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 , 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 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.

Reprogramming Cdr2-Dependent Geometry-Based Cell Size Control in Fission Yeast

“…These nodes have been proposed as an important element in cell size control, as their number scales with cell size, and cdr2 mutants defective in node association are defective in size control [ 3 , 18 ]. Recent studies have suggested that the Cdr1 and Cdr2 kinases in the nodes transiently recruit and inactivate Wee1 by phosphorylation [ 19 , 20 ]. Upstream Cdr2 regulators include an inhibitory kinase Pom1 [ 21 , 22 ] and an activating kinase CaMK Ssp1 [ 4 , 23 ].…”

The regulation of Net1/Cdc14 by the Hog1 MAPK upon osmostress unravels a new mechanism regulating mitosis

“…This prevents Swe1 degradation, leading a transient G2 arrest and, thus, a delay in progression into M phase, thereby allowing adaptation to osmostress. It should be noted that the same mechanism has been proposed in Schizosaccharomyces pombe [60], together with alternative mechanisms where the SAPK sty1 regulates cdc25, the phosphatase involved in inhibiting the mitosis repressor wee1 [61]. Similar to the fission yeast, activated p38 is required for a G2/M checkpoint involving Cdc25B and Cdc25C, which regulate the activity of the CDK-cyclin B1 complex during mitosis.…”

A mechanism for how Cdr1/Nim1 kinase promotes mitotic entry by inhibiting Wee1

“…These nodes are formed by Cdr2 oligomers that are required for Cdr1 and Wee1 recruitment (Martin and Berthelot-Grosjean, 2009;Moseley et al, 2009;Allard et al, 2018). We previously identified a mutant cdr1(∆460-482) that fails to localize to nodes and results in elongated cell size at division like cdr1∆ ( Fig 5A) (Opalko and Moseley, 2017). We tested the effects of artificially recruiting mEGFP-cdr1(∆460-482) back to nodes using cdr2-GBP-mCherry, which contains the GFP-binding peptide (GBP).…”

“…Cells were then lysed in 100µl sample buffer (65mM Tris pH 6.8, 3% SDS, 10% glycerol, 10% 2-mercaptoethanol, 50mM sodium fluoride, 50mM β-gylcerophosphate, 1mM sodium orthovanadate, complete EDTA-free protease inhibitor tablet (Pierce)) in a Mini-beadbeater-16 for 2 minutes. Blots were probed with anti-FLAG M2 (Sigma), anti-GST (Covance), anti-Cdr1 (Opalko and Moseley, 2017), anti-Wee1 (Allard et al, 2018), anti-cdc2 (Santa Cruz Biotechnologies sc-53217), anti-pY15 (Cell Signaling #9111L), anti-His (Santa Cruz Biotechnologies sc-8036), and anti-thiophosphate ester (Abcam ab92570).…”