CDC25 dual-specificity protein phosphatases: detection and activity measurements.
Most cyclin-dependent kinases are negatively regulated by phosphorylation of two residues, a threonine at residue 14 and a tyrosine at residue 15. These residues are dephosphorylated by the cdc25 family of dual-specificity phosphatases leading to cell cycle progression. These phosphatases are inactivated by cellular checkpoint pathways in response to DNA damage leading to cell cycle arrest. Checkpoint pathways regulate the function of these phosphatases by regulating their stability, localization, association with substrate, and their activity. Hence, determining these properties for the cdc25 family of phosphatases becomes crucial for understanding how checkpoint pathways regulate the function of the cdc25 family members and, hence, cell cycle progression. This chapter describes methods to determine the activity, levels, phosphorylation status, and localization of both endogenous and overexpressed cdc25 proteins.
Dalal
Sorab N
SN
Biochemistry and Cell Biology, ACTREC, Navi Mumbai, India.
Volkening
Melanie
M
D001665:Binding Sites; D002453:Cell Cycle / Q000502:physiology; D002458:Cell Fractionation; D002460:Cell Line; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D020687:cdc25 Phosphatases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D018844:Cyclin-Dependent Kinases; D020687:cdc25 Phosphatases