TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes

Mammary epithelial cells undergo widespread lysosomal-mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor-alpha (TNF alpha), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study is to determine how TNF alpha retargets ZnT2 to lysosomes. We tested the hypothesis that TNFa signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C-terminus of ZnT2, allowing adaptor protein complex-3 (AP-3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNF alpha redistributed wild-type (WT) ZnT2 from late endosomes (Pearson's coefficient = 0.202 +/- 0.05 and 0.097 perpendicular to 0.03; P<0.05) to lysosomes (0.292 perpendicular to 0.03 and 0.649 perpendicular to 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 TNF alpha to redistribute ZnT2 from late endosomes to lysosomes, increase lysosomal Zn, or activate LCD. Moreover, TNF alpha increased (P < 0.05) AP-3 binding to wt ZnT2 but not to L294V immunoprecipitates. 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 the C-terminus of ZnT2 may be at risk for inadequate involution and breast disease due the inability to traffic ZnT2 to lysosomes. (C) 2015 Wiley Periodicals, Inc.