Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division

Report

Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division Highlights d

Ssp1 phosphorylates Kin1 activation loop to promote cell polarity

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Phosphoproteomic screen identifies substrates of MARK/ PAR-1-related kinase Kin1

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Authors Mid Eum Lee, Scott F. Rusin, Nicole Jenkins, Arminja N. Kettenbach, James B. Moseley

Correspondence

Interdependent regulation of Kin1 and its substrate Pal1 for cell polarity

[email protected]

Polarity kinases Kin1 and Pom1 have overlapping substrates in polarity and division

Functional connections between protein kinases in complex signaling networks remain poorly understood. Lee et al. report that cell polarity and cytokinesis require two kinase signaling pathways (Ssp1/Kin1 and Pom1) that converge by phosphorylating distinct sites on shared substrates in fission yeast.

Lee et al., 2018, Current Biology 28, 1–9 January 8, 2018 ª 2017 Elsevier Ltd. https://doi.org/10.1016/j.cub.2017.11.034

In Brief

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

Current Biology

Report Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division Mid Eum Lee,1 Scott F. Rusin,1 Nicole Jenkins,1,2 Arminja N. Kettenbach,1,2 and James B. Moseley1,3,* 1Department

of Biochemistry and Cell Biology, The Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA Cotton Cancer Center, The Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA 3Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.cub.2017.11.034 2Norris

SUMMARY

Connections between the protein kinases that function within complex cell polarity networks are poorly understood. Rod-shaped fission yeast cells grow in a highly polarized manner, and genetic screens have identified many protein kinases, including the CaMKK-like Ssp1 and the MARK/PAR-1 family kinase Kin1, that are required for polarized growth and cell shape, but their functional mechanisms and connections have been unknown [1–5]. We found that Ssp1 promotes cell polarity by phosphorylating the activation loop of Kin1. Kin1 regulates cell polarity and cytokinesis through unknown mechanisms [4–7]. We performed a large-scale phosphoproteomic screen and found that Kin1 phosphorylates itself and Pal1 to promote growth at cell tips, and these proteins are interdependent for localization to growing cell tips. Additional Kin1 substrates for cell polarity and cytokinesis (Tea4, Mod5, Cdc15, and Cyk3) were also phosphorylated by a second kinase, the DYRK family member Pom1 [8]. Kin1 and Pom1 were enriched at opposite ends of growing cells, and they phosphorylated largely non-overlapping sites on shared substrates. Combined inhibition of both Kin1and Pom1 led to synthetic defects in their shared substrates Cdc15 and Cyk3, confirming a non-redundant functional connection through shared substrates. These findings uncover a new Ssp1-Kin1 signaling pathway, and define its functional and mechanistic connection with Pom1 signaling for cell polarity and cytokinesis. These kinases are conserved in many eukaryotes including humans, suggesting that similar connections and mechanisms might operate in a broad range of cells. RESULTS AND DISCUSSION Mutations in the fission yeast CaMKK-like protein kinase Ssp1 generate defects in cell-cycle progression, nutrient sensing, and cell polarity [9–11]. Ssp1 directly phosphorylates the activa-

tion loops of the cell-cycle kinase Cdr2 and the metabolic sensor kinase Ssp2 [12, 13], but Ssp1 substrates in cell polarity have been undefined. The activation loop of fission yeast Kin1 is nearly identical both to its MARK/PAR-1 orthologs and to Cdr2 and Ssp2 (Figure 1A). Thus, we hypothesized that Ssp1 might regulate cell polarity by phosphorylating this conserved threonine (T299) within the Kin1 activation loop. We used BiFC (bimolecular fluorescence complementation) as a first test because this assay has the potential to trap transient cellular interactions, such as between a kinase and its substrate. Ssp1 localizes primarily in the cytoplasm [9, 10, 14], and Kin1 localizes to growing cell ends [4, 6]. In BiFC assays, we observed fluorescence at the ends of cells expressing Ssp1-VC and Kin1-VN, but not in negative controls (Figure S1A). Therefore, we raised a phospho-specific antibody against Kin1-pT299. Kin1 was phosphorylated at T299 in wild-type cells, but not in ssp1D cells or a non-phosphorylatable kin1-T299A mutant (Figures 1B, S1B, and S1C). To test direct phosphorylation, we performed in vitro thiophosphate kinase assays using purified analog-sensitive Ssp1-as1 and immunoprecipitated Kin1. Analog-sensitive kinase alleles can use a bio-orthogonal ATPgS molecule in which the g-phosphate is replaced by a thiophosphate moiety [15, 16]. Only direct substrates become thiophosphorylated, and this modification can then be alkylated for detection by a specific anti-thiophosphate ester antibody. Using this assay, we found that Ssp1-as1 directly phosphorylates Kin1 in vitro (Figure 1C). We conclude that Ssp1 phosphorylates the Kin1 activation loop, and this modification might explain the function of Ssp1 in cell polarity. If Ssp1 promotes cell polarity by phosphorylating Kin1-T299, then a non-phosphorylatable kin1-T299A mutant should exhibit defects in cell polarity. kin1-T299A mutant cells displayed increased monopolar growth (Figures 1D and 1E), indicating a defect in the switch to bipolar growth. cdc25-22 mutant cells arrest at 36 in G2 with a bipolar growth pattern [17], as visualized by actin and cell wall staining (Figures 1F–1H, S1D, and S1E). However, kin1-T299A cdc25-22 mutants exhibited monopolar growth with displaced actin patches and cell wall deposition (Figures 1F–1H, S1D, and S1E). Finally, previous work identified synthetic lethality between kin1D and pom1D, a different cell polarity kinase [7]. kin1-T299A pom1D double-mutant cells displayed severe synthetic defects in growth and morphology (Figures S1F and S1G). These results show that phosphorylation of Kin1-T299 by Ssp1 is required for proper cell polarity. Current Biology 28, 1–9, January 8, 2018 ª 2017 Elsevier Ltd. 1

