A tumor-associated K318N variant abolishes AGGF1 nuclear import by disrupting key nucleocytoplasmic transport motifs

Abstract Aberrant nucleocytoplasmic transport is linked to tumorigenesis. AGGF1 exerts nuclear tumor-suppressing functions, yet how its nuclear import is regulated remains unclear. Through deletion mapping and point mutations, we identified three motifs (272–279, 317–328 and 509–519 aa) governing AGGF1 nucleocytoplasmic distribution. Knockdown of importin β1 and inhibition of importin α1 showed that the importin α1/β1 complex is the primary nuclear import receptor. GST pull-down assays revealed that 317–328 aa and 509–519 aa, but not 272–279 aa, are required for efficient binding to importin α1. None of the three motifs alone was sufficient to efficiently drive nuclear import, whereas the combined 272–328 aa region conferred robust nuclear import. The tumor-associated K318N variant, within 317–328 aa, is absent from gnomAD and causes profound, cell-type-independent cytoplasmic mislocalization that is not attributable to enhanced CRM1-mediated nuclear export or 14-3-3α/β-dependent cytoplasmic distribution. Consequently, K318N fails to bind nuclear p53, stabilize p53, or activate its transcription, impairing AGGF1 tumor-suppressing function. RNA-seq showed K318-dependent genes enriched in Rap1 signaling, cytoskeleton regulation, and cardiac muscle morphogenesis, suggesting a predominantly loss-of-function effect. These findings identify key motifs and the nuclear import receptor for AGGF1, and reveal how a single variant disrupts nuclear localization and p53 regulation, potentially contributing to tumorigenesis.