Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction

Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ER alpha interactions remain unclear. For example, we do not know which Hsps are the major or minor ER alpha interactants and whether or not different Hsp isoforms associate equally with ER alpha. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ER alpha in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ER alpha, followed by two Hsp90s (Hsp90 alpha and Hsp90 beta) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90 alpha was found to be 2-3 times more abundant than Hsp90 beta in the ER alpha-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ER alpha-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ER alpha in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ER alpha-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17 beta-estradiol (E2) did not change this. In addition, E2-treatment weakened the ER alpha-Hsc70 interaction but had no effect on the ER alpha-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ER alpha in both forms of the chromatins in MCF7 cells.