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

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Figure 1. Ssp1 Promotes Cell Polarity by Phosphorylating the Activation Loop of Kin1 (A) Sequence alignment of activation loops from the indicated MARK/PAR-1 and AMPK-related kinases. Black letters represent invariant residues; asterisk denotes phosphorylated threonine. (B) Kin1-pT299 is absent in ssp1D mutant. Whole-cell extracts from the indicated strains were separated by SDS-PAGE and probed by western blot with the indicated antibodies. Asterisk denotes background band. (C) In vitro thiophosphate kinase assay showing direct phosphorylation of Kin1 by Ssp1-as1. Ssp1-as1 was purified from bacteria; kin1-mEGFP was immunoprecipitated from kin1-mEGFP ssp1D cells. Phosphorylation is detected by anti-thiophosphate ester antibody (a-ThioP). (D) Actin staining of wild-type and kin1-T299A mutant. F-actin was visualized with Alexa Fluor 488 phalloidin staining. Maximum projection images are shown. Scale bar, 5 mm. (E) Quantification of polarity patterns from actin staining of kin1+ and kin1(T299A) strains. Values are the mean ± SD from three independent experiments (n > 150 cells each). **p < 10 2. (F) Actin staining of cdc25-22 or kin1-T299A cdc25-22 cells grown to log phase at 25 C and then shifted to 36 C for 4 hr. Cells were fixed and stained with Alexa Fluor 488 phalloidin before imaging. Brackets indicate medial 10-mm section used for quantifying actin patch numbers. Scale bar, 5 mm. (G) Quantification of polarity patterns from actin staining of cdc25-22 and kin1-T299A cdc25-22 mutant arrested at 36 C for 4 hr. Values are the mean ± SD from three independent experiments (n > 150 cells each). (H) Quantification of F-actin patches within 10-mm medial region of cells from (F). Values are the mean ± SD from ten cells. ***p < 10 5. See also Figure S1.

The phenotypes of kin1-T299A cells are less severe than kin1D or kinase-dead kin1 mutations. Rather, the kin1-T299A phenotype is reminiscent of ssp1 mutant cells, which have defects in bipolar growth, but not severe morphology defects [9, 10]. These results indicate that Kin1 retains some kinase activity in the absence of activation by Ssp1, unlike the related Ssp1 sub2 Current Biology 28, 1–9, January 8, 2018

strates Cdr2 and Ssp2 [12, 13]. The pleiotropic defects of ssp1 mutants can be explained by these three related substrates, with Kin1 as the key target for cell polarity. However, no substrates of Kin1 are known, raising the question of how Kin1 converts upstream activation by Ssp1 into downstream cell polarity cues.

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

Identification of Kin1 Substrates by Phosphoproteomics To understand how Kin1 regulates cell polarity, we performed an unbiased phosphoproteomic screen. We generated an analogsensitive mutant of Kin1 (F220G) (kin1-as1) based on previous work [18]. kin1-as1 cells phenocopied kin1D cells when grown in the presence of 1-(tert-Butyl)-3-(3-methylbenzyl)-1Hpyrazolo[3,4-d]pyrimidine-4-amine (3-MB-PP1) inhibitor, but not in DMSO (Figure 2A). Kin1 kinase activity is required for its localization to cell tips [18]. Using Kin1 tip localization and SDS-PAGE mobility shifts as a readout for kinase activity, we found that 1 mM 3-MB-PP1 inhibited kin1-as1 activity within 5–15 min (Figures 2B and S2A). We integrated Kin1 inhibition into a pipeline for quantitative phosphoproteomics (see Method Details). In four separate experiments, kin1-as1 cultures were treated with either 3-MBPP1 inhibitor or DMSO control, and then cells were harvested and lysed. We used heavy/light dimethyl labeling with liquid chromatography and mass spectrometry to compare the abundance of tryptic phosphopeptides isolated from 3-MB-PP1 versus DMSO treatment. We identified more than 1,400 phosphopeptides that were >2-fold reduced by Kin1-specific inhibition (Table S1). Gene Ontology (GO) analysis showed that potential Kin1 substrates were enriched for factors that function in polarity establishment and localize at cell tips (Figure S2B). Critical proteins include Kin1 itself, cell polarity proteins (Pal1, Mod5, Tea4, and Spa2), cell division proteins (Cyk3, Cdc15, and Rng10), a cell wall enzyme (Bgs1), and Rho GTPase regulators (Rga2 and Rgf1) (Table S2). We conclude that our screen identified candidate substrates that might be phosphorylated by Kin1, after its activation by Ssp1, to regulate cell polarity and division. Kin1 Autophosphorylation Is Required for Localization and Function Kin1 activity is required for its localization and phosphorylationdependent SDS-PAGE band shift, raising the possibility of autophosphorylation (Figures 2B, S2A, and S2G) [18]. We identified 19 phosphorylation sites on Kin1 that were >1.4-fold reduced upon inhibition. These sites clustered in an uncharacterized central domain (Figure 2C). Immunoprecipitated Kin1-as1-mEGFP autophosphorylated when we performed in vitro thiophosphate kinase assays (Figure 2D). Kin1 autophosphorylation in cells requires its activation by Ssp1, as shown by SDS-PAGE mobility shifts in kin1-T299A and ssp1D mutants (Figures S2C and S2D). To test the function of Kin1 autophosphorylation, we mutated 16 autophosphorylation sites to alanine. Three results showed that kin1(16A) eliminated most, but not all, autophosphorylation sites. First, the kin1-as1(16A) mutation decreased in vitro autophosphorylation 6-fold (Figure S2E). Second, kin1(16A)-mEGFP migrated faster than wild-type Kin1-mEGFP by SDS-PAGE (Figure 2E), similar to the inhibited kin1-as1 mutant (Figure 2B). Third, l-phosphatase treatment induced a larger SDS-PAGE band shift for wild-type Kin1 than for kin1(16A) (Figure S2F). Therefore, we used kin1(16A) to test the function of autophosphorylation in cells. kin1(16A) cells displayed cell polarity and septation defects similar to kin1D, and failed to enrich at cell tips even in rod-shaped cells (Figures 2F and S2H). These data indicate that Kin1 autophosphorylation of its central domain is required for localization and function in cells.

