Article PP2ACdc55 Phosphatase Imposes Ordered Cell-Cycle Phosphorylation by Opposing Threonine Phosphorylation Graphical Abstract Authors Molly Godfrey, Sandra A. Touati, Meghna Kataria, Andrew Jones, Ambrosius P. Snijders, Frank Uhlmann Correspondence [email protected] In Brief The eukaryotic cell cycle comprises a robustly ordered series of phosphorylation events. Godfrey et al. show that the phosphatase PP2ACdc55 counteracts and thereby delays cell-cycle phosphorylation by cyclin-dependent kinase specifically on threonine, but not serine, phosphorylation sites. This reveals an ordering principle that might be also relevant in other signaling networks. Highlights d The phosphatase PP2ACdc55 counteracts Cdk phosphorylation during the cell cycle d It does so specifically on threonine, but not serine, phosphorylation sites d Consequently, threonine phosphorylation is a late event in the cell cycle d Thereby, PP2ACdc55 contributes to setting the timing of mitosis Godfrey et al., 2017, Molecular Cell 65, 393–402 February 2, 2017 ª 2016 The Author(s). Published by Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2016.12.018 Molecular Cell Article PP2ACdc55 Phosphatase Imposes Ordered Cell-Cycle Phosphorylation by Opposing Threonine Phosphorylation Molly Godfrey,1,3 Sandra A. Touati,1 Meghna Kataria,1 Andrew Jones,2 Ambrosius P. Snijders,2 and Frank Uhlmann1,4,* 1Chromosome Segregation Laboratory 2Mass Spectrometry Proteomics Science Technology Platform The Francis Crick Institute, London NW1 1AT, UK 3Present address: Developmental Biology Division, Department of Biology, Utrecht University, 3584 CH Utrecht, the Netherlands 4Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.12.018 SUMMARY before mitosis, is not well understood. Sequential ordering of phosphorylation events is in part thought to arise from In the quantitative model of cell-cycle control, pro- transcriptional waves of G1-, S phase-, and mitosis-specific gression from G1 through S phase and into mitosis cyclins that confer substrate specificity to the kinase. For is ordered by thresholds of increasing cyclin-depen- example, G1- and S phase-specific cyclins recognize hydro- dent kinase (Cdk) activity. How such thresholds phobic substrate motifs that increase kinase-substrate affinity are read out by substrates that respond with the and thereby enhance the phosphorylation rate of certain correct phosphorylation timing is not known. Here, substrates (Bhaduri and Pryciak, 2011; Brown et al., 1999; Ko˜ i- vomagi€ et al., 2011). using the budding yeast model, we show that Cdc55 While cyclin specificity provides an apparently straightforward the abundant PP2A phosphatase counteracts ordering principle, cell-cycle progression is remarkably resilient Cdk phosphorylation during interphase and delays to its abolition. For example, the ordering of S phase and mitosis Cdc55 phosphorylation of late Cdk substrates. PP2A remains uncompromised if S phase cyclins are replaced by specifically counteracts phosphorylation on threo- mitotic cyclins in both budding yeast and vertebrates (Hu and nine residues, and consequently, we find that threo- Aparicio, 2005; Moore et al., 2003). In another example, all nine-directed phosphorylation occurs late in the cell budding yeast G1-specific cyclins can be removed so long as cycle. Furthermore, the late phosphorylation of a their role in upregulating S phase cyclin expression is compen- model substrate, Ndd1, depends on threonine sated for (Tyers, 1996). What is more, ordered cell-cycle pro- identity of its Cdk target sites. Our results support a gression is maintained with a single source of cyclin-Cdk activity model in which Cdk-counteracting phosphatases in fission yeast (Coudreuse and Nurse, 2010; Fisher and Nurse, 1996). This has led to the proposal that the quantitative increase, contribute to cell-cycle ordering by imposing Cdk rather than qualitative differences, in Cdk activity controls or- thresholds. They also unveil a regulatory principle dered Cdk substrate phosphorylation (Stern and Nurse, 1996). based on the phosphoacceptor amino acid, which How such a quantitative increase of Cdk activity is read out by is likely to apply to signaling pathways beyond cell- substrates that respond by phosphorylation at the correct time cycle control. is not known. In budding yeast, Cdk phosphorylates its many substrates with widely varying efficiencies (Loog and Morgan, 2005), but there is no obvious correlation between phosphoryla- INTRODUCTION tion efficiency and phosphorylation timing. Thus, ordered cell- cycle phosphorylation is not simply defined by kinase affinity The cell-division cycle is made up of an ordered series of for its substrates, as the best substrates are not necessarily events that ensure faithful replication and accurate partitioning those that are phosphorylated first. of the genetic material. A major impetus for cell-cycle progres- If Cdk activity is chemically inhibited at various cell-cycle sion stems from the phosphorylation of numerous target pro- stages, Cdk substrate phosphorylation