Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage.
The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.
Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267, USA.
El Mustapha
el M
Bahassi
David L
DL
Myer
Richard J
RJ
McKenney
Robert F
RF
Hennigan
Peter J
PJ
Stambrook
D001260:Ataxia Telangiectasia; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D017930:Genes, Reporter; D006801:Humans; D010766:Phosphorylation; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D000097868:Polo-like Kinases
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D004268:DNA-Binding Proteins; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C486256:PLK3 protein, human; D000097868:Polo-like Kinases; C576324:ATM protein, human; C578634:CHEK2 protein, human