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Nif1, a novel mitotic inhibitor in Schizosaccharomyces pombe
1Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Abstract:
In Schizosaccharomyces pombe, the activity of the M-phase-inducing Cdc2/Cdc13 cyclin-dependent kinase is inhibited by Wee1 and Mik1 tyrosine kinases, and activated by Cdc25 and Pyp3 tyrosine phosphatases. Cdc2/Cdc13 activity is also indirectly regulated by the approximately 70 kDa Nim1 (Cdrl) serine/threonine kinase, which promotes mitosis by inhibiting Wee1 via direct phosphorylation. To understand better the function and regulation of Nim1, the yeast two-hybrid system was used to isolate S.pombe cDNA clones encoding proteins that interact with Nim1. Sixteen of the 17 cDNA clones were derived from the same gene, named nif1 + (nim1 interacting factor-1). Nif1 is a novel approximately 75 kDa protein containing a leucine zipper motif. The Nif1-Nim1 interaction requires a small region of Nim1 that immediately follows the N-terminal catalytic domain. This region is required for Nim1 activity both in vivo and in vitro. delta nif1 mutants are approximately 10% smaller than wild type, indicating that Nif1 is involved in inhibiting the onset of mitosis. Consistent with this proposal, overproduction of Nif1 was found to cause a cell elongation phenotype that is very similar to delta nim1 mutants. Nif1 overproduction causes cell cycle arrest in cells that are partly defective for Cdc25 activity, but has no effect in delta nim1 or delta wee1 mutants. Nif1 also inhibits Nim1-mediated phosphorylation of Wee1 in an insect cell expression system. These observations strongly suggest that Nif1 negatively regulates the onset of mitosis by a novel mechanism, namely inhibiting Nim1 kinase.
Insights
Nim1 interacting factor 1 (Nif1) negatively regulates mitosis onset in Schizosaccharomyces pombe by inhibiting Nim1 kinase activity. This discovery reveals a novel mechanism controlling cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The cell cycle, particularly entry into mitosis, is tightly regulated by kinases and phosphatases.
- In Schizosaccharomyces pombe, Cdc2/Cdc13 kinase activity is modulated by Wee1, Mik1, Cdc25, and Pyp3.
- Nim1 (Cdr1) kinase promotes mitosis by inhibiting Wee1.
Purpose of the Study:
- To identify proteins interacting with Nim1 kinase.
- To elucidate the function and regulatory mechanisms of Nim1.
- To understand the role of novel Nim1-interacting factors in cell cycle control.
Main Methods:
- Yeast two-hybrid system for identifying protein-protein interactions.
- Isolation and characterization of S. pombe cDNA clones.
- Analysis of delta nif1 mutants and Nif1 overproduction phenotypes.
- In vitro kinase assays and in vivo functional studies.
Main Results:
- Identified a novel gene, nif1+, encoding a ~75 kDa protein, Nif1, that interacts with Nim1.
- The Nif1-Nim1 interaction occurs in a region of Nim1 critical for its activity.
- delta nif1 mutants exhibit smaller cell size, suggesting Nif1 inhibits mitosis onset.
- Nif1 overproduction causes cell elongation and cell cycle arrest in specific genetic backgrounds.
- Nif1 inhibits Nim1-mediated phosphorylation of Wee1.
Conclusions:
- Nif1 is a novel negative regulator of mitotic entry in S. pombe.
- Nif1 functions by inhibiting Nim1 kinase activity.
- This represents a new mechanism for controlling the initiation of mitosis.