Extracellular HspBP1 and Hsp72 synergistically activate epidermal growth factor receptor

Background information. Heat-inducible Hsp72 is the founding member of the Hsp70 (heat shock proteins of 70 kDa) family of molecular chaperones. It is localized primarily in cytoplasm and nucleus but is also found extracellularly. The source of e-Hsp72 (extracellular Hsp72) is not precisely identified and may not be the same in every situation. A number of studies demonstrated that e-Hsp72 plays an important role in cell survival, tumour rejection and immune response. However, currently little is known about regulation of e-Hsp72 function. In cells, Hsp72 is controlled by co-chaperones. An abundant co-chaperone, HspBP1 (Hsp72-binding protein 1) was found extracellularly in the serum. In the present study we analysed the secretion and function of e-HspBP1 (extracellular HspBP1). Results. A431 human squamous carcinoma cells accumulated Hsp72 and HspBP1 in chromogranin A-positive granules following heat stress or in the presence of U73122, an inhibitor of phospholipase C. Following these treatments, A431 cells also increased the secretion of both proteins into the culture medium. The secreted e-Hsp72 and e-HspBP1 were co-immunoprecipitated from the conditioned medium. Purified recombinant HspBP1 augmented e-Hsp72-mediated phosphorylation of EGFR (epidermal growth factor receptor) and its down-stream targets, ERK1 (extracellular signal-regulated kinase 1) and ERK2 in a concentration-dependent manner. Finally, a HspBP1 N-terminal domain deletion mutant and boiled recombinant HspBP1 did not affect the e-Hsp72-mediated activity. Conclusions. Heat stress and PLC (phospholipase C) inhibition result in the enhanced secretion of both Hsp72 and HspBP1. In an extracellular environment, the two chaperones interact both physically and functionally, leading to the activation of th EGFR-ERK1/2 signalling pathway. However, the magnitude of EGFR activation depends on the e-HspBP1/e-Hsp72 ratio in the medium. Extracellular chaperone-mediated activation of EGFR can provide a survival advantage to cells under heat shock and other stresses.