Fission yeast cells can expertly coordinate growth and division – they always divide at exactly the same size. This tight regulation of mitotic division depends on two opposing forces that control the activity of the cyclin- dependent kinase cdc2. The identification of these components - an inhibitory kinase and an activaten phosphatase – took over ten years, and it took several more to fully understand the mechanism. This pioneering work from the Nurse group has since been shown be a universal mechanism for mitotic control. In 1975, Paul Nurse first isolated mutants in which the normal controls that coordinate cell growth and division were lost. He identified the temperature-sensitive wee1-50 mutant – initially known as cdc9-50, but re- named to reflect its mutant phenotype (wee is a Scottish term for small)- in a mutagenesis screen for cells that divide at small size. These cells divided at about half the size of wild-type cells at the restrictive temperature, indicating that the normal control over cell division was lost. But did the wee1-50 mutation cause overactivation of a positive regulator of mitosis, or loss of function of a negative regulator? A clue to this was provided by Nurse and Pierre Thuriauxin 1980 – by this time, many more weel alelles,had been isolated. They showed that one allele, wee1-112, was suppressed by the nonsense suppressor sup3-5 – nonsense suppressors allow the translation machinery to read through nonsense mutations. As nonsense mutations are normally loss-of-function, this indicated that the wee phenotype was caused by loss of wee1 activity, and that the wee1 protein acts to inhibit mitosis. Before we continue with the weel story, we must first turn to its nemesis, cdc25. The cdc25-22 mutant was isolated by Nurse and colleagues in a screen for temperature-sensitive cell-division-cycle mutants; cdc25-22 cells arrest in G2 at the restrictive temperature because they cannot execute an essential function required for the onset of mitosis. Peter Eantes showed in 1979 that cdc25-22 mutants were suppressed by the additional mutation of wee1, so it was suggested that the two proteins might act coordinately to regulate the onset of mitosis. The cdc25 gene was cloned by Paul Russell and Nurse in 1986 by complementation of cdc25-22. They went on to show that cdc25 is an inducer of mitosis and, consequently, antagonizes Wee1: increased cdc25 gene dosage resulted in cells that divided at a smaller size, so were entering mitosis prematurely. But what was the relationship between wee1 and cdc25? Did they act independently or by interacting with each other? Cells lacking cdc25 remain viable as long as wee1 is also defective, so wee1 does not inhibit mitosis by inhibiting cdc25. Likewise, cdc25 does not activate mitosis by blocking the inhibitory effect of wee1, as the premature division phenotypes of cdc25 increased dosage and weel mutants are additive. So, how do these two proteins regulate mitosis? When wee1 was cloned by Russell and Nurse – through its ability to rescue the lethal premature mitosis of cdc25-overproducing cells – the gene showed homology to protein kinases, which provided the first insight into a possible mechanism. Their genetic analysis also provided the first indication that cdc2 could be the mutual substrate of wee1 and cdc25. In 1989, Kathleen Gould and Nurse confirmed that cdc2 was, indeed, regulated by phosphorylation. Immunoprecipitation of cdc2 from 32P-labelled extracts showed that cdc2 was phosphorylated, but that the phosphorylation was lost as cells entered mitosis. Surprisingly, this phosphorylation was on a tyrosine residue, Tyr15: this was the first time that a yeast protein had been shown to contain phospbotxrosioe Cells in which Tyr15 was mutated to phenylalanine displayed the wee phenotype. These results indicated that cdc2 was negatively regulated by phosphorylation of Tyr15, and that dephosphorylation of this residue was required for entry into mitosis. Biochemical evidence later proved definitively that weel was the kinase and cdc25 was the phosphatase in question. The broader significance of this work was revealed when higher eukaryotes were found to have not only cdc2 (see Milestone 11), but also the regulatory mechanisms that control it. But our understanding of cdc2's regulation is not matched by knowledge of its function: very few mitotic substrates have been identified to date. Hopefully, this challenge will be overcome in the future.