Kin1 Directly Phosphorylates Polarity Proteins Pal1, Tea4, and Mod5 We identified Kin1-dependent phosphorylation sites on multiple cell polarity proteins, including Pal1, Tea4, and Mod5 (Figure 2G). Similar to Kin1, these proteins all localize to growing cell ends and the division site, and are required for proper cell polarity and shape [19–22]. To test whether Kin1 directly phosphorylates these substrates, we performed in vitro thiophosphate kinase assays. We expressed and purified full-length Pal1 and fragments of Mod5 and Tea4 that contained Kin1-dependent phosphorylation sites from bacteria [8]. Immunoprecipitated Kin1-as1mEGFP directly thiophosphorylated these proteins, and this activity was inhibited by excess 3-MB-PP1 inhibitor (Figures 2H–2J). Next, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify phosphorylation sites that were phosphorylated by wild-type Kin1, but not by kinasedead Kin1(K154R) [23] (Figure 2G; Table S3). For each protein, we detected overlap between these in vitro phosphorylation sites and the sites that were Kin1 dependent in cells (Figure 2G). Thus, Kin1 directly phosphorylates the cell polarity proteins Pal1, Tea4, and Mod5. Interdependent Localization of Kin1 and Its Substrate Pal1 We investigated the connection between Kin1 and its substrate Pal1, a fungal protein that interacts with endocytic proteins [19]. pal1D and kin1D mutants exhibit similar phenotypic defects in cell shape and septation [4, 19]. In addition, Pal1 localizes to growing cell ends and to the cell middle at division, similar to Kin1 [19, 24]. Pal1-mEGFP localization to cell ends was lost in kin1D mutants (Figure 3A) or upon inhibition of kin1-as1 cells (Figure 3B). Next, we generated a pal1(7A) mutant by mutating seven residues phosphorylated by Kin1 to alanine. The pal1(7A)-mEGFP mutant localized uniformly around the cell cortex (Figure 3C), showing that Kin1 phosphorylates Pal1 to promote its localization to growing cell tips. Further, Pal1 localization at cell tips was reduced in the kin1-T299A mutant (Figure S3A). Thus, Ssp1 phosphorylation of the Kin1 activation loop leads to both Kin1 autophosphorylation and Kin1 phosphorylation of Pal1. These modifications promote localization of Kin1 and Pal1 at the cell tips. Despite its localization defect, the morphology defect of pal1(7A) cells is much less severe than pal1D cells (Figure S3B). kin1(16A) and pal1(7A) mutations did not show synthetic growth defects, consistent with a linear pathway (Figure S3C). To test pal1(7A) function more carefully, we combined it with deletion of tea4, another Kin1 substrate. pal1D and tea4D mutations are synthetically lethal at high temperature [8]. pal1(7A) tea4D double-mutant cells failed to grow at high temperature and exhibited severe morphology defects at permissive temperatures (Figures S3D and S3E). We conclude that phosphorylation by Kin1 is required for the localization and full function of Pal1. This mechanism requires activation of Kin1 by Ssp1, thereby defining a novel Ssp1/Kin1/Pal1 pathway for cell polarity. We next tested reciprocal regulation of Kin1 by Pal1. In pal1D mutant cells, Kin1-mEGFP was dispersed throughout the cytoplasm and cortex (Figure 3D). Thus, Kin1 and Pal1 are interdependent for localization, and Pal1 contributes to recruitment of Current Biology 28, 1–9, January 8, 2018 3

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

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Figure 2. Phosphoproteomic Identification of Kin1 Substrates (A) Images of analog-sensitive kin1-as1 cells treated with DMSO (control) or 2 mM 3-MB-PP1 for 14 hr at 32 C. Cells were stained with blankophor to mark cell walls. kin1D cells are shown on the right for comparison. Scale bars, 5 mm. (B) SDS-PAGE band shifts for kin1-as1-mEGFP when inhibited by 3-MB-PP1 for the indicated time points. Samples are whole-cell extracts. Asterisk denotes background bands. The Kin1 intensity profiles along each lane for 15-min time point are shown on the right. a.u., arbitrary units. (C) Schematic summary of Kin1 structure and phosphorylation sites from our phosphoproteomic screen. Black residues were Kin1 dependent; green residues were unaffected by Kin1 inhibition (see also Table S1).KA1, kinase associated-1 domain. (D) In vitro thiophosphate kinase assay using kin1-as1-mEGFP immunoprecipitated from S. pombe. Phosphorylation is detected by a-ThioP; 3-MB-PP1 inhibits Kin1-as1-mEGFP. (E) SDS-PAGE band shift for kin1(16A)-mEGFP. Samples are whole-cell extracts. The Kin1 intensity profiles along each lane are shown on the right. Note that mutant migrates faster than wild-type. (F) Localization and phenotype of kin1(16A)-mEGFP. Both GFP and differential interference contrast (DIC) images are shown. Wild-type kin1-mEGFP cells are shown on the right for comparison (see also Figure S2H). Scale bar, 5 mm. (G) Schematic summary of Kin1 phosphorylation sites on Pal1, Mod5, and Tea4. Black lines mark in vivo Kin1-dependent phosphorylation sites. Green lines mark sites that were phosphorylated by wild-type Kin1, but not by kinase-dead Kin1 in vitro. Blue lines mark sites mapped by both in vivo and in vitro assays (see also Tables S1 and S3). Protein domains are marked with gray bars for each protein, and the fragments purified for Mod5 and Tea4 are underlined in black. (H–J) In vitro thiophosphate kinase assay using kin1-as1-mEGFP immunoprecipitated from S. pombe. Substrates (H) Pal1, (I) Tea4(113–436), and (J) Mod5(28–495) were purified from bacteria. Phosphorylation is detected by a-ThioP; 3-MB-PP1 inhibits Kin1-as1-mEGFP. See also Figure S2 and Tables S1, S2, and S3.

Kin1 to both the cortex and growing cell tips. The reduction of cortical Kin1-mEGFP in pal1D cells was unexpected because Kin1 has a phospholipid-binding kinase associated-1 (KA1) 4 Current Biology 28, 1–9, January 8, 2018

domain [25], which is predicted to associate with the plasma membrane [18]. However, a kin1(DKA1)-mEGFP mutant had no defects in localization or function (Figures 3E and 3F). This result

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

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Figure 3. Interdependent Localization of Kin1 and Its Substrate Pal1 (A) Localization of pal1-mEGFP in wild-type (kin1+) and kin1D cells. (B) pal1-mEGFP localization in kin1-as1 cells treated with DMSO or 15 mM 3-MB-PP1 for 1 hr before imaging. Graph shows quantification of Pal1 localization defect. Values are the mean ± SD from three independent experiments (n > 100 cells each). (C) Localization of pal1(7A)-mEGFP mutant lacking Kin1-dependent phosphorylation sites. See (A) for localization of wild-type pal1-mEGFP. Graph shows quantification of pal1(7A) localization defect. Values are the mean ± SD from three independent experiments (n > 180 cells each). (legend continued on next page)

