Mdm2 and PCAF increase Chk2 ubiquitination and degradation independently of their intrinsic E3 ligase activities.
We have previously shown that Chk2 stability, which is increased after DNA damage in some cells, is regulated in part through phosphorylation at S456 and that a mutant Chk2 (S456A) is hyperubiquitinated and turned over more rapidly than wild-type Chk2.(1) Here we show that the S456A mutant preferentially binds to Mdm2 compared to wild-type Chk2 and downregulation of Mdm2 with siRNA can rescue the observed destabilization of S456A Chk2 following DNA damage. Ubiquitination of wild-type Chk2 is increased by co-expression of Mdm2, and increasing amounts of Mdm2 enhance degradation of both wild-type and mutant S456A Chk2. Surprisingly, however, an E3 ligase defective version of Mdm2 (DeltaC7) also increases ubiquitination and degradation of Chk2 when transfected into cells, suggesting that the ability of Mdm2 to affect Chk2 turnover is independent of its intrinsic E3 ligase function. Recently the acetyltransferase PCAF was shown to have intrinsic E3 ligase activity against itself and Mdm2. We found that PCAF interacts with both Mdm2 and Chk2 and that PCAF increases Chk2 ubiquitination and degradation. Interestingly as well, a PCAF deletion mutant that lacks E3 ligase activity towards Mdm2 also enhances Chk2 degradation. We hypothesize that Mdm2 and PCAF may function as part of a multi-subunit E3 complex in their regulation of Chk2 turnover.
Kass
Elizabeth M
EM
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Poyurovsky
Masha V
MV
Zhu
Yan
Y
Prives
Carol
C
D000818:Animals; D002460:Cell Line; D064447:Checkpoint Kinase 2; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D050880:p300-CBP Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000099224:p300-CBP-Associated Factor
D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; D051736:Proto-Oncogene Proteins c-mdm2; D034741:RNA, Small Interfering; D044767:Ubiquitin-Protein Ligases; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C497206:MDM2 protein, human; C578634:CHEK2 protein, human