Downregulation of ANP32B, a novel substrate of caspase-3, enhances caspase-3 activation and apoptosis induction in myeloid leukemic cells

The acidic leucine-rich nuclear phosphoprotein 32 (ANP32)B has been reported to regulate gene expression by acting as a histone chaperone or modulate messenger RNA trafficking by serving as a HuR ligand. However, its exact cellular functions are poorly understood. By utilizing a proteomics-based approach, in this work, we identify that the human ANP32B protein is cleaved during Apoptosis induction by NSC606985, a novel camptothecin analog. Further investigation shows that various Apoptosis inducers cause a decrease of full-length ANP32B in multiple cell lines with a concomitant increase of an approximately 17 kDa fragment. The proteolytic cleavage of ANP32B is inhibited by a specific Caspase-3 inhibitor Z-DEVD-fmk, and it cannot be seen in NSC606985-induced death of caspase-3-deficient MCF-7 cells. In vitro Caspase cleavage assay and mutagenesis experiment reveal that ANP32B is a direct substrate of Caspase-3 and it is primarily cleaved at the sequence of Ala-Glu-Val-Asp, after Asp-163. Additionally, the reduced expression of endogenous ANP32B by specific small interfering RNA enhances Caspase-3 activation and Apoptosis induction by NSC606985 and etoposide. These results suggest that ANP32B is a novel substrate for Caspase-3 and acts as a negative regulator for Apoptosis, the mechanism of which remains to be explored.