Current Biology 28, 1–9, January 8, 2018 5

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

led us to dissect the requirements for Kin1 localization and function through structure-function experiments (Figures 3E and S3F). Consistent with proper localization of kin1(DKA1), the KA1 domain alone (amino acids [aa] 753 to end) only weakly associated with the cortex. We found a critical role for aa 511–752 within the previously uncharacterized middle region of Kin1, which contains the majority of autophosphorylation sites (Figure 2C). Kin1(1–510), which truncates both the KA1 domain and the middle region, was nonfunctional and localized to the cytoplasm (Figure S3F). Further, the Kin1(D511–752) construct that deletes only the internal region abolished cortical localization and function (Figure S3F). Pal1 localization largely mirrored Kin1 localization in these mutants (Figure S3F), and we did not observe synthetic growth defects when kin1(DKA1) or kin1(D511–752) were combined with pal1(7A) (Figures S3G and S3H). We conclude that Kin1 localization and function require the middle region, but not the KA1 domain. Kin1(DKA1) might localize to the cell cortex by interacting with other proteins. We tested the role of Kin1 substrates Pal1, Tea4, and Mod5 (Figures 3G and S3I). pal1D prevented cortical localization of Kin1(DKA1), but not full-length Kin1 (Figure 3D). This result indicates that cortical targeting by the Kin1 KA1 domain is dispensable in the presence of Pal1. The localization of Kin1(DKA1) at the cell tips and cell cortex was also reduced, but not abolished, in the pal1(7A) mutant (Figure 3G). These combined results suggest that a physical interaction between Kin1 and Pal1 reciprocally promotes their localization for proper cell polarity. In addition to our in vitro kinase assays, we used two independent approaches to test the physical interaction of Kin1Pal1. First, Kin1 and Pal1 exhibited strong interaction in vivo by BiFC (Figure 3H). This interaction was not simply due to colocalization at cell ends because Kin1 did not associate by BiFC with Gef1, an unrelated protein at cell ends. Pal1 interacted with Kin1(511–752), but not Kin1(1–510), by BiFC, indicating that the Kin1 middle region is both necessary and sufficient to interact with Pal1 in this assay (Figure 3H). Second, purified GST-Kin1(511–752) associated with Pal1-mEGFP in yeast cell extracts, but GST alone did not (Figures 3I and 3J). Interestingly, the Pal1(7A)-mEGFP mutant retained physical interaction with GST-Kin1(511–752). We conclude that Kin1 localizes to the cortex and cell ends primarily by associating with its substrate Pal1, whereas the KA1 domain can act to promote membrane association. This two-part mechanism is reminiscent of the C. elegans Kin1-related kinase PAR-1, which localizes due to both its membrane-binding KA1 domain and binding to its ligand PAR-2 [26].

Kin1 and Pom1 Phosphorylate Distinct Sites on Shared Substrates We considered how this new pathway (Ssp1/Kin1/Pal1/Tea4/ Mod5) connects with other cell polarity pathways. Several Kin1 substrates were previously shown to be phosphorylated by the cell polarity kinase Pom1 (Figure 4A) [8], and kin1D and pom1D are synthetically lethal [7]. Interestingly, Kin1 and Pom1 phosphorylated largely distinct residues on shared substrates Pal1, Mod5, and Tea4 (Figure S4A). Consistent with distinct phosphorylation sites, Pal1 localization depends on Kin1 (Figure 3), but not on Pom1 [8]. We found that Kin1 and Pom1 are enriched at opposite ends of growing cells (Figures 4B and S4B). Using a kin1-as1 pom1-as1 double-mutant strain, inhibiting both kinases led to T-shaped cells that were not observed in either kin1-as1 or pom1-as1 single mutants (Figures 4C and 4D). Thus, the combined activities of Kin1 and Pom1 kinases at opposite cell ends restrict polarized growth to the cell ends. These kinases phosphorylate largely distinct residues on shared substrates. We also observed defects in division septa upon inhibition of kin1-as1 pom1-as1 cells (Figures 4C and 4E). Our phosphoproteomics identified Kin1-dependent phosphorylation of two cytokinesis proteins, Cyk3 and Cdc15 (Table S2), which physically interact [27]. Cdc15 scaffolds the cytokinetic ring to the plasma membrane, and Cyk3 coordinates septum assembly with ring constriction [28–30]. Previous phosphoproteomics also identified Pom1-dependent phosphorylation of Cyk3 and Cdc15 in cells [8], and Kin1 physically associates with Cdc15 [29]. Pom1 also regulates Cdc15 localization in S. japonicus [31], and has been functionally linked to Cdc15 in S. pombe [32]. In thiophosphate kinase assays, both Kin1-as1-mEGFP and Pom1-as13HA directly thiophosphorylated Cdc15 and Cyk3 (Figure S4C). We performed a second set of in vitro kinase assays and then used LC-MS/MS to map phosphorylation sites. Kin1 and Pom1 both directly phosphorylated Cdc15 and Cyk3, but at largely non-overlapping sites (Figure S4D; Table S3). We conclude that Kin1 and Pom1 phosphorylate distinct sites on shared substrates for both cell polarity and cytokinesis. Finally, we tested how Kin1 and Pom1 cooperate to regulate their shared cytokinesis targets in cells. Cyk3 localizes at cell ends during interphase and then at the cytokinetic ring during division [28]. Upon inhibition of kin1-as1 pom1-as1 doublemutant cells, Cyk3-GFP did not fully relocalize to the cytokinetic ring during division, exhibited by residual signal at cell ends (Figures 4F and 4G). In movies of inhibited cells, Cyk3-GFP remained partially at cell tips during cell division (Figure S4E). Cyk3 also mislocalized during interphase in both kin1-as1 and kin1-as1 pom1-as1 cells upon inhibition (Figures 4F and S4F).

(D) Localization of kin1-mEGFP in pal1D cells. Graph shows quantification of Kin1 localization defect for the indicated strains. Values are the mean ± SD from three independent experiments (n > 100 cells each). (E) Schematic summary of Kin1 structure-function analysis. The conserved KA1 domain is gray, the kinase domain is black, and the critical middle region is yellow. Numbers indicate the amino acids in each truncation mutant. Each construct was assessed for localization to the cell ends, the cell sides, and the division septum. Cell morphology was used to test function. (F) Localization of kin1-mEGFP and kin1(DKA1)-mEGFP in cells. Kin1(DKA1) consists of aa 1–752. See (D) for quantification. (G) Localization of kin1(DKA1)-mEGFP in pal1D cells and pal1(7A) mutant. (H) Quantification of BiFC experiments for the indicated strains. The percentage of cells with YFP fluorescence was quantified, and data show the mean ± SD from three independent experiments (n > 100 cells each). VC, C-terminal half of Venus; VN, N-terminal half of Venus. (I) GST-kin1(511–752) and GST alone expressed and purified from bacteria, as described in STAR Methods. CBB, Coomassie brilliant blue. (J) In vitro binding assay shows Pal1-mEGFP binds to GST-kin1(511–752), but not to GST alone. pal1(7A) mutation does not impair this interaction. Scale bars, 5 mm. See also Figure S3.

