Biochemical Investigation of Zinc Transporters to Discover their Functional Mechanism in Cells

Abstract

Zinc is one of the most abundant micronutrients in the human body and it plays a vital role in many normal cellular processes. The cellular zinc level is tightly controlled by zinc transporters, including the ZIP family, which function to increase cytosolic zinc levels. The alteration of this function has been associated with human diseases, including cancer. ZIP7 belongs to the ZIP family of zinc transporters and resides on the endoplasmic reticulum store. It is responsible for releasing zinc from stores after it has been phosphorylated by CK2 on residues S275 and S276. This ZIP7-mediated zinc release inhibits tyrosine phosphatases and activates cellular tyrosine kinases, several of which are associated with progression of cancer. Moreover, two other ZIP transporters, namely ZIP6 and ZIP10, have been demonstrated to be involved in cell growth and proliferation by importing zinc across biological membranes to cause cell rounding and detachment, essential for migration and the first step of mitosis. In order to achieve this, the N-terminus of ZIP6 has to be cleaved before these transporters relocate to the plasma membrane. The present study generated novel constructs to understand the functional mechanisms of these transporters. Firstly, the activation of ZIP7 was investigated by mutating all four residues S275, S276, S293 and T294 predicted to be phosphorylated. This study found that all four of these residues were required for ZIP7 maximal activation. Secondly, the role of N-terminal cleavage of ZIP6 and ZIP10 was investigated by making chimera constructs replacing the usually cleaved N-terminus with the ZIP7 N-terminus, known not to be cleaved. This study found that the N-terminal cleavage of ZIP6 and ZIP10 was required to enable the cells to round up and detach, indicating a critical role for the N-terminus of ZIP6 and ZIP10 in this mechanism. These findings not only help us to understand the mechanism for these transporters but also enable new tools to be discovered for diseases, such as cancer, that are exacerbated by these transporters.