is rapidly lost (Holt teins by the master cyclin-dependent kinase (Cdk) (Morgan, et al., 2009; Liakopoulos et al., 2003). This suggests that 2007). Cdk substrates whose phosphorylation triggers DNA potent Cdk-counteracting phosphatase(s) are active in cells replication (Tanaka et al., 2007; Zegerman and Diffley, 2007), at most times. By reversing phosphorylation, such Cdk-coun- as well as many substrates that are important for entry into teracting phosphatases could set thresholds that Cdk activity and progression through mitosis (Errico et al., 2010; Ubersax must overcome to achieve net substrate phosphorylation (Uhl- et al., 2003), are known, but how the correct substrate phos- mann et al., 2011). Whether and how Cdk-counteracting phos- phorylation timing is defined, such that DNA replication occurs phatases indeed act during interphase to order cell-cycle Molecular Cell 65, 393–402, February 2, 2017 ª 2016 The Author(s). Published by Elsevier Inc. 393 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A Vertebrates Budding yeast Cdc25 ? Mih1 Cdk1- P Cdk1 Cdc28 P Cdc28 PP2AB55 PP2ACdc55 Cyclin B Cyclin B Clb2 Clb2 Wee1 Swe1 ? ? Cell cycle Cell cycle substrate phosphorylation substrate phosphorylation B swe1Δ swe1Δ cdc55Δ swe1Δ swe1Δ cdc55Δ Time after min G1 release % spindles % spindles G1 10 20 30 40 50 60 70 80 90 105 120 G1 10 20 30 40 50 60 70 80 90 105 120 (min) Ask1-HA 4 17 120 Tubulin 12 38 105 24 61 swe1Δ swe1Δ cdc55Δ 90 66 79 80 G1 10 20 30 40 50 60 70 80 90 105 120 G1 10 20 30 40 50 60 70 80 90 105 120 min 90 87 70 Sli15-HA 54 60 60 Tubulin 10 16 50 1 1 swe1Δ swe1Δ cdc55Δ 40 0 2 30 G1 10 20 30 40 50 60 70 80 90 G1 10 20 30 40 50 60 70 80 90 min 1 2 20 Ndd1-HA 0 0 10 0 0 G1 Tubulin 1c 2c 1c 2c Figure 1. PP2ACdc55 Delays Cdk Substrate Phosphorylation Independently of Impacting Cdk Tyrosine Phosphorylation (A) Simplified schematics of how PP2ACdc55 is thought to affect Cdk activity in vertebrates and budding yeast (Chica et al., 2016; Harvey et al., 2011; Kinoshita et al., 1993; Lee et al., 1991; Minshull et al., 1996). See also Figure S1 for a comparison of cell-cycle progression in wild-type and swe1D strains. (B) PP2ACdc55 delays cell-cycle phosphorylation of a subset of Cdk substrates. swe1D and swe1D cdc55D cells were arrested in G1 by pheromone a-factor treatment and released to progress through a synchronous cell cycle before re-arrest in the next G1 phase by a-factor re-addition. Protein extracts were prepared at the indicated times from strains in which Ask1, Sli15, or Ndd1 was fused to a hemagglutinin (HA) epitope tag. Cell-cycle progression was monitored by fluorescence-activated cell sorting (FACS) analysis of DNA content. Shown is a representative profile of the Sli15-HA experiment, as well as representative scores of cells containing mitotic (short or long) spindles. See also Figure S2A for FACS analyses of the other experiments, Figure S2B for a repeat of the Ndd1 phosphorylation analysis, including internal timing controls, and Figure S3 for cell-cycle phosphorylation analysis using antibodies raised against phosphorylated Cdk consensus peptides. phosphorylation is not yet known. It is also unknown how such RESULTS phosphatases might differentiate early from late phosphory- lated Cdk targets to set a higher Cdk threshold for phosphor- PP2ACdc55 Phosphatase Imposes Late Cdk Substrate ylation of the latter. Here, we analyze the contribution of the Phosphorylation budding yeast PP2ACdc55 phosphatase to the dynamics of We analyzed a possible contribution of the abundant PP2ACdc55 cell-cycle phosphorylation. We find that PP2ACdc55 indeed phosphatase to the regulation of Cdk substrate phosphoryla- counteracts and thereby delays Cdk phosphorylation, but tion during the budding yeast cell cycle. PP2ACdc55 and its only on a subset of substrates. These are enriched for threo- orthologs in vertebrates have been studied for their impact on nines as the phosphoacceptor sites, consistent with the cell-cycle progression by controlling inhibitory Cdk tyrosine known biochemical PP2A substrate preference. Our results phosphorylation (Chica et al., 2016; Harvey et al., 2011; Kinosh- portray cell-cycle phosphorylation as a dynamic process in ita et al., 1993; Lee et al., 1991; Minshull et al., 1996)(Figure 1A). which the steady-state level of phosphorylation is under con- Budding yeast cells lacking PP2ACdc55 show gross morpholog- trol of the changing activity ratio of Cdk and its counteracting ical defects and poor growth due to attenuated Cdk activity phosphatases. (Wang and Burke, 1997). To disambiguate a possible role of 394 Molecular Cell 65, 393–402, February 2, 2017 PP2ACdc55 in dephosphorylating Cdk substrates, we therefore G1, S, and G2 phases. Numerous phosphosites adhering to employed a budding yeast strain background lacking the the minimal (pS/pTP) or full (pS/pTPxR/K; Figure S4B) Cdk Cdk tyrosine kinase Swe1 (swe1D). Without inhibitory Cdk consensus sites were among those enriched in the absence of phosphorylation, swe1D cells enter mitosis slightly earlier than PP2ACdc55.