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Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

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Figure 4. Kin1 and Pom1 Kinase Share Cell Polarity and Cytokinesis Substrates

(A) Selected substrates of Kin1 and Pom1, identified from independent phosphoproteomic screens. Overlapping substrates are shown in the middle in green. (B) Localization of Kin1 and Pom1 in an interphase kin1-mEGFP pom1-tdTomato cell. Graphs show fluorescence intensity for each protein along the cell length (see also Figure S4B). (C) Phenotypes of kin1-as1 pom1-as1 double mutant treated for 4 hr at 32 C with DMSO control D E C or 10 mM 3-MB-PP1. Cells were stained with blankophor before imaging. Arrowheads indicate T-shaped cells; asterisks indicate aberrant septa. Scale bar, 5 mm. (D) Quantification of T-shaped cells in the indicated strains treated for 4 hr at 32 C with 10 mM 3-MB-PP1. Values are the mean ± SDs from three independent experiments (n > 200 cells each). G F (E) Quantification of septation defects in the indicated strains treated as in (C). Categories of septation defects are shown on the right. Values are the mean ± SD from three independent experiments (n > 100 cells each). (F) Localization of cyk3-GFP in kin1-as1 pom1-as1 mutants after treatment with DMSO or 15 mM 3-MB-PP1 for 1 hr. Arrowheads indicate Cyk3GFP signals at the cell tips with a Cyk3 ring in the I H middle. Scale bar, 5 mm. (G) Quantification of cyk3-GFP localization pattern in dividing cells of the indicated strains, treated as in (F). Values are the mean ± SD from three independent experiments (n > 35 dividing cells each). *p < 0.02; **p < 10 6. (H) Localization of GFP-cdc15 in kin1-as1 pom1-as1 mutants after treatment with DMSO or J 15 mM 3-MB-PP1 for 1 hr. Arrowheads indicate aberrant GFP-cdc15 rings. Scale bar, 5 mm. (I) Left: maximum projection images from deconvolved z series of aberrant GFP-cdc15 ‘‘lasso’’ K rings from two kin1-as1 pom1-as1 cells treated with 15 mM 3-MB-PP1 for 1 hr. Scale bar, 3 mm. Cells are outlined in red. Middle: percentage of cells with GFP-cdc15 lassos from the indicated strains treated with 15 mM 3-MB-PP1 for 1 hr. Values are the mean ± SD from three independent experiments (n > 40 cells each). Right: percentage of cells with GFP-cdc15 off-centered rings from the indicated strains treated with 15 mM 3-MB-PP1 for 1 hr. Values are the mean ± SD from three independent experiments (n > 50 cells). (J) Time-lapse images of GFP-cdc15 and rlc1-mCherry in kin1-as1 pom1-as1 cells treated with 15 mM 3-MB-PP1 treatment for 1 hr before imaging. Time is indicated in minutes. Scale bar, 3 mm. (K) Schematic diagram for convergence of Ssp1/Kin1 pathway and Pom1 pathway in cell polarity and cytokinesis signaling. See also Figure S4 and Table S3.

In addition to Cyk3, organization of Cdc15 at cell division required both protein kinases. We inhibited GFP-cdc15 kin1-as1 pom1-as1 cells with 3-MB-PP1 and observed a strand of GFPCdc15 trailing off the cytokinetic ring, resembling a ‘‘lasso’’ (Figures 4H and 4I). Lassos were not observed in single as mutants or DMSO controls (Figures 4H, 4I, S4G, and S4H). Lasso strands emerged during constrictions of the cytokinetic ring and contained Rlc1, a marker of the actomyosin ring (Figure 4J, lasso observed in seven out of nine cells). Cytokinetic rings were also positioned off-center in inhibited kin1-as1 pom1-as1 double-

mutant cells due to Pom1 [2, 31, 32] (Figure 4I). We conclude that Kin1 works with Pom1 to organize cytokinesis proteins including Cyk3 and Cdc15. Their combined activities relocalize Cyk3 from the cell tips to the cytokinetic ring and generate a coherent cytokinetic ring through regulation of Cdc15. Conclusions We uncovered a multi-step signaling pathway (Ssp1/Kin1/ Pal1/Tea4/Mod5/Cyk3/Cdc15) that intersects with Pom1 as part of a larger cell polarity signaling network (Figure 4K). Ssp1 Current Biology 28, 1–9, January 8, 2018 7

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

promotes bipolar growth by phosphorylating the activation loop of MARK/PAR-1 kinase Kin1, which is the third known substrate of Ssp1. Kin1 and its substrate Pal1 are interdependent for localization and function. Pal1 is one of several proteins also phosphorylated by the DYRK Pom1. Kin1 and Pom1 phosphorylate these shared substrates at largely non-overlapping sites, and inhibition of Kin1 and Pom1 results in synthetic defects in these substrates. The cell polarity program plays a critical role in cell division and cytokinesis [33], but the signaling pathways that distinguish cell polarity during growth versus division are poorly understood. Our work shows that polarity kinases Kin1 and Pom1 promote localization of Cyk3 to the division site and organize Cdc15 in the contractile ring. The functional connection between Kin1 and Pom1 in cell polarity and cytokinesis may represent a general theme for MARK/PAR-1 and DYRK kinases in other organisms. For example, polarity in the one-cell C. elegans embryo requires Kin1-related PAR-1 and Pom1related MBK-2. These two kinases phosphorylate distinct sites on the polarity protein MEX-5, leading to regulation of MEX-5 localization (by PAR-1) [34, 35] and function (by MBK-2) [36]. Large-scale studies examining additional substrates in C. elegans and other systems may reveal more broad overlap between these kinase families, as we have shown in fission yeast. Functional connections between protein kinases in complex signaling networks can be direct, as for Ssp1-Kin1, or indirect, as for the shared substrates of Kin1 and Pom1. These connections are most likely required to coordinate the many events of cell polarity and to ensure robustness and fidelity of the cell polarity control system. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS METHOD DETAILS B Yeast Strains and Growth B Large-Scale Phosphoproteomic Screens B Immunoprecipitation B In Vitro Kinase Assays and Western Blots B In Vitro Binding Assay B Imaging and Data Analysis QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION

was supported by grants from the American Cancer Society (RSG-15-14001-CCG) and NIH (R01 GM099774 to J.B.M.); the V Foundation for Cancer Research (V2016-022) and NIH (R35 GM119455 to A.N.K.); and the NIH (T32 GM008704 to S.F.R.).

