Abstract: Objective： To investigate the mechanisms of nuclear export signal of androgen receptor (NESAR) in the regulation of androgen receptor (AR) protein expression and stability in prostate cancer. Methods: The green fluorescent protein fusion protein expression vectors pEGFP-AR(1-918aa), pEGFP-NESAR (743-817aa), pEGFP-NAR (1-665aa) and pEGFP-NAR-NESAR, and lysine mutants of NESAR pEGFP-NESAR K776R, pEGFP-NESAR K807R and pEGFPNESAR K776R/K807R, were transiently transfec-ted into prostate cancer cell line PC3. Fluorescence microscopy, Western blot and immunoprecipitation were used to detect NESAR regulation of androgen receptor stability. Results: Under the fluorescence microscope, NESAR-containing fusion proteins were cytoplasmic localization, and their fluorescence intensities were much weaker than those without NESAR. The expression levels of NESAR-containing fusion proteins were significantly lower than those without NESAR. The half-lives of GFP-NESAR and GFP-NAR-NESAR were less than 6 h, while the expression of GFP and GFP-NAR was relatively stable and the half-life was more than 24 h in the presence of cycloheximide. The expression levels of GFP-NESAR were significantly increased by proteasome inhibitor MG132 treatment in a dose-dependent manner; in contrast, MG132 did not show any significant effect on the protein levels of GFP. When new protein synthesis was blocked, MG132 could also prevent the degradation of GFP-NESAR in the transfected cells in the presence of cycloheximide, while it had no significant effect on GFP protein stability in the parallel experiment. GFP immunoprecipitation showed that the ubiquitination level of GFP-NESAR fusion protein was significantly higher than that of the GFP control. The mutations of lysine sites K776 and K807 in NESAR significantly reduced the level of ubiquitination, and showed increased protein stability, indicating that they were the key amino acid residues of NESAR ubiquitination. Conclusion: NESAR was unstable and decreased the stability of its fusion proteins. NESAR was the target of polyubiquitination and mediated the degradation of its fusion proteins through the ubiquitin-proteasome pathway in prostate cancer cells. Our research provides a new way to regulate the level and/or activity of AR proteins, thus helping us understand the molecular mechanisms of AR degradation and strict control of AR in the progression to castration-resistance.

Key words: Androgen receptor, Nuclear export signal of androgen receptor, Ubiquitin proteasome system, Degradation