A Substrate Trapping Method for Identification of Direct Cdc14 Phosphatase Targets.
Mitotic exit requires the inactivation of cyclin-dependent kinase (Cdk) activity and reversal of Cdk-mediated phosphorylation events by protein phosphatases. In Saccharomyces cerevisiae the mitotic exit network (MEN) leads to activation and dispersal of the Cdc14 phosphatase throughout the cell following successful chromosome segregation. MEN-released Cdc14 is required for both full Cdk inactivation and dephosphorylation of Cdk substrates. While Cdc14 originally was thought to act broadly on mitotic Cdk substrates, recent biochemical studies revealed that Cdc14 possesses a strong preference for a subset of Cdk phosphorylation sites. This intrinsic specificity appears well conserved across fungi and animals. Identifying the direct physiological substrates of Cdc14 is an important step in fully understanding its biological functions, both in yeast and other species. Despite its strict specificity for phosphoserine Cdk sites, Cdc14 is structurally and mechanistically related to protein tyrosine phosphatases (PTPs). Like other PTPs, mutation of catalytic residues in the Cdc14 active site creates an inactive enzyme that retains high affinity substrate binding. Here we describe a protocol for using such "substrate trap" variants to biochemically isolate and detect direct substrates by co-immunopurification. The protocol is written for use in S. cerevisiae, but should be easily adaptable to other research organisms.
Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, IN, 47907, USA.
Brendan L
BL
Powers
Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, IN, 47907, USA.
Hana
H
Hall
Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, IN, 47907, USA.
Harry
H
Charbonneau
Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, IN, 47907, USA. mchall@purdue.edu.
Mark C
MC
Hall
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015151:Immunoblotting / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D008938:Mitosis; D016296:Mutagenesis; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
C074860:CDC14 protein, S cerevisiae; D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D017027:Protein Tyrosine Phosphatases
Cdc14; Cdc14 substrate identification; Co-immunoprecipitation; Co-immunopurification; Cyclin-dependent kinase; Protein phosphatase; Substrate interaction assay; Substrate trap