Exploring the intramolecular phosphorylation sites in human Chk2.
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.
Olsen
Birgitte B
BB
Biomedical Research Group, Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Larsen
Martin R
MR
Boldyreff
Brigitte
B
Niefind
Karsten
K
Issinger
Olaf-Georg
OG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D064447:Checkpoint Kinase 2; D004268:DNA-Binding Proteins / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics
D000596:Amino Acids; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D010455:Peptides; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases