Negative regulation of the weel protein kinase by direct action of the nim1/cdr1 mitotic inducer

doi:10.1016/0092-8674(93)90580-J

Cell

Volume 72, Issue 6, 26 March 1993, Pages 919-929

https://doi.org/10.1016/0092-8674(93)90580-J Get rights and content

Abstract

The wee1 protein kinase suppresses the entry into mitosis by mediatingthe inhibitory tyrosine phosphorylation of p34^cdc2. Genetic studies have suggested that the nim1 protein kinase (also known as cdr1) acts as a positive regulator of mitosis by down-regulating the wee1 pathway in yeast cells. We have overexpressed the nim1 protein in both bacteria and insect cells. The recombinant nim1 protein autophosphorylates on both tyrosine and serine residues and can phosphorylate the isolated wee1 protein directly in a cell-free system. The nim1-catalyzed phosphorylation of the wee1 protein occurs in its C-terminal region and leads to a substantial drop in its activity as a cdc2-specific tyrosine kinase. This nim1-dependent inhibition of the wee1 protein kinase can be reversed readily in vitro by treatment with a protein phosphatase. These experiments provide direct biochemical evidence that the wee1 protein is subject to negative regulation by phosphorylation and indicate that the niml protein acts as an inhibitory, wee1-specific kinase.

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Wee1 activity in cells is regulated in a size-dependent manner by the protein kinases Cdr1 and Cdr2 (9), which are conserved SAD family kinases. Cdr1 (also called Nim1) directly phosphorylates Wee1 to inhibit its kinase activity (10–13). Cdr1 acts in a dosage-dependent manner: loss-of-function causes elongated cells due to over-active Wee1, while cdr1+ overexpression reduces cell size at division (14).
Many cell cycle regulatory proteins catalyze cell cycle progression in a concentration-dependent manner. In the fission yeast Schizosaccharomyces pombe, the protein kinase Cdr2 promotes mitotic entry by organizing cortical oligomeric nodes that lead to inhibition of Wee1, which itself inhibits the cyclin-dependent kinase Cdk1. cdr2Δ cells lack nodes and divide at increased size due to overactive Wee1, but it has not been known how increased Cdr2 levels might impact Wee1 and cell size. It also has not been clear if and how Cdr2 might regulate Wee1 in the absence of the related kinase Cdr1/Nim1. Using a tetracycline-inducible expression system, we found that a 6× increase in Cdr2 expression caused hyperphosphorylation of Wee1 and reduction in cell size even in the absence of Cdr1/Nim1. This overexpressed Cdr2 formed clusters that sequestered Wee1 adjacent to the nuclear envelope. Cdr2 mutants that disrupt either kinase activity or clustering ability failed to sequester Wee1 and to reduce cell size. We propose that Cdr2 acts as a dosage-dependent regulator of cell size by sequestering its substrate Wee1 in cytoplasmic clusters, away from Cdk1 in the nucleus. This mechanism has implications for other clustered kinases, which may act similarly by sequestering substrates.
Dynamic regulation of Cdr1 kinase localization and phosphorylation during osmotic stress
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We sought to identify mechanisms that might regulate the protein kinase Cdr1 according to different environmental and growth conditions. Cdr1 controls the timing of mitotic entry and has been reported to autophosphorylate in vitro (19–21). To investigate Cdr1 phosphorylation in fission yeast cells, we integrated a 5×FLAG epitope tag at the carboxyl terminus of endogenous Cdr1; this tag included a nine-glycine linker and did not interfere with Cdr1 function, as tested by cell length at division.
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.
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Cell size is an important adaptive trait that influences nearly all aspects of cellular physiology. Despite extensive characterization of the cell-cycle regulatory network, the molecular mechanisms coupling cell growth to division, and thereby controlling cell size, have remained elusive. Recent work in yeast has reinvigorated the size control field and suggested provocative mechanisms for the distinct functions of setting and sensing cell size. Further examination of size-sensing models based on spatial gradients and molecular titration, coupled with elucidation of the pathways responsible for nutrient-modulated target size, may reveal the fundamental principles of eukaryotic cell size control.
Fission yeast receptor of activated C kinase (RACK1) ortholog Cpc2 regulates mitotic commitment through Wee1 kinase
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Citation Excerpt :
In fission yeast this task is performed at least by two SAD kinases, Nim1/Cdr1 and Cdr2, which phosphorylate and inactivate Wee1 (8–14). As a result, deletion of either nim1+/cdr1+ or cdr2+ causes a G2/M delay due to lack of Wee1 down-regulation (8–14). Importantly, we have found that protein levels of Cdr2 are down-regulated in the absence of Cpc2, supporting that Cpc2 exerts its function as a positive translational regulator of the mRNA encoding Cdr2.
In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G₁/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G₂/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G₁/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.
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