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. Author manuscript; available in PMC: 2015 Feb 17.
Published in final edited form as: Curr Biol. 2014 Feb 6;24(4):428–433. doi: 10.1016/j.cub.2014.01.009

Dueling kinases regulate cell size at division through the SAD kinase Cdr2

Lin Deng 1, Suzanne Baldissard 1, Arminja N Kettenbach 1,2, Scott A Gerber 2, James B Moseley 1
PMCID: PMC4055034  NIHMSID: NIHMS555073  PMID: 24508166

Summary

Cell size control requires mechanisms that integrate cell growth and division. Key to this integration in fission yeast is the SAD family kinase Cdr2, which organizes a set of cortical nodes in the cell middle to promote mitotic entry through Wee1 and Cdk1 [1-3]. Cdr2 is inhibited by a spatial gradient of the DYRK kinase Pom1 emanating from cell tips in a cell size-dependent manner [2, 3], but how the Pom1 gradient inhibits Cdr2 activity during cell growth is unknown. Here, we show that Pom1 acts to prevent activation of Cdr2 kinase activity by the CaMKK Ssp1. We found that Ssp1 activates Cdr2 through phosphorylation of a conserved threonine residue (Thr166) in the activation loop of the Cdr2 N-terminal kinase domain both in vitro and in cells. The levels of this activating phosphorylation increased with cell cycle progression, and genetic epistasis demonstrated that Ssp1 promotes mitotic entry through Cdr2. Intriguingly, Pom1 phosophorylated the C-terminal domain (CTD) of Cdr2, and this modification reduced Cdr2-T166 phosphorylation by Ssp1. These findings show how activation of the conserved mitotic inducer Cdr2 is integrated with an inhibitory spatial gradient to ensure proper cell size control at mitosis.

Results and Discussion

Many cell types delay cell cycle transitions until reaching a critical size threshold, but the mechanisms that sense and transmit these signals are largely unknown. Fission yeast cells have a band of cortical nodes in the cell middle; these nodes contain conserved cell cycle regulators including Wee1 and its inhibitors Cdr1 and Cdr2 [1-3]. These proteins are key components of the system that prevents mitotic entry until cells reach a critical and reproducible size. Prior to mitotic entry, the SAD family kinase Cdr2 organizes node formation and represents the most upstream component of these structures [2]. Thus, Cdr2 regulatory factors might represent input signals for the mitotic size control system [4]. Indeed, the DYRK kinase Pom1 forms a spatial gradient that is proposed to link cell size with mitotic entry through inhibition of Cdr2. This Pom1-Cdr2 system has collectively been referred to as the ‘cell geometry network’ [5, 6]. However, it has been unclear how Pom1 inhibits Cdr2 function in cells, and how this inhibition might relate to the control of cell size at mitosis by other Cdr2 regulatory factors. Here, we address these questions through the identification of a novel activation step for Cdr2 in mitotic entry that is inhibited by Pom1.

Cdr2 is activated through T166 phosphorylation

We identified 16 in vivo phosphorylation sites on Cdr2 as part of a large-scale SILAC-based phosphoproteomic screen for substrates of Pom1 (Figures 1A and Table S1). Briefly, Pom1 kinase activity was inhibited for 15 minutes using an analog-sensitive pom1-as strain. Changes in the phosphoproteome were quantified using phosphopeptide enrichment followed by nanoscale microcapillary LC-MS/MS as previously described (see Experimental Procedures, the full results of this screen will be published as a separate study). The C-terminal tail domain (CTD) of Cdr2 was phosphorylated in a manner that heavily depended on Pom1, suggesting that this cluster of serine residues is the primary site of Pom1-dependent regulation in cells. Consistent with this possibility, purified Pom1 phosphorylated the Cdr2-CTD directly in vitro (Figure S1A). In contrast, we found a single residue (Thr166) in the Cdr2 kinase domain that was more heavily phosphorylated upon inhibition of Pom1 (Table S1). This suggests that Thr166 is modified by a different kinase, and may be antagonized by Pom1. Thr166 was the only phosphorylation site that we found in the Cdr2 kinase domain, and represents a well-conserved feature of the activation loop (T-loop) among AMPK/SNF1-related kinases from yeast through humans (Figure 1B). This T-loop Thr is present in three additional fission yeast members of the AMPK/SNF1 protein kinase family, but not in the Cdr2-related protein kinase Cdr1. T-loop phosphorylation by upstream kinases is required for the catalytic activity of members of this protein kinase family [7-11]. Therefore, we decided to investigate the role of Cdr2-T166 phosphorylation in cells.

