TNFα Post‐Translationally Targets ZnT2 to Lysosomes to Activate Lysosomal‐Mediated Cell Death and Mammary Gland Involution

Abstract

Mammary epithelial cells undergo widespread lysosomal‐mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that TNFα, a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The current objective was to determine how TNFα retargets ZnT2 to lysosomes. We hypothesized that TNFα signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in its C‐terminus, allowing adaptor protein complex‐3 (AP‐3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNFα redistributed wildtype (wt) ZnT2 from late endosomes (Pearson's coefficient=0.202±0.05 and 0.097±0.03; P<0.05) to lysosomes (0.292±0.03 and 0.649±0.03; P<0.0001), which increased lysosomal Zn (P<0.0001) and activated LCD (P<0.0001) compared to untreated cells. Mutation of the dileucine motif (L294V) eliminated the ability of TNFα to redistribute ZnT2 from late endosomes to lysosomes, increase lysosomal Zn, or activate LCD. Moreover, TNFα increased (P<0.05) AP‐3 binding to wt‐ZnT2 but not to L294V. Finally, using phospho‐ and dephospho‐mimetics of predicted phosphorylation sites (T281, T288, and S296), we found that dephosphorylated S296 was required to target ZnT2 to accumulate Zn in lysosomes and activate LCD. Our findings suggest that women with variation in ZnT2′s C‐terminus may be at risk for inadequate involution and breast disease due to the inability to target ZnT2 to lysosomes.Supported in part by R01HD058614.