Autophosphorylated residues involved in the regulation of human chk2 in vitro.
Human checkpoint kinase 2 is a major actor in checkpoint activation through phosphorylation by ataxia telangiectasia mutated in response to DNA double-strand breaks. In the absence of de novo DNA damage, its autoactivation, reported in the event of increased Cds1/checkpoint kinase 2 (Chk2) expression, has been attributed to oligomerization. Here we report a study performed on autoactivated recombinant Chk2 proteins that aims to correlate kinase activity and phosphorylation status. Using a fluorescence-based technique to assay human checkpoint kinase 2 catalytic activity, slight differences in the ability to phosphorylate Cdc25C were observed, depending on the recombinant system used. Using mass spectrometry, the phosphorylation sites were mapped to identify sites potentially involved in the kinase activity. Five phosphorylated positions, at Ser120, Ser260, Thr225, Ser379 and Ser435, were found to be common to bacteria and insect cells expression systems. They were present in addition to the six known phosphorylation sites induced by ionizing radiation (Thr68, Thr432, Thr387, Ser516, Ser33/35 and Ser19) detected by immunoblotting. After phosphatase treatment, Chk2 regained activity via autorephosphorylation. The determination of the five common sites and ionizing-radiation-inducible positions as rephosphorylated confirms that they are potential positive regulators of Chk2 kinase activity. For Escherichia coli's most highly phosphorylated 6His-Chk2, 13 additional phosphorylation sites were assigned, including 7 novel sites on top of recently reported phosphorylation sites.
CEA, DSV, iBEB, Service de biochimie et toxicologie nucléaire, Centre de Marcoule, BP 17171, F-30207 Bagnols-sur-Cèze Cedex, France.
Guillaume
G
Gabant
Alain
A
Lorphelin
Nathalie
N
Nozerand
Charles
C
Marchetti
Laurent
L
Bellanger
Alain
A
Dedieu
Eric
E
Quéméneur
Béatrice
B
Alpha-Bazin
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D064447:Checkpoint Kinase 2; D019295:Computational Biology / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008954:Models, Biological; D019913:Nuclear Localization Signals / Q000737:chemistry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D020687:cdc25 Phosphatases / Q000378:metabolism
D000596:Amino Acids; D019913:Nuclear Localization Signals; D011994:Recombinant Proteins; D012694:Serine; D005982:Glutathione Transferase; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases