Production of a soluble cyclin B/cdc2 substrate for cdc25 phosphatase.
Study of the function and regulation of the important cell cycle regulator cdc25 phosphatase has been hampered by the lack of a sensitive and specific substrate and assay. Here we report the production of a specific and sensitive substrate for the cdc25 phosphatase. The substrate is human cyclin B1/cdc2 phosphorylated on the inhibitory Thr14 and Tyr15 residues and activating Thr161 on cdc2, and is relatively simple to produce from readily available materials. The assay is based on the cdc25-specific dephosphorylation and activation of the phosphorylated cyclin B1/cdc2 substrate (PY15), using the increased histone H1 kinase activity of the activated PY15 as a read-out of cdc25 activity.
Clark
J M
JM
Queensland Cancer Fund Research Laboratory, Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Australia.
Gabrielli
B G
BG
D000818:Animals; D016203:CDC2 Protein Kinase / Q000096:biosynthesis / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002458:Cell Fractionation; D019926:Cyclin B / Q000096:biosynthesis / Q000378:metabolism*; D056744:Cyclin B1; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D014981:Xenopus; D020687:cdc25 Phosphatases
C534699:CCNB1 protein, human; D018797:Cell Cycle Proteins; D019926:Cyclin B; D056744:Cyclin B1; D011993:Recombinant Fusion Proteins; D010769:Phosphothreonine; D019000:Phosphotyrosine; D005982:Glutathione Transferase; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D020687:cdc25 Phosphatases