AUTHOR CONTRIBUTIONS M.E.L. and J.B.M. conceived the research; S.F.R., N.J., and A.N.K. performed and analyzed mass spectrometry experiments; M.E.L. performed all other experiments; M.E.L. and J.B.M. analyzed the data and wrote the manuscript; all authors edited the manuscript. Received: June 29, 2017 Revised: October 18, 2017 Accepted: November 15, 2017 Published: December 14, 2017 REFERENCES 1. Verde, F., Mata, J., and Nurse, P. (1995). Fission yeast cell morphogenesis: identification of new genes and analysis of their role during the cell cycle. J. Cell Biol. 131, 1529–1538. €hler, J., and Pringle, J.R. (1998). Pom1p, a fission yeast protein kinase 2. Ba that provides positional information for both polarized growth and cytokinesis. Genes Dev. 12, 1356–1370. 3. Koyano, T., Kume, K., Konishi, M., Toda, T., and Hirata, D. (2010). Search for kinases related to transition of growth polarity in fission yeast. Biosci. Biotechnol. Biochem. 74, 1129–1133. 4. Drewes, G., and Nurse, P. (2003). The protein kinase kin1, the fission yeast orthologue of mammalian MARK/PAR-1, localises to new cell ends after mitosis and is important for bipolar growth. FEBS Lett. 554, 45–49. 5. Levin, D.E., and Bishop, J.M. (1990). A putative protein kinase gene (kin1+) is important for growth polarity in Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA 87, 8272–8276. 6. La Carbona, S., Allix, C., Philippe, M., and Le Goff, X. (2004). The protein kinase kin1 is required for cellular symmetry in fission yeast. Biol. Cell 96, 169–179. 7. La Carbona, S., and Le Goff, X. (2006). Spatial regulation of cytokinesis by the Kin1 and Pom1 kinases in fission yeast. Curr. Genet. 50, 377–391. 8. Kettenbach, A.N., Deng, L., Wu, Y., Baldissard, S., Adamo, M.E., Gerber, S.A., and Moseley, J.B. (2015). Quantitative phosphoproteomics reveals pathways for coordination of cell growth and division by the conserved fission yeast kinase pom1. Mol. Cell. Proteomics 14, 1275–1287. 9. Matsusaka, T., Hirata, D., Yanagida, M., and Toda, T. (1995). A novel protein kinase gene ssp1+ is required for alteration of growth polarity and actin localization in fission yeast. EMBO J. 14, 3325–3338. 10. Rupes, I., Jia, Z., and Young, P.G. (1999). Ssp1 promotes actin depolymerization and is involved in stress response and new end take-off control in fission yeast. Mol. Biol. Cell 10, 1495–1510. 11. Hanyu, Y., Imai, K.K., Kawasaki, Y., Nakamura, T., Nakaseko, Y., Nagao, K., Kokubu, A., Ebe, M., Fujisawa, A., Hayashi, T., et al. (2009). Schizosaccharomyces pombe cell division cycle under limited glucose requires Ssp1 kinase, the putative CaMKK, and Sds23, a PP2A-related phosphatase inhibitor. Genes Cells 14, 539–554.

Supplemental Information includes four figures and four tables and can be found with this article online at https://doi.org/10.1016/j.cub.2017.11.034.

12. Deng, L., Baldissard, S., Kettenbach, A.N., Gerber, S.A., and Moseley, J.B. (2014). Dueling kinases regulate cell size at division through the SAD kinase Cdr2. Curr. Biol. 24, 428–433.

ACKNOWLEDGMENTS

13. Deng, L., Lee, M.E., Schutt, K.L., and Moseley, J.B. (2017). Phosphatases generate signal specificity downstream of Ssp1 kinase in fission yeast. Mol. Cell. Biol. 37, e00494-16.

We thank members of the Moseley laboratory for the comments; Charles Barlowe for shared equipment; Erik Griffin for discussion and comments; Jian-Qiu Wu, Fred Chang, and Mohan Balasubramanian for strains; Stuart MacNeill for BiFC plasmids; and William Wickner for pMBP-parallel1 plasmid. This work

8 Current Biology 28, 1–9, January 8, 2018

14. Freitag, S.I., Wong, J., and Young, P.G. (2014). Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast. Open Biol. 4, 130127.

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Current Biology 28, 1–9, January 8, 2018 9

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit polyclonal anti-GFP

[37]

N/A

Mouse monoclonal anti-myc (9E10)

Santa Cruz Biotechnology

Cat #: SC-40; RRID: AB_627268

Mouse monoclonal anti-HA (16B12)

Biolegend

Cat #: 901513; RRID: AB_2565335

Rabbit polyclonal anti-Kin1-pT299 phospho antibody

This study; 21st Century Biochemicals, Inc

N/A

Antibodies

IRDye 680RD Goat anti-Mouse IgG

LI-COR Biosciences

Cat #: 925-68070; RRID: AB_2651128

IRDye800CW Goat anti-Rabbit IgG

LI-COR Biosciences

Cat #: 925-32211; RRID: AB_2651127

Rabbit monoclonal anti-thiophosphate ester

abcam

Cat #: ab92570; RRID: AB_10562142

3-MB-PP1 (1-(tert-Butyl)-3-(3-methylbenzyl)-1Hpyrazolo[3,4-d]pyrimidine-4-amine

Millipore Sigma

Cat #: 529582

iodoacetamide

Sigma-Aldrich

Cat #: I6125

p-nitrobenzyl mesylate (PNBM)

Abcam Biochemicals

Cat #: ab138910

PDX accession PXD006782

http://proteomecentral.proteomexchange.org/ cgi/GetDataset?ID=PXD006782

This study

N/A

Chemicals, Peptides, and Recombinant Proteins

Deposited Data ProteomeXchange Consortium Experimental Models: Organisms/Strains Schizosaccharomyces pombe strains, see Table S4 Recombinant DNA pGEX-6P-1

