The stress-inducible transcription factor ATF4 accumulates at specific rRNA-processing nucleolar regions after proteasome inhibition.

Functional protein homeostasis is essential for the maintenance of normal cellular physiology, cell growth, and cell survival. Proteasome inhibition in cancer cells can disturb protein homeostasis in such a way that synthetic proteasome inhibitors like bortezomib may selectively kill myeloma cells. Solid cancer cells appear to respond less to bortezomib which may in part be due to a rescue mechanism of the unfolded protein response/endoplasmic reticulum stress mechanism which leads to a temporary shutdown of protein biosynthesis at the translational level. Here we show that proteasome inhibition by bortezomib may also interfere with general protein biosynthesis already at the stage of nucleolar ribosome biogenesis. Ultrastructural analysis revealed not only that bortezomib induces conspicuous changes in cytoplasmic morphology but also pronounced morphological changes of the nucleolar ultrastructure, associated with an accumulation of the transcription factor ATF4 at nucleolar sites. Stress-induced intra-nucleolar ATF4 accumulation was observed in cancer cells in a dose and time dependent manner and ultrastructural studies revealed that ATF4 is preferentially localized inside the dense fibrillar and granular component of nucleoli. Furthermore, bortezomib affected not only the number of nucleoli, but also the volume and distribution of nucleolar components. The localization of ATF4 in the granular component of nucleoli together with its association with nascent RNA transcripts in cells undergoing proteotoxic cell stress could suggest a new function for ATF4 in cell stress management.