Three different human Cdc25 family members exist. Cdc25A regulates the G1/S transition and
Cdc25B and Cdc25C are involved in G2/M progression. Evidence suggests that two critical amino acids
located within the cyclin-dependent kinases, threonine 14 and tyrosine 15, represent the major targets for
the Cdc25 family of protein phosphatases. Dephosphorylation of these two critical amino acid residues is
essential for proper cell cycle progression and the subsequent association of cyclin-dependent kinases
with their associated cyclins (1).
Given their crucial role in cell cycle progression and checkpoint control, the regulation of the activity
of the various Cdc25 family members has been the subject of numerous investigations. For the case of
Cdc25C, enzymatic activity has been demonstrated to be low during interphase, in part because the
phosphatase is phosphorylated on serine 216. In response to DNA damage, various intracellular kinases
including Chk1, Chk2, and C-TAK1 (Cdc25C-associated protein kinase), appear to phosphorylate
Cdc25C on this residue (2–6).
One of the important functional consequences of phosphorylation of Ser-216 is to create a consensus
binding site for 14-3-3 protein binding (4). A variety of evidence suggests that in human cells, the
binding of 14-3-3 increases the cytoplasmic localization of the protein (7–9). In addition to 14-3-3
binding, Cdc25C is also actively transported from the nucleus through a leptomycin B-sensitive pathway
that requires an N-terminal nuclear export sequence (9).
Measurement of checkpoint kinase (Chk1, Chk2, and C-TAK1) activity
The protocol generally regarded as most sensitive for the quantitative measurement of checkpoint
kinase activity involves incubation of the checkpoint kinase sample with substrate (either a natural or
synthetic polypeptide such as Chktide substrate peptide), in the presence of Mg2+and 32P-labeled ATP.
The reaction is terminated by "spotting" a sample onto a filter paper disc, followed by immersion in acid
to precipitate the radiolabeled product. The filter papers are then washed extensively to remove
unincorporated radiolabel and the radioactivity is counted. While sensitive, this method is
labor-intensive, generates hazardous radioactive waste and depends on a radioisotope with a short
half-life. It is particularly unsuitable when kinase assays are only performed on an infrequent basis. The
CycLex Checkpoint Kinase Assay/Inhibitor Screening Kit-1 uses a peroxidase coupled
anti-phospho-Cdc25C S216 monoclonal antibody as a reporter molecule in a 96-well ELISA format.
This assay provides a non-isotopic, sensitive and specific method to measure the activities of checkpoint
kinases.