A mechanism for how Cdr1/Nim1 kinase promotes mitotic entry by inhibiting Wee1
Hannah E Opalko1, Isha Nasa1,2, Arminja N Kettenbach1,2
1Department of Biochemistry and Cell Biology, The Geisel School of Medicine at Dartmouth, Hanover, NH 03755.
Abstract:
To enter into mitosis, cells must shut off the cell cycle inhibitor Wee1. SAD family protein kinases regulate Wee1 signaling in yeast and humans. In Schizosaccharomyces pombe, two SAD kinases (Cdr1/Nim1 and Cdr2) act as upstream inhibitors of Wee1. Previous studies found that S. pombe Cdr1/Nim1 directly phosphorylates and inhibits Wee1 in vitro, but different results were obtained for budding yeast and human SAD kinases. Without a full understanding of Cdr1 action on Wee1, it has been difficult to assess the in vivo relevance and conservation of this mechanism. Here, we show that both Cdr1 and Cdr2 promote Wee1 phosphorylation in cells, but only Cdr1 inhibits Wee1 kinase activity. Inhibition occurs when Cdr1 phosphorylates a cluster of serine residues linking α-helices G and H of the Wee1 kinase domain. This region is highly divergent among different Wee1 proteins, consistent with distinct regulatory mechanisms. A wee(4A) mutant that impairs phosphorylation by Cdr1 delays mitotic entry and causes elongated cells. By disrupting and retargeting Cdr1 localization, we show that Cdr1 inhibition of Wee1 occurs in cells at cortical nodes formed by Cdr2. On the basis of our results, we propose a two-step model for inhibition of Wee1 by Cdr1 and Cdr2 at nodes.
Insights
Cell cycle regulators Wee1 and SAD kinases are crucial for mitosis. In fission yeast, Cdr1 and Cdr2 kinases regulate Wee1, with Cdr1 directly inhibiting Wee1 activity at specific cellular locations.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The cell cycle inhibitor Wee1 must be inactivated for cells to enter mitosis.
- SAD family kinases regulate Wee1 signaling in yeast and humans.
- In *Schizosaccharomyces pombe*, Wee1 is inhibited by SAD kinases Cdr1/Nim1 and Cdr2.
Purpose of the Study:
- To elucidate the mechanism by which Cdr1 inhibits Wee1 kinase activity in *S. pombe*.
- To investigate the in vivo relevance and conservation of Wee1 regulation by SAD kinases.
- To understand the spatial regulation of Wee1 inhibition by Cdr1 and Cdr2.
Main Methods:
- In vivo and in vitro kinase assays.
- Site-directed mutagenesis to create *wee(4A)* mutant.
- Cellular localization studies using disrupted and retargeted Cdr1.
- Analysis of cell cycle progression and cell morphology.
Main Results:
- Both Cdr1 and Cdr2 promote Wee1 phosphorylation in cells, but only Cdr1 inhibits Wee1 kinase activity.
- Cdr1 inhibits Wee1 by phosphorylating serine residues in the G-H linker of the Wee1 kinase domain.
- A *wee(4A)* mutant impairs Cdr1-mediated Wee1 phosphorylation, leading to delayed mitotic entry and cell elongation.
- Cdr1 inhibition of Wee1 occurs at cortical nodes formed by Cdr2.
Conclusions:
- Cdr1 directly inhibits Wee1 kinase activity through phosphorylation at a specific site.
- The Wee1 G-H linker region is a key regulatory site, though divergent across species.
- Cdr1 and Cdr2 cooperate at cortical nodes to regulate Wee1, proposing a two-step inhibition model.
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