Graph shows quantification of Pal1 localization defect (n 100 cells each). (C) Localization of mutant lacking Kin1-dependent phosphorylation sites. related to Figures 2 and ?and4.4. Selected cell polarity and cell division targets of Kin1 kinase activity in cells. For each phosphopeptide, the average log2-transformed heavy/light ratio is given for kin1-as1. Phospho-peptides identified in 2 fold reduction changes in kin1-as1 experiments are shown. Abbreviations: # = phosphorylation; * = oxidation Table S3, related to Figures 2 and S4. Phospho-peptides identified from the indicated in vitro kinase assays. column B: the sequence of phosphopeptide, residues immediately before LHR2A antibody # are phosphorylated; * indicates the oxidized methionine residues; . indicates the cleavage sites. Phosphorylated peptides were identified in parallel reactions containing kinase-dead Kin1 in column K, wildtype Kin1 in column M, kinase-dead Pom1 in column O and wild type Pom1 in column Q. Table S4, Related to Figures 1C4, S1-4, and STAR METHODS. Yeast strains and plasmids used in this study. NIHMS922239-supplement-1.pdf (1.7M) GUID:?BB27A9DF-00F9-48A2-985D-2047385BC7FB 2. NIHMS922239-supplement-2.xlsx (2.4M) GUID:?37A10A3E-D01A-4171-AD65-729B92BA9E98 3. NIHMS922239-supplement-3.xlsx (14K) GUID:?DEEC636C-9C33-48A1-8F33-3A7E9DEF01E8 4. NIHMS922239-supplement-4.xlsx (30K) GUID:?8ED5095B-87EA-4924-B7E3-EE34AD0875FF 5. NIHMS922239-supplement-5.xlsx (74K) GUID:?AA94FD36-7FC6-4896-B0CA-03A0E3AC2C22 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 [1C5]. We found that Ssp1 promotes cell polarity by phosphorylating the activation Levoleucovorin Calcium loop of Kin1. Kin1 regulates cell polarity and cytokinesis through unknown mechanisms [4C7]. 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 . 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 Kin1 and Pom1 led to synthetic defects in their shared substrates Cdc15 and Levoleucovorin Calcium 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 [9C11]. Ssp1 directly phosphorylates the activation loops of the cell cycle kinase Cdr2 and the metabolic sensor kinase Ssp2 [12, 13], Levoleucovorin Calcium Levoleucovorin Calcium 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. Open in a separate window 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 thiophosphate kinase assay showing direct phosphorylation of Kin1 by Ssp1-as1. Ssp1-as1 was purified from bacteria; was immunoprecipitated from mutant. F-actin was visualized with Alexa Fluor-488 phalloidin staining. Maximum projection images are shown. Scale bar, 5m. (E) Quantification of polarity patterns from actin staining of strains. Values are mean standard deviation from 3 independent experiments (n 150 cells each). **, P 10?2. (F) Actin staining of and mutant arrested at 36C for 4 hours. Values are mean standard deviation from 3 independent experiments (n 150 cells each). (H) Quantification of F-actin patches within 10 m medial region of cells from panel F. Values are mean standard deviation from 10 cells. ***, P 10?5. See also Figure S1. 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, 16] and Kin1 localizes to growing cell ends [4, 6]. Levoleucovorin Calcium 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.