Figure 1. Cdr2 promotes mitotic entry through activation of T166 phosphorylation.

Figure 1

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(A) Schematic of Cdr2 structure and phosphorylation map. Pom1-dependent sites are highlighted. KA1: kinase associated-1 domain; CTD: C-terminal tail domain. (B) Sequence alignment of activation loops for Cdr2 and related kinases. The asterisk marks the conserved threonine; identical residues are shown in black. Sc. Saccharomyces cerevisiae; Sp. Schizosaccharomyces pombe; Dm. Drosophila melanogaster; Ce. Caenorhabditis elegans; Hs. Homo sapiens. (C) Length of dividing, septated cells of the indicated strains (mean ± SD; n > 50 for each value). (D) Localization of indicated Cdr2 mutant proteins. Images are inverted maximum projections from deconvolved z-series. Scale bar, 5 μm. (E) Detection of Cdr2-T166 phosphorylation using a phospho-specific α-pT166 antibody. Arrowheads indicate the Cdr2-pT166. Note the doublets in wild type. (F) Synchronization of 6His2HA-cdr2 cells grown in YE4S at 30°C following centrifugal elutriation. Two cell cycles were tracked; the septation index (n>700) and cell size (n>250) were measured on blankophor-stained cells; the dash lines indicate the septation peaks. (G) Western blots of synchronized cells from 120 min to 300 min post elutriation. A wild type sample (no tag) was loaded as control. Total Cdr2 level was blotted by α-HA, and Tubulin (α-TAT1) was used as loading control. (H) Quantification of total Cdr2 and Cdr2-pT166 levels during cell cycle progression in Figure 1H. Values were normalized by TAT1.

To test the physiological role of Cdr2-T166 phosphorylation, we generated a non-phosphorylatable cdr2-T166A mutant. We integrated wild type cdr2+, non-phosphorylatable cdr2-T166A, and kinase-dead cdr2-E177A constructs at the endogenous locus using gene replacement. Fission yeast cells reproducibly enter into mitosis and divide at ~14 μm in length, due in part to regulation of Cdr2. We found that cdr2-T166A cells divided at a larger size than wild type cells, reflecting a delay in mitotic entry. This defect phenocopied the kinase-dead cdr2-E177A mutant (and a separate kinase-dead mutant cdr2-K39R, Figure S1B) but was not as severe as full cdr2Δ mutant (Figure 1C), consistent with the previously demonstrated kinase-independent scaffolding function for Cdr2 [1, 2]. Using integrated GFP tags, we found that both mEGFP-Cdr2-T166A and Cdr2-E177A-mEGFP localized to cortical nodes in the cell middle, similar to wild type mEGFP-Cdr2 (Figure 1D). This contrasts with budding yeast Cdr2-like kinase Hsl1, which requires autophosphorylation for proper localization [12]. Similar to kinase-dead mutations [2], cdr2-T166A did not disrupt localization of downstream node components (Figure S1C). These combined results suggest that Thr166 phosphorylation is required for Cdr2 kinase activity in cells, similar to other members of the AMPK/SNF1 protein kinase family. Loss of kinase activity in the cdr2-T166A or cdr2-E177A mutants does not affect Cdr2 localization but delays mitotic entry leading to increased cell size at division.

Cell cycle oscillation of Cdr2-T166 phosphorylation

To probe the cell cycle timing of Cdr2-T166 phosphorylation, we generated a phospho-specific antibody against this activating modification. In western blots using whole-cell extracts, the α-pT166 antibody detected a double band at the correct molecular weight for Cdr2 in wild type cells but not in cdr2Δ or cdr2-T166A cells (Figure 1E). A size-shifted band was detected with α-pT166 for mEGFP-Cdr2 due to the GFP tag, but not for mEGFP-Cdr2-T166A. Further, we performed in vitro phosphatase assays with immunoprecipitated Cdr2 to confirm the phospho-specificity of α-pT166 (Figure S1D).

