Heat Shock Protein 90 Regulates The Stability Of C-Jun In HEK293 Cells

The 90-kDa Heat Shock Protein (HSP90) is one of the most abundant cytosolic proteins in eukaryotes. It normally functions as a molecular chaperone to participate in folding of newly synthesized proteins, refolding of denatured proteins after stress and regulating of client proteins' stability and activity. Up to now, over 100 HSP90 client proteins have been found, which mainly include transcription factors, protein kinases, polymerases and so on. Unlike other heat shock proteins, there are specific inhibitors for HSP90. Geldanamycin (GA) and its ramifications, radicicol (RAD) are the familiar ones. They have been routinely used as a powerful research tool to probe the molecular chaperone function of HSP90. These drugs compete with ATP and bind tightly to the HSP90 N terminal ATP/ADP pocket to inhibit its chaperone activity, then the immature or misfolded client proteins are targeted for degradation by proteasome, ultimately a lot of biologic activities such as transcriptional regulation and signal transduction were influenced. On the other hand, as a regular peptide aldehyde proteasome inhibitor, MG132 (Z-Leu-Leu-Leu-CHO) blocks the specific degradation of substance proteins by proteasome. In addition, c-Jun is a significant component protein of the important nucleolus transcription factor AP-1, which is widely involved in transcriptional regulation and signal transduction in cells. In this present study, we tried to identify a potential candidate for client protein that HSP90 could regulate. It has been reported that heat shock remarkably induces elevated expression of hsp90, c-jun genes while notably decreases expression of integrin alpha-4 and transforming growth factor beta in Jurkat cells. Thus, the c-Jun protein is worthy of our note.Here we show that GA declines the level of endogenous c-Jun in Human embryonic kidney 293 (HEK293) cells in a time and dose dependent manner, and this decrease can be reversed by transfection of HSP90 plasmids. Transfection of HSP90 plasmids alone also increases the protein level of endogenous c-Jun, but does not obviously affect c-Jun mRNA level in HEK293 cells. Besides, we find that HSP90 prolongs the half-life of c-Jun by stabilizing c-Jun protein; the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocks degradation of c-Jun promoted by GA. On the other hand, transfection of HSP90 plasmids does not obviously alter phosphorylation of c-Jun, and Jun-2 luciferase activity assay indicates that over-expression of HSP90 elevates the total protein activity of c-Jun in HEK293 cells. In conclusion, all the evidences support that the molecular chaperone HSP90 up-regulates the stability of c-Jun protein, which influences the total protein activity of c-Jun in HEK293 cells.