A comparative biochemical analysis was performed using recombinant human protein 
kinase Chk2 (checkpoint kinase 2) expressed in bacteria and insect cells. 
Dephosphorylated, inactive, recombinant human Chk2 could be reactivated in a 
concentration-dependent manner. Despite distinct time-dependent 
autophosphorylation kinetics by monitoring the phosphorylation of amino acid 
residues T68, S19, S33/35, T432, in Chk2 wildtype and Chk2 mutants (T68A, T68D 
and Q69E) they gave identical specific activities. However, upon gel filtration 
of Chk2 wildtype and the mutants, only Chk2 wildtype and the T68D mutant led to 
the formation of a 'pure' dimer; dephosphorylated wildtype Chk2 eluted as a 
monomer. Transfection of HEK293 cells with Chk2 wildtype and Chk2 mutants in the 
absence or presence of DNA damage showed significant T68 phosphorylation already 
in the absence of DNA damaging reagents. Upon DNA damage, phosphorylation of 
additional Chk2 sites was observed (S19, S33/35). A comparison of ATM+/+ and 
ATM-/- cells with respect to phosphorylation of residues T68, S19, S33/35 in the 
absence and presence of DNA damage showed in all cases phosphorylation of T68, 
although signal intensity was increased ca. three-fold after DNA damage. Mass 
spectrometric analyses of human recombinant Chk2 isolated from bacteria and 
insect cells showed distinct differences. The number of phosphorylated residues 
in human recombinant Chk2 isolated from bacteria was 16, whereas in the case of 
the recombinant human Chk2 from insect cells it was 8. Except for phosphorylated 
amino acid T378 which was not found in the Chk2 isolated from bacteria, all 
other phosphorylated residues identified in human Chk2 from insect cells were 
present also in Chk2 from bacteria.