(hpm) mutation, indicating that specific docking of Clb6-Cdk1 fulfilled the minimal CDK threshold for phosphorylation of the Far1(1–150 WT) (Fig 3E, Appendix Fig S1C). To further analyze the functional differences of the two motifs, we tested the importance of the docking on the double-negative feedback loop between Far1 and Clb5-Cdk1, by using a halo assay for pheromone sensitivity combined with overexpression of CLB5. Overexpression of CLB5 causes lethality in sic1Δ background (Jacobson et al, 2000), presumably due to inhibition of replication origin licensing by Clb5-Cdk1 activity (Lengronne & Schwob, 2002). Activation of Far1 inhibitory activity toward CDK by pheromone, however, rescues the effect of CLB5 overexpression in sic1Δ cells, presumably by inhibition of the excess Clb5-Cdk1 activity, thus enabling the cells to grow only in the presence of pheromone (Fig 3F). Disruption of the NLxxxL docking by mutation of N130A, L131A, or L135A decreases the ability of the CLB5 overexpressing cells to grow in the presence of pheromone, as the cells barely grow only in a specific concentration range of diffusing pheromone (Fig 3F, Appendix Fig S1D). This could be because of the inability of Clb5-Cdk1 to degrade Far1 in these cells. Furthermore, substitution of the NLxxxL motif with RxL motif results in a similar phenotype as L135A mutation, showing that RxL cannot substitute for NLxxxL motif in this case. The contribution of Cks1 to the degron phosphorylation depends on the cyclin docking motif In our previous studies of multisite phosphorylation of a number of key Cdk1 targets (Sic1, Cdc6, Swe1, Ndd1, etc.), we have described a general mechanism of Cks1-mediated N-to-C-terminally directed sequential phosphorylation that can have varying degrees of processivity (Kõivomägi et al, 2011b, 2013; McGrath et al, 2013; Örd et al, 2019a). To study how Cks1 docking affected phosphorylation of Far1(1–150), we mutated the

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