Dynamic regulation of Cdr1 kinase localization and phosphorylation during osmotic stress

Abstract

Environmental conditions modulate cell cycle progression in many cell types. A key component of the eukaryotic cell cycle is the protein kinase Wee1, which inhibits the cyclin-dependent kinase Cdk1 in yeast through human cells. In the fission yeast Schizosaccharomyces pombe, the protein kinase Cdr1 is a mitotic inducer that promotes mitotic entry by phosphorylating and inhibiting Wee1. Cdr1 and Wee1 both localize to punctate structures, termed nodes, on the medial cortex, but it has been unknown whether node localization can be altered by physiological signals. Here we investigated how environmental conditions regulate Cdr1 signaling for cell division. Osmotic stress induced hyperphosphorylation of the mitotic inducer Cdr1 for several hours, and cells delayed division for the same time period. This stress-induced hyperphosphorylation required both Cdr1 autophosphorylation and the stress-activated protein kinase Sty1. During osmotic stress, Cdr1 exited cortical nodes and localized in the cytoplasm. Using a series of truncation mutants, we mapped a C-terminal domain that is necessary and sufficient for Cdr1 node localization and found that Sty1 directly phosphorylates this domain in vitro Sty1 was not required for Cdr1 exit from nodes, indicating the existence of additional regulatory signals. Both Cdr1 phosphorylation and node localization returned to basal levels when cells adapted to osmotic conditions and resumed cell cycle progression. In summary, we identified a mechanism that prevents Cdr1 colocalization with its inhibitory target Wee1 during osmotic stress. Dynamic regulation of protein localization to cortical nodes might represent a strategy to modulate entry into mitosis under differing environmental conditions.

Keywords: Cdr1; Schizosaccharomyces pombe; Wee1; cell cycle; mitosis; osmotic stress; protein kinase; protein phosphorylation; yeast.

Figure 1.

Hyperphosphorylation of Cdr1 during osmotic stress response. A, Cdr1 is a phosphoprotein in vivo. Immunoprecipitated Cdr1-FLAG was treated with λ phosphatase or mock-treated and then analyzed by SDS-PAGE and Western blotting. B, modification of Cdr1 by different environmental stresses. Cells were exposed to the indicated conditions for 15 min, and then whole-cell extracts were analyzed by SDS-PAGE and Western blotting. C, the change in Cdr1 mobility is due to phosphorylation. Cells were exposed to EMM4S alone or to EMM4S + 1 m KCl for 15 min. Immunoprecipitated (IP) Cdr1 was treated with λ phosphatase and analyzed by SDS-PAGE and Western blotting. Note that slower-migrating bands collapse into a single band when dephosphorylated. D, hyperphosphorylation of Cdr1 during sorbitol treatment. Cells were exposed to the indicated condition for 15 min, and then whole-cell extracts were analyzed by SDS-PAGE and Western blotting. E, time course of asynchronous cells exposed to 1 m KCl. Whole-cell extracts were blotted with anti-FLAG. F, timing of septation delay by osmotic stress of wild-type cells. Cell cycle progression was synchronized by centrifugal elutriation, and then cells were released into YE4S or YE4S + 1 m KCl. Percent septation was used to monitor cell cycle progression; n > 100 cells for each time point. G, timing of septation delay by osmotic stress in cdc25–22 cdr1-FLAG cells, which were arrested in G₂ phase by incubation at 37 °C and then released into synchronized cell cycle progression by switching to 25 °C in YE4S or YE4S + 1 m KCl. H, change in Cdr1 phosphorylation during mitotic arrest induced by osmotic stress. Shown is a Western blot of samples from G; Cdr1 mobility was analyzed by SDS-PAGE at the indicated time points. I, timing of septation delay by osmotic stress of cdr1Δ cells, analyzed using centrifugal elutriation as in F.

Figure 2.

Cdr1 exits nodes during osmotic stress. A, inverted contrast single focal plane images of Cdr1-GFPγ grown in EMM4S to mid-log phase and then split into medium containing 1 m KCl or into control medium (EMM4S). Insets are enlarged images of the medial cortex; yellow boxes indicate the enlarged area. Scale bar = 5 μm. B, quantification of cells containing Cdr1 localization to nodes (n > 100 cells for each time point; error bars represent standard deviation).

Figure 3.