Next, we tested the levels of Cdr2-pT166 during cell cycle progression. We synchronized cell cycle progression in cultures of 6His2HA-cdr2 cells by centrifugal elutriation (Figures 1F and S1E). The level of Cdr2 protein was constant throughout the cell cycle, consistent with previous work [13]. In contrast, the level of phosphorylated Cdr2-pT166 was low in small newborn cells, and then increased during G2 as cells grew and approached division (Figure 1G). Cdr2-pT166 levels were highest 20 minutes prior to the peak of septation, which occurred at 240 minutes (Figure 1H). This peak in Cdr2-pT166 coincides with loss of Cdk1-Tyr15 phosphorylation (Figure S1F), which occurs at mitotic entry. We conclude that Cdr2 activation by Thr166 phosphorylation increases as cells grow during G2 phase of the cell cycle and peaks as cells enter mitosis and divide. This suggests the existence of a dynamic system that links this activating modification with the mitotic size control system.

The CaMKK Ssp1 phosphorylates Cdr2-Thr166 to promote mitotic entry

To identify the upstream kinase that activates Cdr2 by phosphorylation of Thr166, we screened a collection of 32 non-essential protein kinase deletion mutants enriched for potential links to cell cycle regulation. We probed whole cell extracts for Cdr2-pT166 and found a single mutant, ssp1Δ, which abolished this modification (Figure 2A). Ssp1 is a conserved CaMKK (Ca2+/calmodulin-dependent protein kinase kinase) that has been suggested to promote mitotic entry through unknown pathways [14, 15]. In diverse cell types and organisms, CaMKK proteins activate AMPK-related kinases through phosphorylation. To test the role of Ssp1 kinase activity, we generated the kinase-dead mutant ssp1-KD with K164A mutation (Figure S2A). In both ssp1Δ and ssp1-KD mutants, Cdr2-pT166 was absent but total protein levels of Cdr2 were unchanged from wild type (Figure 2B). These results indicate that Ssp1 kinase activity is required for phosphorylation of Cdr2-T166 in cells. To determine if Ssp1 directly phosphorylates Cdr2, we purified recombinant full-length Ssp1 and Cdr2 kinase domain (aa. 8-263) from bacteria (Figure S2B), and then performed in vitro kinase assays. We detected Cdr2-pT166 in the presence but not in the absence of Ssp1 (Figure 2C), demonstrating that Ssp1 can directly phosphorylate this residue. Further, the T166A mutation abolished phosphorylation of Cdr2 by Ssp1 in vitro, as detected by 32P incorporation (Figure 2D and S2C). Based on these combined results, we conclude that Ssp1 phosphorylates Cdr2-T166 both in vitro and in cells.

Figure 2. CaMKK Ssp1 promotes mitotic entry by activating Cdr2.

Figure 2

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(A) Ssp1 is required for Cdr2-pT166 in cells. Kinase mutant strains were grown in YE4S, and whole cell lysates were blotted by α-pT166 antibody. Note the absence of Cdr2-pT166 in ssp1Δ mutant. Asterisk marks background band. (B) Cdr2-pT166 is absent in ssp1Δ and ssp1-KD mutants. The indicated whole cell lysates were probed by α-pT166 antibody. α-TAT1 was used as loading control. (C) In vitro kinase assays using purified recombinant GST-Cdr2N (aa. 1-400) and Ssp1 with γ-32P-ATP. Note loss of 32P incorporation for T166A mutant. (D) In vitro kinase assays using purified recombinant Cdr2 kinase domain (aa. 8-263) and full-length Ssp1. (E) Length of dividing, septated cells of the indicated strains (mean ± SD; n > 50 for each value). The p values of two-tailed Student’s t tests were indicated in brackets for specific pairs. (F) Localization of Cdr2 and Cdr2-T166A in ssp1Δ cells. Images are inverted maximum projections from deconvolved z-series. Scale bar, 5 μm.

