Regulation of Chk2 phosphorylation by interaction with protein phosphatase 2A via its B' regulatory subunit.
Chk2 is a key player of the DNA damage signalling pathway. To identify new regulators of this kinase, we performed a yeast two-hybrid screen and found that Chk2 associated with the B' regulatory subunit of protein phosphatase PP2A. In vitro GST-Chk2 pulldowns demonstrated that B'gamma isoforms bound to Chk2 with the strongest apparent affinity. This was confirmed in cellulo by co-immunoprecipitation after overexpression of the respective partners in HEK293 cells. The A and C subunits of PP2A were present in the complexes, suggesting that Chk2 was associated with a functionnal PP2A. In vitro kinase assays showed that B'gamma3 was a potent Chk2 substrate. This phosphorylation increased the catalytic phosphatase activity of PP2A measured on MAP kinase-phosphorylated myelin basic protein as well as on autophosphorylated Chk2. Finally, we demonstrated that overexpressing B'gamma3 in HEK293 suppressed the phosphorylation of Chk2 induced by a genotoxic treatment, suggesting that PP2A may counteract the action of the checkpoint kinase in living cells.
Dozier
Christine
C
Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération, UMR 5088 CNRS, Institut Fédératif de Recherche 109, Université Paul Sabatier, Bât 4R3-B1, 118 route de Narbonne, 31062 Toulouse, France. dozier@cict.fr
Bonyadi
Mortaza
M
Baricault
Laurent
L
Tonasso
Laure
L
Darbon
Jean-Marie
JM
D002460:Cell Line; D064447:Checkpoint Kinase 2; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D020798:Two-Hybrid System Techniques
D021122:Protein Subunits; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2