TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes

Stephen R. Hennigar, Shannon Kelleher

Research output: Contribution to journalArticle

14 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2345-2350
Number of pages6
JournalJournal of Cellular Physiology
Volume230
Issue number10
DOIs
StatePublished - Oct 1 2015

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Lysosomes
Cell death
Zinc
Tumor Necrosis Factor-alpha
Adaptor Protein Complex 3
Cell Death
Endosomes
Phosphorylation
Breast Diseases
Human Mammary Glands
Breast
Cytokines
Epithelial Cells
Mutation

All Science Journal Classification (ASJC) codes

  • Physiology
  • Clinical Biochemistry
  • Cell Biology

Cite this

Hennigar, Stephen R. ; Kelleher, Shannon. / TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes. In: Journal of Cellular Physiology. 2015 ; Vol. 230, No. 10. pp. 2345-2350.
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TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes. / Hennigar, Stephen R.; Kelleher, Shannon.

In: Journal of Cellular Physiology, Vol. 230, No. 10, 01.10.2015, p. 2345-2350.

Research output: Contribution to journalArticle

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AB - 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.

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