We next used genetic epistasis to test the functional role of Ssp1 in activating Cdr2 for mitotic entry (Figure 2E). Both ssp1Δ and cdr2-T166A single mutants were elongated at division, consistent with previous work for Ssp1 [14, 15]. These effects were not additive: the double mutant ssp1Δ cdr2-T166A phenocopied the single mutant ssp1Δ. In contrast, the mitotic delay of both ssp1Δ and cdr2-T166A mutants was additive with mutations in Cdc25, which promotes mitotic entry through a separate, parallel pathway [13]. This suggests that Ssp1 promotes mitosis through phosphorylation of Cdr2-T166. To test this possibility further, we generated a phosphomimetic cdr2-T166E mutant, which exhibited a small but reproducible increase in size at division, suggesting that it is not fully functional. Nonetheless, cdr2-T166E partially suppressed the cell size defect of ssp1Δ cells (Figure 2E). Interestingly, the degree of suppression (3 μm) by phosphomimetic cdr2-T166E correlated with the increase in cell size by non-phosphorylatable cdr2-T166A. These combined results indicate that Ssp1 promotes mitotic entry in cells by phosphorylating Cdr2-T166 to ensure proper cell size control.

Three additional results from our genetic experiments offer insights into the Ssp1-Cdr2 relationship in cells. First, we observed a small additive defect in the cdr2Δ ssp1Δ double mutant (Figure 2E), which contrasts with the non-additive ssp1Δ cdr2-T166A double mutant. Second, ssp1Δ did not impair the localization of Cdr2 or Cdr2-T166A (Figure 2F). These two results suggest that Ssp1 acts through Cdr2 kinase activity independent from Cdr2 scaffolding activity. Finally, the ssp1Δ defects are exacerbated under stress conditions such as high temperature, while cdr2-T166A defects are not (Figure S2D). This indicates that Ssp1 is likely to activate additional mitotic regulators beyond Cdr2 under environmental stress. Ssp1 may link to environmental sensing through its activation of AMPK Ssp2 [11, 16], as well as other potential targets.

Spatial control of the Ssp1-Cdr2 mitotic entry system

We next investigated the spatial requirements for Ssp1-Cdr2 regulation in cells. Cdr2 localizes to cortical nodes in the cell middle [1]; endogenous Ssp1 has not been examined, but plasmid-expressed Ssp1 was present in the cytoplasm of unstressed cells [14]. We integrated a C-terminal mEGFP tag at the endogenous ssp1+ locus, and found that Ssp1-mEGFP localized in a diffuse pattern in the cytoplasm (Figure 3A). Recruitment of Ssp1 to the cell cortex by a lipid-modified CAAX motif did not increase the level of Cdr2-pT166 (Figures 3A and 3B), suggesting that cytoplasmic Ssp1 performs this modification.

Figure 3. Membrane localization is not required for Cdr2 activation by Ssp1.

Figure 3

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(A) Localization of Ssp1-mEGFP and Ssp1-mEGFP-CAAX. Images are inverted, single focal planes. Scale bar, 5 μm. (B) Western blots for Cdr2-pT166 for whole cell lysates from indicated strains. Panel on the right shows quantification of pT166 levels from three independent experiments with values normalized to wild type. The error bar indicates standard deviation. (C) Localization of mEGFP-tagged Cdr2 mutant proteins. Images are inverted, single focal planes. Scale bar, 5 μm. (D) Detection of Cdr2-pT166 in whole cell lysates from Cdr2 mutants. The asterisk denotes background bands. Panel on the right shows quantification of pT166 levels in three independent experiments with values normalized to wild type. The error bar indicates standard deviation. (E) Localization of Ssp1-mEGFP and Cdr2-GBP-mCherry in the same strain. GBP: GFP binding peptide. (F) Detection of Cdr2-pT166 in the indicated strains. In Figures 3B and 3F, α-TAT1 was used as loading control.

