The integrity of the DNA damage response pathway is essential for preventio
n of neoplastic transformation. Several proteins involved in this pathway i
ncluding p53, BRCA1, and ATM are frequently mutated in human cancer. Checkp
oint kinase 2 (Chk2) is a DNA damage-activated protein kinase that lies dow
nstream of ATM in this pathway. Recently, heterozygous germline mutations i
n Chk2 have been identified in a subset of patients with Li-Fraumeni syndro
me, a highly penetrant familial cancer phenotype, suggesting that Chk2 is a
tumor suppressor gene. In this study, we have reported the biochemical cha
racterization of the four tumor-associated Chk2 mutants. Two of the reporte
d Chk2 mutations identified in Li-Fraumeni syndrome result in loss of Chk2
kinase activity, Whereas one mutation within the Chk2 forkhead homology-ass
ociated (FHA) domain, R145W, retains some basal kinase activity, this mutan
t cannot be phosphorylated at an ATM-dependent phosphorylation site (Thr-68
) and cannot be activated following gamma radiation. Wild-type Chk2 exists
mainly in a protein complex of M-r similar to 200,000 whereas the R145W mut
ant forms a larger, presumably inactive complex in the cell. The other FHA
domain mutant, I157T, behaves as wild-type Chk2 in all the assays used here
. Because the FHA domain is involved in protein-protein interactions, this
mutation may affect associations of Chk2 with other proteins. Additionally,
we have shown that Chk2 can also be inactivated by down-regulation of its
expression in cancer cells. Thus, Chk2 may be inactivated by multiple mecha
nisms in the cell.