Abstract
The zinc transporter ZnT2 imports zinc into secretory vesicles and regulates zinc export from the mammary epithelial cell. Mutations in ZnT2 substantially impair zinc secretion into milk. The lactogenic hormone prolactin (PRL) transcriptionally increases ZnT2 expression through the Jak2/STAT5 signaling pathway, increasing zinc accumulation in secretory vesicles and zinc secretion. Herein, we report that PRL post-translationally stimulated ZnT2 ubiquitination, which altered ZnT2 trafficking and augmented vesicular zinc accumulation and secretion from mammary epithelial cells in a transient manner. Ubiquitination then down-regulated zinc secretion by stimulating degradation of ZnT2. Mutagenesis of two N-terminal lysine residues (K4R and K6R) inhibited ZnT2 ubiquitination, vesicular zinc accumulation and secretion, and protein degradation. These findings establish that PRL post-translationally regulates ZnT2-mediated zinc secretion in a multifactorial manner, first by enhancing zinc accumulation in vesicles to transiently enhance zinc secretion and then by activating ubiquitin-dependent ZnT2 degradation. This provides insight into novel mechanisms through which ZnT2 and zinc transport is tightly regulated in mammary epithelial cells.
Highlights
ZnT2 regulates zinc export from mammary cells
PRL Transiently Stimulates ZnT2-mediated Zinc Secretion from mammary epithelial cells (MECs)—We first established the effects of PRL stimulation on zinc secretion in MECs preloaded with 65Zn
Our data demonstrated that PRL treatment significantly increased zinc secretion ϳ2-fold (0.1802 Ϯ 0.004 area under the curve (AUC) units) in an acute and transient manner compared with untreated cells
Summary
ZnT2 regulates zinc export from mammary cells. Results: Prolactin stimulates ZnT2 ubiquitination, targeting ZnT2 to vesicles and activating zinc accumulation to transiently enhance zinc secretion dependent upon Lys4/Lys, after which ZnT2 is degraded. These findings establish that PRL post-translationally regulates ZnT2-mediated zinc secretion in a multifactorial manner, first by enhancing zinc accumulation in vesicles to transiently enhance zinc secretion and by activating ubiquitin-dependent ZnT2 degradation This provides insight into novel mechanisms through which ZnT2 and zinc transport is tightly regulated in mammary epithelial cells. Ubiquitination and degradation of the zinc transporters ZRT1 [23, 24] and ZIP4 [25] modulate zinc transport in yeast and mammalian cells, respectively This suggests that PRL-mediated ubiquitination may play a role in post-translationally regulating zinc transport in MECs. in the present study, we demonstrated that PRL activates the ubiquitination of ZnT2, stimulating ZnT2 trafficking and zinc accumulation in vesicles and secretion, followed by protein degradation. We determined that two N-terminal lysine residues (Lys and Lys6) are required for PRL-induced ubiquitination and regulation of ZnT2
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