We tested the role of Cdr2 localization to cortical nodes by using a truncated cdr2(1-600) construct that lacks the membrane-targeting KA1 domain (kinase associated-1 domain [17]) and the CTD. Cdr2(1-600)-mEGFP was absent from cortical nodes, and instead localized to the cytoplasm and nucleus. This contrasts with full-length Cdr2-mEGFP and mEGFP-Cdr2-T166A, which were present at cortical nodes and in the cytoplasm (Figure 3C). Despite its removal from the cell cortex, Cdr2(1-600)-mEGFP was still phosphorylated at Thr166, though at slightly reduced levels (Figure 3D). We note that loss of Cdr2-CTD phosphorylation by Pom1 could also affect pT166 levels in the Cdr2(1-600) construct. These results suggest a location-independent activation event, similar to the mechanism proposed for T-loop activation of the related protein kinase Kin4 in budding yeast [7]. We conclude that Cdr2 cortical localization is not required for activation by Ssp1. Rather our results indicate that this modification likely occurs in the cytoplasm, with the potential to activate Cdr2 for additional layers of regulation.

As a final test of these spatial requirements, we forced the interaction of Ssp1 and Cdr2 using the GBP (GFP-binding peptide) system [4, 18, 19]. Ssp1 was recruited to cortical nodes in cdr2-GBP-mCherry ssp1-mEGFP cells, leading to increased levels of Cdr2-pT166 (Figures 3E and 3F). This suggests that, in the absence of a forced interaction, cellular mechanisms exist to limit Cdr2 activation by Ssp1. Such a mechanism may also be linked to the increase of Cdr2-pT166 levels as cells progress through the cell cycle.

Phosphorylation of Cdr2 tail by Pom1 inhibits Cdr2 activation by Ssp1

Our results suggest that Ssp1 progressively activates Cdr2 as cells increase in size and approach mitosis and division. This contrasts with the mitotic inhibitor Pom1, which forms a spatial gradient that is proposed to inhibit Cdr2 in small cells but not in large cells. Pom1 can phosphorylate Cdr2 both in vitro and in cells [3], but the mechanisms that link this modification with mitotic entry are unknown. To probe the connection between Pom1 and Ssp1 in regulating Cdr2, we used α-pT166 to test a panel of cell polarity mutants including pom1Δ. In pom1Δ samples, the double Cdr2-pT166 band was collapsed to a single band, consistent with an upper Pom1-dependent phospho-isoform (Figures 2A and S3A). The upper band also depended on Tea4 (Figure S3A) which is required for formation of the Pom1 cortical gradient [20]. More importantly, we consistently found increased signal for Cdr2-pT166 in pom1Δ versus wild type cell extracts. Using epitope-tagged Cdr2 to normalize for total Cdr2 protein levels, we measured a three-fold increase in the level of Cdr2-pT166 in pom1Δ versus wild type cells (Figures 4A and 4B). This suggests the possibility that phosphorylation of the Cdr2 C-terminal tail domain by Pom1 inhibits the activating pT166 modification in the Cdr2 kinase domain.

Figure 4. Phopshorylation of Cdr2 C-terminal tail by Pom1 inhibits Cdr2 activation by Ssp1.

Figure 4

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(A) Detection of Cdr2-pT166 in the indicated strains. (B) Quantification of Cdr2-pT166 levels for strains in panel A. Background was subtracted from non-tagged strain and values were normalized to wild type. Error bars show standard deviation in three biological repeats. (C) Length of dividing, septated cells of the indicated strains (mean ± SD; n > 50 for each value). The p values of two-tailed Student’s t tests were indicated in brackets for interested pairs. (D) Schematic of genetic pathway for Ssp1 and Pom1. (E) Model for cell-size dependent regulation of Cdr2-pT166 through activating cytoplasmic Ssp1 and inhibitory Pom1 gradient.