GE Healthcare

Cat #: 28-9546-48

pQE30

QIAGEN

Cat #: 33203

gBlocks Gene Fragment

Integrated DNA Technologies

N/A

Software and Algorithms SoftWoRx software

Applied Precision

N/A

ImageJ 1.46 r

NIH

https://imagej.nih.gov/ij/

Image Studio Lite version 5.2

LI-COR

https://www.licor.com/bio/products/software/ image_studio_lite/

Other Protease Inhibitor Cocktail Tablets

Roche

Cat #: 11836170001

GFP-nAb Magnetic Beads

Allele

Cat #: ABP-NAB-GFPM025

Pierce Anti-HA Magnetic Beads

ThermoFisher

Cat #: 88837

glutathione-agarose

Sigma-Aldrich

Cat #: G4510

AcTEV Protease

Invitrogen

Cat #: 12575015

Gibson Assembly Cloning Kit

New England Biolabs

Cat #: E5510S

l-phosphatase

New England Biolabs

Cat #: P0753S

Ni-NTA Agarose

QIAGEN

Cat #: 30210

Blankophor

MP Biochemicals

Cat #: 196040

6-Bn-ATP-g-S

Axxora

Cat #: BLG-B072-05

QuikChange II mutagenesis kit

Stratagene

Cat #: 200514

Alexa Fluor 488 Phalloidin

ThermoFisher

Cat #: A12379

16% paraformaldehyde

Fisher scientific

Cat #: 50-980-487

Nitrocellulose membrane

Biorad

Cat #: 162-0115

e1 Current Biology 28, 1–9.e1–e4, January 8, 2018

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, James B. Moseley ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS The fission yeast Schizosaccharomyces pombe strains used in this study are listed in Table S4. For microscopy experiments, cells were grown in shaking flasks in EMM4S medium at 32 C to mid-log phase unless otherwise indicated. METHOD DETAILS Yeast Strains and Growth Standard S. pombe media and methods were used [38]. Gene tagging and deletion were performed using PCR and homologous recombination [39], and correct integrations were verified by colony PCR, fluorescence, or backcross as needed. The non-phosphorylatable kin1 mutants, kinase-dead kin1(K154R) and analog-sensitive kin1-as1 (F220G) alleles were generated by site-directed mutagenesis using QuikChange II mutagenesis (Stratagene) kit according to the manufacturer’s protocol, and integrated into kin1D::kanR strain at the ura4 locus using pJK210 [40]. The wild-type pJK210-kin1+ plasmid integrated in this manner fully rescued all kin1D mutant phenotypes. The kin1(16A) mutant allele was synthesized as a gBlocks Gene Fragment (Integrated DNA Technologies), and then inserted into the PCR-linearized fragment from pJK210-kin1-mEGFP plasmid by Gibson Assembly Cloning Kit (New England Biolabs) according to the manufacturer’s protocol. The non-phosphorylatable mutants of Pal1 were also generated with the same method except that these alleles were integrated at the leu1 locus using pJK148 [40]. Double mutants were generated by tetrad dissections. To generate Kin1 truncations, Kin1 fragments were cloned into the pJK210 vector using XhoI and NotI sites, GFP was inserted at the C terminus of Kin1 using NotI, and integrated into the kin1D::kanR strain at the ura4 locus. All plasmids were sequenced for verification. For growth assays in Figures S1F, S3C, S3D, S3G, and S3H, cells were spotted by 10-fold serial dilutions (started at OD595 = 0.1) on YE4S plates and were incubated at 32 C or 37 C for 3-4 days before scanning. Large-Scale Phosphoproteomic Screens The strain used for phosphoproteomic screen was JM4023 (kin1D::kanR ura4::[pJK210-Pkin1-kin1-as1(F220G)-Tkin1]). The experimental procedure was similar to previous work [8], except cells were grown in EMM2 (Edinburgh Minimal Medium with supplements) or 2X YE (Yeast Extract). After 10 generations of growth at 32 C, cell cultures were treated for 10 min with final concentration of 2 mM 3-MB-PP1 (1-(tert-Butyl)-3-(3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine (Toronto Research Chemicals), while control culture was treated with an equal volume of DMSO. 5 min was the minimal time needed to observe Kin1-as1 inhibition by 3-MB-PP1, and we used 10 min inhibition for these large-scale experiments to ensure maximal identification of sites. Each culture volume was 1.5 l in 2X YE or 3 l in EMM2. The cultures were then harvested by 6-min centrifugation at 8,000 g at 4 C, washed once with 200 mL ice-cold PBS, and then centrifuged again. The pellet was lysed as previously described [8]. Lysed powder was resuspended in ice-cold lysis buffer (8 M urea, 25 mM Tris-HCl pH8.6, 150 mM NaCl, phosphatase inhibitors (2.5 mM beta-glycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium molybdate, 1 mM sodium tartrate) and protease inhibitors (1 mini-Complete EDTA-free tablet per 10 mL lysis buffer; Roche Life Sciences), followed by three rounds of sonication (15 s each) with intermittent cooling on ice. Lysates were then spun for 30 min at 2851 x g, and the supernatants were transferred to a new tube. Protein concentration was determined using a BCA assay (Pierce/ThermoFisher Scientific). Phosphopeptide enrichment followed by dimethyl-labeling was performed as previously described [41]. After labeling, samples were combined and fractionated by pentafluorophenyl (PFP)-RP chromatography [42]. Samples were analyzed by LC-MS/MS on a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with an Easy-nLC 1000 (Thermo Fisher Scientific) and nanospray source (Thermo Fisher Scientific) as previously described [43]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [44] via the Mass spectrometry Interactive Virtual Environment (MassIVE) partner repository. PDX accession PXD006782 (http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD006782). Immunoprecipitation Fission yeast cells were grown at least eight generations at 32 C in rich medium. For Kin1-mEGFP, kin1-as1-mEGFP, Pom1-mEGFP, pom1-as1-3HA pull-down experiments, 25 OD595 cells were harvested and lysed in 200 mL IP buffer (20 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid [HEPES], pH 7.4, 1 mM EDTA, 500 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride [PMSF], complete EDTA-free protease inhibitor tablets [Roche, Indianapolis, IN], 25 mM NaF, 50 mM b-glycerophosphate, 1mM sodium orthovanadate) with 400 mL glass beads using a Mini-beadbeater-16 (Biospec, Bartlesville, OK; two cycles of 2 min at maximum speed) at 4 C. Lysates were then spun at 16,000 x g for 10 min at 4 C and supernatants were incubated at 4 C for 1.5 h with anti-GFP magnetic beads (Allele Biotech) or anti-HA magnetic beads (Thermo Scientific Pierce). For in vitro phosphatase assay, immunoprecipitated Kin1-mEGFP, kin1-16A-mEGFP, and kin1-as1-mEGFP with/without 3-MBPP1 inhibitor were treated with/without l-phosphatase (New England Biolabs) at 30 C for 30 min according to the manufacturer’s Current Biology 28, 1–9.e1–e4, January 8, 2018 e2

