Abstract
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 (Hsp90α) 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 Hsp90α 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 Hsp90α phosphorylation following IR have implications for the design of novel radiosensitizing agents that specifically target the Hsp90α isoform. Here we show that ATM phosphorylates Hsp90α at the T5/7 residues immediately after IR. The kinetics of Hsp90α T5/7 phosphorylation correlate with the kinetics of H2AX S139 phosphorylation (γH2AX). Although Hsp90α is located in both the cytoplasm and nucleus, only nuclear Hsp90α is phosphorylated by ATM after IR. The siRNA mediated knockdown of Hsp90α sensitizes head and neck squamous cell carcinoma cells, lung cancer cells and lung fibroblasts to IR. Furthermore, MEF cells that are Hsp90α null have reduced levels of γH2AX indicating that Hsp90α is important for the formation of γH2AX. Thus, this study provides evidence that Hsp90α is a component of the signal transduction events mediated by ATM following IR, and that Hsp90α loss decreases γH2AX levels. This work supports additional investigation into Hsp90α T5/7 phosphorylation with the goal of developing targeted radiosensitizing therapies.
Highlights
The 90 kDa heat shock protein (Hsp90) is an abundant and essential stress protein expressed in all eukaryotic cells that aids in the post-translational folding and stabilization of a wide variety of proteins called Hsp90 “clients”, and it is an important regulator of protein homeostasis [1, 2]
Hsp90α T5/7 phosphorylation correlated with H2AX S139 phosphorylation
Hsp90α, Hsp90β, and heat shock protein 70 (Hsp70) protein levels did not change upon treatment with increased doses of ionizing radiation (IR) in any of these cell lines
Summary
The 90 kDa heat shock protein (Hsp90) is an abundant and essential stress protein expressed in all eukaryotic cells that aids in the post-translational folding and stabilization of a wide variety of proteins called Hsp90 “clients”, and it is an important regulator of protein homeostasis [1, 2]. The two isoforms of human Hsp, Hsp90α (gene Hsp90AA1) and Hsp90β (Hsp90AB1), are 86 per cent homologous in amino acid sequence, are expressed from separate genes and are together referred to as Hsp90 [3]. The functions of Hsp90α and Hsp90β are thought to be largely redundant, but there are some differences. Mouse embryos lacking Hsp90β die at implantation [5]. These differences make Hsp90α an attractive pharmacological target and highlights the importance of studying the consequences of Hsp90α specific posttranslational modifications. Dimerization of Hsp through the C-terminus is required for in vivo function [6]
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