We tested this possibility further by mutating Pom1-dependent phosphorylation sites in the Cdr2 CTD. We first generated a cdr2-5A construct by mutating four Pom1 sites (S758, S760, S761 and S762), and also a nearby site (S755) that matches our Pom1 consensus (RXXpS). In addition, we deleted these sites by generating a truncated cdr2ΔCTD construct. Similar to pom1Δ, these mutations increased the levels of Cdr2-pT166 at least three-fold over wild type (Figures 4A and 4B). We conclude that Pom1 prevents mitotic entry at least in part by inhibiting the levels of activated Cdr2-pT166. If Pom1 acts through Cdr2-pT166, then Pom1 effects on cell size at division should require this modification. Indeed, pom1Δ cells exhibit a semi-wee size defect, but we found that pom1Δ had no effect on cell size when combined with cdr2-T166A. Similarly, pom1Δ did not reduce the division size of ssp1Δ cells (Figure 4C), which lack phosphorylation of Cdr2-T166. These effects are not due to changes in protein localization, as no localization dependency was observed between Pom1 and Ssp1 (Figure S3B). Thus, Pom1 prevents the activating Cdr2-pT166 modification by phosphorylating the Cdr2 C-terminus, and Pom1 cannot regulate cell size at division in the absence of Cdr2-pT166 (Figure 4D).

These results indicate that Cdr2 integrates input signals from Pom1 and Ssp1, leading to increased Cdr2-pT166 as cells grow. We propose that Ssp1 activates Cdr2 kinase activity by phosphorylating Cdr2-T166 to promote mitotic entry. In contrast, Pom1 phosphorylates the Cdr2 CTD, and this modification reduces phosphorylation of Cdr2-T166 in the kinase domain. These findings lead to a model where the level of Cdr2-pT166 is low in small G2 cells due to overlap between Cdr2 nodes and the Pom1 polar gradient. As cells grow during G2, reduced overlap between Pom1 and Cdr2 leads to increased levels of Cdr2-pT166 and higher mitotic potential (Figure 4E). An important next step will be to determine how phosphorylation of the Cdr2 CTD by Pom1 inhibits phosphorylation at T166. Phosphorylation by Pom1 might generate a docking site for phosphatases to reverse Cdr2-T166 phosphorylation. We also note that the CTD of several Cdr2-related proteins inhibits kinase activity [21-24], raising the possibility of Pom1-dependent autoinhibition. It is also possible that phosphorylation of Cdr2-CTD by Pom1 regulates binding by Ssp1. These models are not mutually exclusive and indicate that additional proteins are likely to contribute to this regulatory cascade.

Our data identify the conserved kinases Pom1 and Ssp1 as master regulators in the coordination of cell growth and division. The cytosolic localization of CaMKK Ssp1 is consistent with a role in activating downstream targets for additional layers of regulation. In contrast, the spatial gradient of Pom1 can tune the activity of downstream targets with changes in cell growth and shape. These signals converge on a single target in the SAD kinase Cdr2. This mechanism bears striking similarity to the regulation of SAD kinases in axonal arbor formation in mammals [25]. In these neurons, Cdk5 phosphorylates the CTD of SAD-A and SAD-B to inhibit activation by T-loop phosphorylation, although the activating kinase in these neurons is unknown. Thus, CTD phosphorylation prevents T-loop activation of SAD kinases in diverse biological systems such as neuronal morphogenesis and the yeast cell cycle. We anticipate that future work will uncover additional inputs and mechanisms that are integrated by this conserved class of signaling molecules.

Supplementary Material

01

Highlights.

  • CaMKK Ssp1 phosphorylates Cdr2 at Thr166 to promote mitotic entry

  • Cdr2-pT166 levels increase during cell cycle progression

  • DYRK kinase Pom1 phosphorylates Cdr2 C-terminal tail to prevent activation by Ssp1

  • Pom1 acts through Cdr2-pT166 to regulate cell size at mitosis

Acknowledgements

We thank members of the Moseley laboratory and the Biochemistry Department for discussions; Larry Myers and Youjun Wu for technical assistance; and Bill Wickner, Charlie Barlowe, and Dean Madden for reagents and shared instruments. We also thank Paul Nurse, Pilar Perez, Kenneth Sawin and Fred Chang for strains; Amy Gladfelter, Harry Higgs, and Duane Compton for discussion and comments on the manuscript. This work was supported by National Institutes of Health grants R01-GM099774 (J.B.M.) and R01-CA155260 (S.A.G.). J.B.M. is a Pew Scholar in the Biomedical Sciences.

Footnotes

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