Promyelocytic Leukemia Activates Chk2 by Mediating Chk2 Autophosphorylation

Pier Paolo Pandolfi,Chang-Hun Lee,Jay H Chung,Shutong Yang,Myung K Kim,Alexandra L Brown,Jae-Hoon Jeong

doi:10.1074/jbc.m604391200

Abstract

Chk2 is a kinase critical for DNA damage-induced apoptosis and is considered a tumor suppressor. Chk2 is essential for p53 transcriptional and apoptotic activities. Although mutations of p53 are present in more than half of all tumors, mutations of Chk2 in cancers are rare, suggesting that Chk2 may be inactivated by unknown alternative mechanisms. Here we elucidate one such alternative mechanism regulated by PML (promyelocytic leukemia) that is involved in acute promyelocytic leukemia (APL). Although p53-inactivating mutations are extremely rare in APL, t(15;17) chromosomal translocation which fuses retinoic acid receptor (RARalpha) to PML is almost always present in APL, while the other PML allele is intact. We demonstrate that PML interacts with Chk2 and activates Chk2 by mediating its autophosphorylation step, an essential step for Chk2 activity that occurs after phosphorylation by the upstream kinase ATM (ataxia telangiectasia-mutated). PML/RARalpha in APL suppresses Chk2 by dominantly inhibiting the auto-phosphorylation step, but inactivation of PML/RARalpha with alltrans retinoic acid (ATRA) restores Chk2 autophosphorylation and activity. Thus, by fusing PML with RARalpha, the APL cells appear to have achieved functional suppression of Chk2 compromising the Chk2-p53 apoptotic pathway.

Highlights

Chk2 is a kinase critical for DNA damage-induced apoptosis and is considered a tumor suppressor
Mutations of p53 are present in more than half of all tumors, mutations of Chk2 in cancers are rare, suggesting that Chk2 may be inactivated by unknown alternative mechanisms. We elucidate one such alternative mechanism regulated by PML that is involved in acute promyelocytic leukemia (APL)
P53inactivating mutations are extremely rare in APL, t(15;17) chromosomal translocation which fuses retinoic acid receptor (RAR␣) to PML is almost always present in APL, while the other PML allele is intact

Summary

EXPERIMENTAL PROCEDURES

Mice and Cells—129Sv PMLϪ/Ϫ mice [19](Pandolfi PP, Sloan-Kettering, NY) and 129Sv/C57BL/6 Chk2Ϫ/Ϫ mice Expression vectors for GST-PML proteins, HA-PML IV and V5-Chk have been described elsewhere [6]. For the solution binding assays shown, the strep-tagged Chk proteins were incubated with GST-PML IV extracts (100 ul) or GST tag control extract (100 ul) in binding buffer at 4 °C, overnight. The immunoprecipitating anti-HA or anti-V5 antibodies were conjugated with protein A-agarose beads in cell lysis buffer for 2 h at 4 °C. The cells were incubated with monoclonal anti-human PML (PG-M3, SC-966, 1:100 dilution, Santa Cruz Biotechnology), polyclonal anti-Chk (FL) or anti-Chk (H-300, SC-9064, 2 ␮g/ml, Santa Cruz), polyclonal anti-HA (SC-805, 1:100 dilution, Santa Cruz Biotechnology) or monoclonal anti-V5 antibodies (1:500 dilution, Invitrogen) at 4 °C overnight. Sion regarding Chk localization, we performed immunofluorescence confocal microscopy with several commercially available anti-Chk antibodies (see “Experimental Procedures”) as well as our anti-Chk cells.

RESULTS

Confocal microscopy revealed that

Findings

DISCUSSION