Fission yeast cells can expertly coordinate growth and division – they always divide at exactly the same size. This tight regulation of mitotic division depends on two opposing forces that control the activity of the cyclin- dependent kinase cdc2. The identification of these components - an inhibitory kinase and an activaten phosphatase – took over ten years, and it took several more to fully understand the mechanism. This pioneering work from the Nurse group has since been shown be a universal mechanism for mitotic control. In 1975, Paul Nurse first isolated mutants in which the normal controls that coordinate cell growth and division were lost. He identified the temperature-sensitive wee1-50 mutant – initially known as cdc9-50, but re- named to reflect its mutant phenotype (wee is a Scottish term for small)- in a mutagenesis screen for cells that divide at small size. These cells divided at about half the size of wild-type cells at the restrictive temperature, indicating that the normal control over cell division was lost. But did the wee1-50 mutation cause overactivation of a positive regulator of mitosis, or loss of function of a negative regulator? A clue to this was provided by Nurse and Pierre Thuriauxin 1980 – by this time, many more weel alelles,had been isolated. They showed that one allele, wee1-112, was suppressed by the nonsense suppressor sup3-5 – nonsense suppressors allow the translation machinery to read through nonsense mutations. As nonsense mutations are normally loss-of-function, this indicated that the wee phenotype was caused by loss of wee1 activity, and that the wee1 protein acts to inhibit mitosis. Before we continue with the weel story, we must first turn to its nemesis, cdc25. The cdc25-22 mutant was isolated by Nurse and colleagues in a screen for temperature-sensitive cell-division-cycle mutants; cdc25-22 cells arrest in G2 at the restrictive temperature because they cannot execute an essential function required for the onset of mitosis. Peter Eantes showed in 1979 that cdc25-22 mutants were suppressed by the additional mutation of wee1, so it was suggested that the two proteins might act coordinately to regulate the onset of mitosis. The cdc25 gene was cloned by Paul Russell and Nurse in 1986 by complementation of cdc25-22. They went on to show that cdc25 is an inducer of mitosis and, consequently, antagonizes Wee1: increased cdc25 gene dosage resulted in cells that divided at a smaller size, so were entering mitosis prematurely. But what was the relationship between wee1 and cdc25? Did they act independently or by interacting with each other? Cells lacking cdc25 remain viable as long as wee1 is also defective, so wee1 does not inhibit mitosis by inhibiting cdc25. Likewise, cdc25 does not activate mitosis by blocking the inhibitory effect of wee1, as the premature division phenotypes of cdc25 increased dosage and weel mutants are additive. So, how do these two proteins regulate mitosis? When wee1 was cloned by Russell and Nurse – through its ability to rescue the lethal premature mitosis of cdc25-overproducing cells – the gene showed homology to protein kinases, which provided the first insight into a possible mechanism. Their genetic analysis also provided the first indication that cdc2 could be the mutual substrate of wee1 and cdc25. In 1989, Kathleen Gould and Nurse confirmed that cdc2 was, indeed, regulated by phosphorylation. Immunoprecipitation of cdc2 from 32P-labelled extracts showed that cdc2 was phosphorylated, but that the phosphorylation was lost as cells entered mitosis. Surprisingly, this phosphorylation was on a tyrosine residue, Tyr15: this was the first time that a yeast protein had been shown to contain phospbotxrosioe Cells in which Tyr15 was mutated to phenylalanine displayed the wee phenotype. These results indicated that cdc2 was negatively regulated by phosphorylation of Tyr15, and that dephosphorylation of this residue was required for entry into mitosis. Biochemical evidence later proved definitively that weel was the kinase and cdc25 was the phosphatase in question. The broader significance of this work was revealed when higher eukaryotes were found to have not only cdc2 (see Milestone 11), but also the regulatory mechanisms that control it. But our understanding of cdc2's regulation is not matched by knowledge of its function: very few mitotic substrates have been identified to date. Hopefully, this challenge will be overcome in the future.

Biology 2e

2nd Edition

ISBN:9781947172517

Author:Matthew Douglas, Jung Choi, Mary Ann Clark

Publisher:Matthew Douglas, Jung Choi, Mary Ann Clark

Chapter10: Cell Reproduction

Section: Chapter Questions

Problem 39CTQ: Rb is a negative regulator that blocks the cell cycle at the G1 checkpoint until the cell achieves a...

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