ATM is the primary kinase responsible for phosphorylation of Hsp90α after ionizing radiation.

Heat shock protein 90 is a chaperone that plays an essential role in the stabilization of a large number of signal transduction molecules, many of which are associated with oncogenesis. An Hsp90 isoform (Hsp90a) has been shown to be selectively phosphorylated on two N-terminal threonine residues (threonine 5 and 7) and is involved in the DNA damage response and apoptosis. However, the kinase that phosphorylates Hsp90a after ionizing radiation (IR) and its role in post-radiation DNA repair remains unclear. Inasmuch as several proteins of the DNA damage response machinery are Hsp90 clients, the functional consequences of Hsp90a phosphorylation following IR have implications for the design of novel radiosensitizing agents that specifically target the Hsp90a isoform. Here we show that ATM phosphorylates Hsp90a at the T5/7 residues immediately after IR. The kinetics of Hsp90a T5/7 phosphorylation correlate with the kinetics of H2AX S139 phosphorylation (gamma H2AX). Although Hsp90a is located in both the cytoplasm and nucleus, only nuclear Hsp90a is phosphorylated by ATM after IR. The siRNA mediated knockdown of Hsp90a sensitizes head and neck squamous cell carcinoma cells, lung cancer cells and lung fibroblasts to IR. Furthermore, MEF cells that are Hsp90a null have reduced levels of gamma H2AX indicating that Hsp90a is important for the formation of gamma H2AX. Thus, this study provides evidence that Hsp90a is a component of the signal transduction events mediated by ATM following IR, and that Hsp90a loss decreases gamma H2AX levels. This work supports additional investigation into Hsp90a T5/7 phosphorylation with the goal of developing targeted radiosensitizing therapies.