Skorupa, Jennifer A.. IGFBP-3 induced by ribotoxic stress is not secreted prior to nuclear localization in mammary epithelial cells. Retrieved from https://doi.org/doi:10.7282/T3D50R5W
DescriptionRibotoxic stressors such as anisomycin (ANS) and deoxynivalenol (DON) induce apoptosis in MAC-T cells. These agents also increase IGFBP-3 expression and knockdown of IGFBP-3 mitigates the apoptotic effects of these toxins. IGFBP-3 contains both a signal sequence and a nuclear localization sequence (NLS) and is thus both secreted and localized to the nucleus. Nuclear IGFBP-3 has been proposed to be important in its apoptotic effect. Following treatment with DON and ANS, nuclear IGFBP-3 is glycosylated, a hallmark of the secretory pathway. However, how it escapes the secretory pathway to traffic to the nucleus is unknown. Some studies have reported that extracellular IGFBP-3 is rapidly internalized and delivered to the nucleus, suggesting IGFBP-3 may require secretion and re-internalization prior to nuclear localization. To study trafficking of the endogenous protein, MAC-T cells were treated with ANS or DON. Fluorescent microscopy and Western immunoblot analysis demonstrated that ANS and DON induced nuclear localization of IGFBP-3. Treatment of nuclear IGFBP-3 with the deglycosylation enzyme Endoglycosidase H (Endo H) resulted in a lower molecular weight band indicating nuclear IGFBP-3 contains a mannose or hybrid type glycan. In contrast, the sugar of secreted IGFBP-3 was not truncated using Endo H, but was deglycosylated using PNGase indicating complex-type glycosylation. Cells treated with Brefeldin A (BFA), an inhibitor of anterograde transport from the ER to the Golgi, still showed nuclear movement of IGFBP-3. Glycosylation and BFA data indicate that IGFBP-3 is not secreted and re-internalized prior to nuclear localization during ribotoxic stress.