Checkpoint kinase 2 (Chk2) monomers or dimers phosphorylate Cdc25C after DNA damage regardless of threonine 68 phosphorylation.
We have purified and characterized human Chk2 both from baculovirus-infected insect cells and from either untreated or DNA damage-stressed human HCT116 cells. Chk2 from unstressed human cells is largely monomeric and inactive in phosphorylating its substrate, Cdc25C. It is also unphosphorylated at Thr-68, a site that is the target of the ataxia telangiectasia-mutated protein kinase. After treatment of HCT116 cells with a radiomimetic compound neocarzinostatin, active Chk2 exists as stable Thr-68-phosphorylated dimers as well as interconvertable Thr-68-unphosphorylated monomers and dimers. Interestingly, Chk2 from insect cells behaves by all criteria tested like active Chk2 from neocarzinostatin-treated HCT116 cells. Based on Stokes radius and sedimentation coefficient values, Chk2 monomers and dimers have asymmetric rather than globular shapes. Both Thr-68-phosphorylated and Thr-68-unphosphorylated forms of active Chk2 are capable of phosphorylating Cdc25C. Thus, although phosphorylation of Thr-68 may be required for initial oligomerization and activation of Chk2, it is not needed for maintenance of dimerization or kinase activity.
Ahn
Jinwoo
J
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Prives
Carol
C
D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D019281:Dimerization; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*; D020687:cdc25 Phosphatases / Q000378:metabolism*
D018797:Cell Cycle Proteins; D011993:Recombinant Fusion Proteins; D013912:Threonine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases