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Heat shock protein 90α (HSP90α), a substrate and chaperone of DNA-PK necessary for the apoptotic response.

Proceedings of the National Academy of Sciences of the United States of America (2012-07-04)
Stéphanie Solier, Kurt W Kohn, Bradley Scroggins, Wanping Xu, Jane Trepel, Leonard Neckers, Yves Pommier
ABSTRAKT

The "apoptotic ring" is characterized by the phosphorylation of histone H2AX at serine 139 (γ-H2AX) by DNA-dependent protein kinase (DNA-PK). The γ-H2AX apoptotic ring differs from the nuclear foci patterns observed in response to DNA-damaging agents. It contains phosphorylated DNA damage response proteins including activated Chk2, activated ATM, and activated DNA-PK itself but lacks MDC1 and 53BP1, which are required to initiate DNA repair. Because DNA-PK can phosphorylate heat shock protein 90α (HSP90α) in biochemical assays, we investigated whether HSP90α is involved in the apoptotic ring. Here we show that HSP90α is phosphorylated by DNA-PK on threonines 5 and 7 early during apoptosis and that both phosphorylated HSP90α and DNA-PK colocalize in the apoptotic ring. We also show that DNA-PK is a client of HSP90α and that HSP90α is required for full DNA-PK activation, γ-H2AX formation, DNA fragmentation, and apoptotic body formation. In contrast, HSP90 inhibition by geldanamycin markedly enhances TRAIL-induced DNA-PK and H2AX activation. Together, our results reveal that HSP90α is a substrate and chaperone of DNA-PK in the apoptotic response. The response of phosphorylated HSP90α to TRAIL and its localization to the γ-H2AX ring represent epigenetic features of apoptosis that offer insights for studying and monitoring nuclear apoptosis.

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Sigma-Aldrich
Anti-γ-Tubulin antibody, Mouse monoclonal, clone GTU-88, purified from hybridoma cell culture
Sigma-Aldrich
Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301, clone JBW301, Upstate®, from mouse
Sigma-Aldrich
Anti-DNA-PK (Ab-2) Mouse mAb (18-2), liquid, clone 18-2, Calbiochem®