Cdr2 nodes remain intact during osmotic stress. A, single focal plane inverted images of a time course with or without exposure to 1 m KCl. Scale bar = 5 μm. DIC, differential interference microscopy. B, quantification of cells containing Cdr2 nodes (n > 100 cells for each time point, error bars represent standard deviation). C, phosphorylation of Cdr1 is independent of Cdr2 nodes and Cdr2 kinase activity. cdr2(E177A) is a catalytically inactive mutation. Shown is a Western blot of whole-cell extracts after 15-min exposure to 1 m KCl. Note the presence of hyperphosphorylated Cdr1 in Cdr2 mutants.

Figure 4.

Identification of the domain that targets Cdr1 to nodes. A, schematic of Cdr1 and truncations. Kinase, kinase domain. Localization to nodes is indicated for each strain. B, representative inverted contrast images of Cdr1 truncations expressed as GFPγ fusions at the endogenous locus in cdr2+ (left panel) or cdr2Δ (right panel) background. Images are middle and top focal planes from a deconvolved Z series. Black arrows indicate nodes. Scale bar = 5 μm.

Figure 5.

Node localization is required for Cdr1 function. A, measurement of cell size at division for the indicated cdr1 alleles (n > 50 cells/strain, values are mean ± S.D.). B, hyperphosphorylation of the indicated Cdr1 mutants upon osmotic stress.

Figure 6.

Hyperphosphorylation requires Cdr1 kinase activity. A, α-FLAG Western blot of whole-cell extracts from the indicated strains after KCl treatment. K41A is kinase-dead mutation of Cdr1 (20, 21). B, Cdr1(K41A)-GFPγ does not exit nodes during osmotic stress. Shown are middle focal plane inverted images of a time course with or without 1 m KCl. Insets show enlarged images of Cdr1 present at nodes; yellow boxes indicate the enlarged area. Scale bar = 5 μm. C, quantification of cells with Cdr1(K41A)-GFPγ localization at nodes (n > 100 for each time point, error bars represent standard deviation).

Figure 7.

Intramolecular autophosphorylation by Cdr1. A, Western blots of whole-cell extracts from strains expressing the indicated combinations of wild-type and kinase-dead (K41A) alleles of Cdr1. Strains were treated with 1 m KCl for 15 min. Note that expression of wild-type Cdr1 does not induce hyperphosphorylation of kinase-dead Cdr1(K41A). B, middle focal plan images of Cdr1(K41A)-GFPγ leu1::[6his-2HA-cdr1] in control EMM4S media or in EMM4S + 1 m KCl. Insets are enlarged images of the medial cortex. Scale bar, 5 μm.

Figure 8.

Activated SAPK Sty1 is necessary and sufficient for Cdr1 hyperphosphorylation. A, tests for hyperphosphorylation of Cdr1 upon 15-min osmotic stress in srk1Δ and sty1Δ mutants. B, Sty1 kinase activity is necessary for full hyperphosphorylation of Cdr1. Analog-sensitive sty1(T97A) cells were treated with BrB-PP1 inhibitor or with a methanol control as indicated and then exposed to 1 m KCl for 15 min. C, time course of Sty1 activation during 1 m KCl treatment. Activation was monitored by phosphorylation of Thr-171/Tyr-173 in the activation loop; total Sty1 protein was detected with α-FLAG. D, Cdr1 hyperphosphorylation by SISA. Sty1 was activated by removal of 3-BrB-PP1 inhibitor, and whole-cell extracts were analyzed by SDS-PAGE and Western blotting. E, quantification of Cdr1-GFPγ localization in wild-type or analog-sensitive sty1(T97A) mutant cells. Cells were treated as in B (n > 100 cells for each condition; error bars represent standard deviation). F, inverted contrast single focal plane images of Cdr1-GFPγ localization in the indicated strain before or after 30 min of osmotic stress. See E for quantification and controls. Insets are enlarged images of the medial cortex; yellow boxes indicate the enlarged area. Scale bar = 5 μm. G, localization of Cdr1-GFPγ in sty1Δ cells after osmotic stress. Insets are enlarged images of the medial cortex; yellow boxes indicate the enlarged area. Scale bar = 5 μm.

Figure 9.

Sty1 directly phosphorylates Cdr1 in vitro. Shown is an in vitro thiophosphate kinase assay using ATPγS. Sty1-FLAG and Sty1(T97A)-FLAG were immunoprecipitated and then added to thiophosphate kinase assays with substrate GST-Cdr1(341–593) or GST alone. Proteins were detected by Western blotting with the indicated antibodies, and phosphorylation was detected using an antibody against thiophosphate ester.

Figure 10.

Working model for the regulation of Cdr1 during osmotic stress response. See the text for discussion.