Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Aug 22;289(34):23653-61.
doi: 10.1074/jbc.M113.531145. Epub 2014 Jul 11.

Prolactin (PRL)-stimulated ubiquitination of ZnT2 mediates a transient increase in zinc secretion followed by ZnT2 degradation in mammary epithelial cells

Young Ah Seo  1 , Sooyeon Lee  2 , Stephen R Hennigar  3 , Shannon L Kelleher  4
Affiliations

Prolactin (PRL)-stimulated ubiquitination of ZnT2 mediates a transient increase in zinc secretion followed by ZnT2 degradation in mammary epithelial cells

Young Ah Seo et al. J Biol Chem. .

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.

Keywords: Mammary Gland; Prolactin; Protein Degradation; Secretion; Ubiquitination; Zinc Transporter; ZnT2.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
ZnT2 was required for PRL-stimulated acute zinc secretion in MECs. Cells expressing endogenous levels of ZnT2 (A), cells overexpressing ZnT2 (B), or ZnT2-attenuated cells (C) were preincubated in serum-free medium containing 1.0 μm ZnSO4 and 0.1 μCi of 65Zn for 3 h. Medium was replaced with serum-free medium containing diethylenetriamine pentaacetic acid (50 μm) with or without PRL, and 65Zn was measured for up to 8 h. Values represent mean pmol of zinc/μg of protein ± S.D. (n = 4 samples/time point). Analysis of AUC indicates a significant difference of PRL treatment in cells expressing endogenous levels of ZnT2 (A) and overexpressing ZnT2 (B), p < 0.05. No effect of PRL in ZnT2-attenuated cells was detected (NS). Experiments were repeated three times. D, cells were transfected with thymidine kinase promoter-linked Renilla luciferase vector (internal control) and 4×MRE-pGL3 and either pGL3 empty vector (EV), scrambled siRNA (Mock), or ZnT2 siRNA. Luminescence was measured as an index of changes in cytoplasmic zinc pools. Data represent mean luminescence ± S.D. (error bars) (n = 4 samples/time point). *, significant effect of ZnT2KD on luciferase activity, p < 0.05. Experiments were repeated two times.
FIGURE 2.
FIGURE 2.
PRL stimulated ZnT2 ubiquitination. A, cells were transfected to express ZnT2-HA and Myc-ubiquitin (Myc-UB WT) alone or in combination or to co-express ZnT2-HA and Myc-ubiquitin K48R (Myc-UB K48R). Following a 24-h recovery, cells were pretreated with CHX to inhibit protein synthesis and with MG132 to inhibit proteasomal degradation and then treated for an additional 8 h in medium (−) or medium containing PRL (+). Cell lysates were passed over anti-HA affinity matrix (IP:HA), and the retained fraction was separated by 4–20% SDS-polyacrylamide gradient gels. Membranes were immunoblotted with anti-Myc antibody (IP:HA and IB:Myc) and stripped and reprobed for anti-HA antibody (IP:HA and IB:HA) as a loading control. Total cell lysates from the same samples were used to detect total abundance of ZnT2-HA protein and β-actin. Experiments were repeated more than three times. B, cells were transfected to express ZnT2-HA and Myc-ubiquitin (Myc-UB) alone or in combination or to co-express ZnT2-HA and K4R/K6R-HA. Following a 24-h recovery, cells were pretreated with CHX to inhibit protein synthesis and with MG132 to inhibit proteasomal degradation and then treated for an additional 8 h in medium (−) or medium containing PRL (+). Cell lysates were passed over anti-HA affinity matrix (IP:HA), and the retained fraction was separated by 4–20% SDS-polyacrylamide gradient gels. Membranes were immunoblotted with anti-Myc antibody (IP:HA and IB:Myc) and stripped and reprobed for anti-HA antibody (IP:HA and IB:HA) as a loading control. Total cell lysates from the same samples were used to detect total abundance of ZnT2-HA protein and β-actin. Experiments were repeated more than three times.
FIGURE 3.
FIGURE 3.
Inability to ubiquitinate ZnT2 in response to PRL impaired ZnT2 trafficking, zinc accumulation, and secretion. A, representative confocal images of ZnT2-HA (green) and K4R/K6R-HA (green) and Rab3A (red) in MECs treated with (+PRL) or without prolactin (−PRL) for 2 h. Nucleus was stained with TOPRO and pseudocolored blue. Pearson's coefficient (R) was determined by correlation analysis using an Olympus FluoView viewer. Shown are values (−1 (no overlap of pixels) and +1 (100% overlap)) from five fields of view from two independent experiments. B, representative confocal images of FluoZin-3 (green) in cells expressing ZnT2-HA or K4R/K6R-HA in MECs treated with or without prolactin for 2 h. C, representative immunoblots of ZnT2-HA and K4R/K6R-HA detected at the cell surface by cell surface biotinylation (+) treated with and without prolactin over 8 h. Infrared images were normalized across immunoblots according to the manufacturer's instructions. D, zinc secretion from MECs expressing ZnT2-HA or K4R/K6R-HA treated with or without PRL. Data represent mean pmol of zinc/μg of protein ± S.D. (error bars) (n = 4 samples/time point). Experiments were repeated two times.
