Posttranslational Regulation of the Menkes Copper ATPase (Atp7a) in Intestinal Epithelial Cells

Abstract

Atp7a pumps copper into the trans‐Golgi for cuproenzyme synthesis and also functions to export copper from enterocytes. It was noted that copper treatment of rat intestinal epithelial (IEC‐6) cells increased Atp7a protein abundance without effecting mRNA levels. We thus hypothesized that intracellular copper stabilizes the Atp7a protein and studies were designed to test this possibility. Differentiated IEC‐6 cells were treated with a protein translation inhibitor (cyclohexamide [CHX]) at different concentrations for different times to estimate Atp7a protein half‐life. CHX at 10 μg/ml for 48 hours led to >50% reduction in immunoreactive Atp7a protein. Next, IEC‐6 cells were exposed to 10 μg/ml CHX in the presence or absence of added copper (150 μM) for 48 hours and Atp7a protein abundance was determined. Results showed that copper stabilized the Atp7a protein, as protein abundance in CHX/Cu treated cells was similar to control cells treated with vehicle while CHX alone led to >50% protein reduction. To determine the mechanism, further studies will be designed to assess the role of the 6 metal binding domains of Atp7a, as not all of them are required for Atp7a copper transport function. We conclude that Atp7a is stabilized by copper, which could be particularly relevant during iron deficiency when the copper content of the intestinal mucosa increases and Atp7a protein abundance in enterocytes increases dramatically.Grant Funding Source : 1R01 DK074867