PRODUCTION OF A SOLUBLE CYCLIN-B CDC2 SUBSTRATE FOR CDC25 PHOSPHATASE

Study of the function and regulation of the important cell cycle regul ator cdc25 phosphatase has been hampered by the lack of a sensitive an d specific substrate and assay. Here we report the production of a spe cific and sensitive substrate for the cdc25 phosphatase. The substrate is human cyclin B1/cdc2 phosphorylated on the inhibitory Thr14 and Ty r15 residues and activating Thr161 on cdc2, and is relatively simple t o 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. (C) 1 997 Academic Press.