FIGURE 4.
FIGURE 4.
PRL induced degradation of ZnT2. A, representative immunoblot of ZnT2-HA detected in the total membrane fraction of cells transfected with pcDNA3.1 (Mock) or cells expressing ZnT2-HA. Cells were treated with cycloheximide (100 μg/ml) alone (CHX) to inhibit new protein synthesis or in combination with PRL (CHX + PRL) for the indicated times. Total membrane fractions from the cells were separated by SDS-PAGE, and membranes were immunoblotted with HA antibody and then stripped and reprobed for β-actin as a loading control. B, ZnT2 protein abundance in response to PRL stimulation. Band intensity of ZnT2-HA was normalized to β-actin and expressed as a percentage of untreated cells. Data represent the mean percentage of ZnT2 protein level at time 0 ± S.D.; the experiment was repeated twice. C, representative immunoblot of ZnT2-HA detected in the total membrane fraction of cells transfected with ZnT2-HA. Cells were pretreated with cycloheximide (100 μg/ml) in the presence of MG132 (proteasome inhibitor) or chloroquine (CQ; lysosome inhibitor). Cells were then treated for an additional 8 h in medium (−) or medium containing PRL (+) in the presence of the above inhibitors. Total membrane fractions from the cells were separated by SDS-PAGE, and membranes were immunoblotted with anti-HA antibody and stripped and reprobed for β-actin as a loading control. Experiments were repeated more than three times. D, data represent the mean ratio of ZnT2/β-actin ± S.E. (error bars) from untreated cells (Con), untreated cells stimulated with PRL (Con+), cells pretreated with MG132 and stimulated with PRL (MG132+), or cells pretreated with chloroquine (CQ) and stimulated with PRL (CQ+) from two independent experiments. *, significant effect of PRL treatment on the ratio of ZnT2/β-actin, p < 0.05.
FIGURE 5.
FIGURE 5.
PRL-stimulated ZnT2 degradation requires two lysine residues in the N terminus. A, representative immunoblot of ZnT2-HA in cells expressing ZnT2-HA, Lys4-HA, or Lys6-HA treated with PRL (+) compared with control (−) cells. Transfected cells were pretreated with cycloheximide and then treated with or without PRL for an 8 h, and the levels of each mutant protein were detected by anti-HA antibody. C, representative immunoblot of ZnT2-HA in cells expressing ZnT2-HA or K4R/K6R-HA treated with PRL (+) compared with control (−) cells. Transfected cells were pretreated with cychloheximide and then treated with or without PRL for 8 h, and the levels of each mutant protein were detected with anti-HA antibody. B and D, band intensity of ZnT2-HA normalized to β-actin expressed as a percentage of untreated cells. Data represent the mean percentage of ZnT2 protein level relative to untreated cells expressing ZnT2-HA ± S.D. (error bars) from two independent experiments. *, significant effect of PRL treatment on ZnT2 protein level, p < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chowanadisai W., Lönnerdal B., Kelleher S. L. (2006) Identification of a mutation in SLC30A2 (ZnT-2) in women with low milk zinc concentration that results in transient neonatal zinc deficiency. J. Biol. Chem. 281, 39699–39707 - PubMed
    1. Lasry I., Seo Y. A., Ityel H., Shalva N., Pode-Shakked B., Glaser F., Berman B., Berezovsky I., Goncearenco A., Klar A., Levy J., Anikster Y., Kelleher S. L., Assaraf Y. G. (2012) A dominant negative heterozygous G87R mutation in the zinc transporter, ZnT-2 (SLC30A2), results in transient neonatal zinc deficiency. J. Biol. Chem. 287, 29348–29361 - PMC - PubMed
    1. Itsumura N., Inamo Y., Okazaki F., Teranishi F., Narita H., Kambe T., Kodama H. (2013) Compound heterozygous mutations in SLC30A2/ZnT2 results in low milk zinc concentrations: a novel mechanism for zinc deficiency in a breast-fed infant. PloS One 8, e64045. - PMC - PubMed
    1. Lopez V., Kelleher S. L. (2009) Zinc transporter-2 (ZnT2) variants are localized to distinct subcellular compartments and functionally transport zinc. Biochem. J. 422, 43–52 - PMC - PubMed
    1. Seo Y. A., Kelleher S. L. (2010) Functional analysis of two single nucleotide polymorphisms in SLC30A2 (ZnT2): implications for mammary gland function and breast disease in women. Physiol. Genomics 42A, 219–227 - PMC - PubMed

Publication types

LinkOut - more resources