A C-terminal cytoplasmic retention motif and nuclear localization signal regulates nuclear import of TP53INP2.

Tumor protein p53 inducible nuclear protein 2 (TP53INP2; also known as DOR) is a multifunctional protein involved in transcriptional coactivation, ribosomal RNA synthesis and autophagy, regulated by subcellular localization. Using CRISPR/Cas9-generated TP53INP2-knockout HeLa cells reconstituted with EGFP-TP53INP2, we show that TP53INP2 is predominantly degraded by nuclear proteasomes under basal conditions. Under stress, including starvation and various chemical stress inducers, TP53INP2 accumulates in the cytoplasm independently of ATG5, CRM1-mediated export, phosphorylation, ubiquitylation or acetylation. We identify a nuclear localization signal (NLS) overlapping a nucleolar localization signal (NoLS) in the C-terminus, which mediates nuclear import and nucleolar enrichment. Deletion of this region redirects TP53INP2 to LC3B-positive puncta. A conserved nine-amino-acid cytoplasmic retention motif (CRM) in the C-terminus prevents nuclear re-entry under stress. This motif and regulation of subcellular localization is conserved in the related TP53INP1 protein. Fluorescence recovery after photobleaching (FRAP) and importin-binding assays show that nutrient starvation disrupts nuclear import of TP53INP2. Finally, we show that starvation enhances TP53INP2 translation via the m6A demethylase FTO, without altering mRNA stability. These findings uncover coordinated regulation of TP53INP2 localization and turnover by cellular stress.