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

protocol. Immunoprecipitated Kin1-mEGFP migrated as a double band for unknown reasons that were not affected by l-phosphatase treatment. In Vitro Kinase Assays and Western Blots Full length Ssp1-as1 and the fragments of Tea4 (aa 113-436), Mod5 (aa 28-495), and Cyk3 (aa 81-388) were cloned into pGEX6P1 (GST tag) vector (GE Healthcare), expressed in BL21 (Rosetta) E. coli strain, and purified with glutathione-agarose resin (Sigma-Aldrich) as previously described [8]. Purified proteins were released from resin by overnight incubation with 3C protease at 4 C or by elution with glutathione. Full length Pal1 was cloned into pQE30 vector (QIAGEN), expressed in BL21(Rosetta) E. coli, and purified with Ni-NTA beads (QIAGEN). Purified protein was eluted with 250 mM imidazole [8]. The fragment of Cdc15 (358-928) was cloned into pMBP-parallel1 [45], expressed in BL21(Rosetta) E. coli, and purified with Amylose resin (New England Biolabs) according to the manufacturer’s protocol. Purified proteins were released from resin by overnight incubation with AcTEV Protease (Invitrogen) at 4 C. For in vitro kinase assays, the purified proteins were incubated with immunoprecipitated wild-type Kin1 or catalytically inactive Kin1(K154R) or wild-type Pom1 or catalytically inactive Pom1 (K728R) in kinase assay buffer (30 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 10% glycerol) supplemented with 20 mM ATP in 40 mL reactions at 30 C for 30 min. Reactions were stopped by boiling for 5 min at 80 C in buffer (1% SDS, 15% glycerol, 50mM Tris-HCl, pH 8.7, 100mM NaCl), reduced with 5 mM DTT, and alkylated with 15 mM iodoacetamide (Sigma-Aldrich) prior to boiling in SDS-PAGE sample buffer for 5 min at 99 C. Reactions were separated by SDS-PAGE analysis. Bands were excised, destained, trypsin-digested, and peptides were extracted and analyzed by LC-MS/MS as described above. We detected some phosphorylation of substrates by kinase-dead Kin1 and kinase-dead Pom1 immunoprecipitates, likely due to contaminating kinases. Therefore, we determined the degree of phosphorylation by label-free quantification. The peak intensity of each peptide was determined for wild-type Kin1 and kinase-dead Kin1 reactions. Sites were considered to be phosphorylated by Kin1 if the peptide intensity was two-fold or more increased upon treatment with wild-type Kin1 versus kinase-dead Kin1, or if they were present in reactions with wild-type but absent from reactions with kinase-dead. The same criteria were applied for Pom1 substrates. For in vitro thiophosphate kinase assays, the kinase reactions were performed using 6-Bn-ATP-g-S (Axxora) in place of ATP. The reactions were stopped by adding EDTA at 20mM final concentration. Subsequently, alkylation of substrates was performed by adding p-nitrobenzyl mesylate for a final concentration of 2.5mM (Abcam Biochemicals) and incubated at room temperature for 2 hr. After alkylation, samples were resuspended in SDS-PAGE sample buffer and boiled for 5 min at 99 C. Western blots were performed using thiophosphate ester specific RabMAb (ab92570) according to manufacturer’s protocol. Whole-cell extracts were prepared as described [13]. Western blots were probed with anti-GFP [37], anti-myc (Santa Cruz), and anti-HA (Covance) antibodies. Rabbit anti-Kin1-pT299 antibody was generated against the phosphopeptide YRRQSRLR[pT] FCGSLYF (21st Century Biochemicals). Secondary antibodies were conjugated to IRDye680RD or IRDye800CW (LI-COR Biosciences). Blots were developed by Odyssey CLx Imaging System (LI-COR), and bands were quantified in Image Studio Lite (LI-COR) for Figure S2E graph. In Vitro Binding Assay GST control and GST-kin1 (aa 511–752) were expressed in E. coli BL21(Rosetta) cells using pGEX6P1 vector. Cells were grown to OD600 = 0.3 at 37 C, and then expression was induced for 3 hr at 37 C by addition of isopropyl-b-d-thiogalactoside to 0.4 mM. To isolate GST fusion proteins, cells were harvested by centrifugation, resuspended in lysis buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 300 mM NaCl, Roche complete protease inhibitors, and 1mM PMSF), and then lysed by French press. The lysate was clarified by centrifugation, and then incubated with glutathione-agarose (Sigma-Aldrich) for 2 hr at 4 C. After incubation, beads were washed extensively using the same lysis buffer, and then washed into binding buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, Roche complete protease inhibitors, and 1mM PMSF). Cell lysates containing Pal1-mEGFP or pal1-7AmEGFP from S. pombe were prepared using the lysis buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 100 mM NaCl, 1% Triton, Roche complete protease inhibitors, and 1mM PMSF), and incubated with GST-kin1(511-752) or GST on agarose resin for 1 hr at 4 C by rocking. Agarose beads were washed with the binding buffer, and proteins were eluted with SDS-PAGE sample buffer, and analyzed for SDS-PAGE and immunoblotting. Imaging and Data Analysis Microscopy was performed at room temperature with a DeltaVision Imaging System (Applied Precision), equipped with an Olympus IX-71 inverted wide-field microscope, a Photometrics CoolSNAP HQ2 camera, and Insight solid-state illumination unit. All images are single focal planes positioned in the cell middle unless indicated. All fluorescence images are shown with inverted look-up table. All image analysis was performed on ImageJ (National Institutes of Health). For quantification of Pal1 or Kin1 mislocalized cells, the absence of strong fluorescent signal at cell ends was considered mislocalization. For time-lapse imaging, cells were placed onto agarose pads containing the same growth medium and 2% agarose. For Figure 4I, 25 z stacks were acquired with 0.2 mm step size and iteratively deconvolved in SoftWoRx software (Applied Precision). All statistical differences were determined by a two-tailed Student’s t test using Excel (Microsoft). For gene ontology analysis, proteins with phosphopeptides that were >16-fold reduced by Kin1-as1 inhibition were selected. From this list, any proteins containing phosphopeptides that were 1.5-fold reduced by 3-MB-PP1 treatment in wild-type strain [8] were removed for the analysis to eliminate non-specific inhibition by the drug. The process and component analysis were performed using e3 Current Biology 28, 1–9.e1–e4, January 8, 2018

Please cite this article in press as: Lee et al., Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division, Current Biology (2017), https://doi.org/10.1016/j.cub.2017.11.034

Princeton GO term finder (http://go.princeton.edu/), using all S. pombe proteins as a background. For actin staining, cells were grown to log phase in YE4S and fixed by adding 16% paraformaldehyde (Fisher scientific) at the growing temperature for 10 min. Cells were washed with PEM buffer (0.1 M Na Pipes, pH 6.8, 1 mM EGTA, and 1 mM MgCl2) [46] and permeabilized with 1% Triton X-100 for 2min, and washed in PEM buffer for three times. Alexa Fluor 488 Phalloidin (ThermoFisher) was dissolved in MeOH and dried according to manufacturer’s protocol, and resuspended in PEM buffer and used to stain cells for 30 min in dark. After staining, cells were washed once in PEM buffer prior to imaging. For images in Figures 1D and 1F, maximum projections were generated using 0.2-mm focal planes throughout the entire cell (25 steps, 5 mm total) using ImageJ. QUANTIFICATION AND STATISTICAL ANALYSIS For all statistical analyses, two-tailed unpaired t test (unequal variances) was performed using Microsoft Excel. p values are described in the figure legends. Total n refers to the number of cells analyzed. Analysis of phosphoproteomic data is described in the Method Details. DATA AND SOFTWARE AVAILABILITY The full list of proteins identified in the phosphoproteomic screen is available with the online version of the paper as Table S1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [44] via the Mass spectrometry Interactive Virtual Environment (MassIVE) partner repository. PDX accession PXD006782 (http://proteomecentral.proteomexchange.org/cgi/ GetDataset?ID=PXD006782).

Current Biology 28, 1–9.e1–e4, January 8, 2018 e4