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A Substrate Trapping Method for Identification of Direct Cdc14 Phosphatase Targets
Brendan L Powers1, Hana Hall1, Harry Charbonneau1
1Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, IN, 47907, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 10, 2016
Summary
Researchers developed a new method to identify direct substrates of the Cdc14 phosphatase, crucial for cell division. This technique uses "substrate trap" variants to isolate and detect proteins phosphorylated by cyclin-dependent kinases (Cdks).
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Mitotic exit depends on inactivating cyclin-dependent kinase (Cdk) activity and reversing Cdk-mediated phosphorylation.
- The mitotic exit network (MEN) in Saccharomyces cerevisiae activates and disperses the Cdc14 phosphatase after chromosome segregation.
- Cdc14 is essential for Cdk inactivation and dephosphorylation of Cdk substrates, with conserved specificity across species.
Purpose of the Study:
- To identify the direct physiological substrates of Cdc14, essential for understanding its functions in cell division.
- To present a novel protocol for isolating and detecting Cdc14 substrates using substrate trap variants.
Main Methods:
- Utilizing "substrate trap" variants of Cdc14, which are catalytically inactive but retain substrate binding affinity.
- Employing co-immunopurification to biochemically isolate and detect direct substrates bound to the trap variants.
- Developing a protocol adaptable for Saccharomyces cerevisiae and potentially other research organisms.
Main Results:
- Demonstrated the utility of substrate trap variants for identifying direct Cdc14 substrates.
- Established a robust co-immunopurification method for capturing transient protein-protein interactions.
- Provided a foundational protocol for future substrate identification studies.
Conclusions:
- The substrate trap approach is effective for identifying direct Cdc14 substrates.
- Understanding Cdc14 substrate specificity is key to elucidating its role in mitotic exit.
- The described protocol offers a valuable tool for cell cycle research in yeast and beyond.
