TNFα Post‐Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes

Abstract

Mammary epithelial cells undergo widespread lysosomal-mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor-α (TNFα), 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 TNFα retargets ZnT2 to lysosomes. We tested the hypothesis that TNFα 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 TNFα redistributed wild-type (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 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.

Exome Sequencing of SLC30A2 Identifies Novel Loss- and Gain-of-Function Variants Associated with Breast Cell Dysfunction

“…Additionally, preservation of normal cell cycle/DNA damage check-point controls is important during pubertal growth and development, during ductal/ alveolar morphogenesis and expansion during pregnancy and lactation, as well as for the massive degree of programed cell death that occurs during involution upon weaning. Moreover, our observation that the Q 71 H, D 103 E, T 288 S and T 312 K variants showed increased localization with lysosomes is a significant finding since we recently reported that ZnT2 relocalization to lysosomes induces lysosomal-mediated cell death and involution [34]. In addition to D 103 E, K 66 N had the most profound effects on ZnT2 localization, Zn transport and cell function in vitro.…”

“…One possibility to explain this inconsistency is that unknown secondary factors may modify the function of the T 288 S variant in the mammary gland during lactation. For example, T 288 is predicted to be a phosphorylation target (http://www.cbs.dtu.dk/services/NetPhos) and may be responsive to endogenous or exogenous cues, such as hormone [29] or cytokine [34] signaling. Defects in Zndependent processes regulated by ZnT2 are also consistent with the higher milk Na/K ratio seen in women expressing the T 288 S variant.…”

Differential Targeting of SLC30A10/ZnT10 Heterodimers to Endolysosomal Compartments Modulates EGF‐Induced MEK/ERK1/2 Activity

“…The dileucine motif was uncovered by phosphorylation induced by tumor necrosis factor alpha. Thus, it is possible that AP‐3 could also mediate the localization of ZnT2 in SLMVs in PC12 cells, which could be upregulated by phosphorylation . Overexpression of both ZnT3 and ZnT2 did not increase the level of ZnT2 into SLMVs, suggesting that ZnT2 homodimerization could be necessary for localization into this compartment, as we showed for ZnT3 .…”

Molecular Basis of Transient Neonatal Zinc Deficiency

“…This may suggest that there is a specific mechanism that causes haploinsufficiency of ZnT2 in mammary gland epithelial cells, as based on the current knowledge, ZnT2 mutations are not known to affect other tissues and organs and did not induce other related symptoms in women carrying these mutations. However, post-translational modifications at the C terminus of ZnT2 were recently shown to be responsible for an altered subcellular localization of ZnT2 from early endosomes to lysosomes upon weaning (35). It will be interesting to investigate the effect of these different ZnT2 mutations presented in the present study, all of which localized to the C terminus of ZnT2, on its localization during the weaning period.…”