A ZIP6-ZIP10 heteromer controls NCAM1 phosphorylation and integration into focal adhesion complexes during epithelial-to-mesenchymal transition

Abstract

The prion protein (PrP) evolved from the subbranch of ZIP metal ion transporters comprising ZIPs 5, 6 and 10, raising the prospect that the study of these ZIPs may reveal insights relevant for understanding the function of PrP. Building on data which suggested PrP and ZIP6 are critical during epithelial-to-mesenchymal transition (EMT), we investigated ZIP6 in an EMT paradigm using ZIP6 knockout cells, mass spectrometry and bioinformatic methods. Reminiscent of PrP, ZIP6 levels are five-fold upregulated during EMT and the protein forms a complex with NCAM1. ZIP6 also interacts with ZIP10 and the two ZIP transporters exhibit interdependency during their expression. ZIP6 contributes to the integration of NCAM1 in focal adhesion complexes but, unlike cells lacking PrP, ZIP6 deficiency does not abolish polysialylation of NCAM1. Instead, ZIP6 mediates phosphorylation of NCAM1 on a cluster of cytosolic acceptor sites. Substrate consensus motif features and in vitro phosphorylation data point toward GSK3 as the kinase responsible, and interface mapping experiments identified histidine-rich cytoplasmic loops within the ZIP6/ZIP10 heteromer as a novel scaffold for GSK3 binding. Our data suggests that PrP and ZIP6 inherited the ability to interact with NCAM1 from their common ZIP ancestors but have since diverged to control distinct posttranslational modifications of NCAM1.

Zip6 Transporter Is an Essential Component of the Lymphocyte Activation Machinery

“…Zip10 is also a plausible candidate considering the regulation observed in human T cells and murine lymphocytes, and also because we found that Zip10 expression depends on the presence or absence of zinc in the extracellular medium. Moreover, Zip10 and Zip6 form heteromers in other cellular systems (7). However, in Jurkat cells, we did not observe upregulation under activation, which argues against the relevance of the heteromer in this cell type.…”

“…6C, 6D), as this drug potentially allows unspecific zinc entry and activates T cells downstream of the TCR. Remarkably, the inhibition of GSK-3 kinase could not rescue Zip6KO activation, so we discard that the underlying defect in our KO cells is a dysregulation on this enzyme, a hypothesis based on the interaction between Zip6 and GSK-3b previously described (7,23) and the required kinase inactivation for the correct activation of in ZIP6KO, ZIP6KO2, and Cas9-WT cells activated for 4 and 24 h with PMA/ionomycin (n 5 3). Bonferroni-corrected ANOVA, *p , 0.05, ***p , 0.001 compared with basal conditions, # p , 0.05, ### p , 0.001 between cell lines.…”

“…Despite the compensatory mechanisms observed by upregulation of other cell transporters in Zip6KO cell line, those proteins were unable to restore the activation program. Zip6, together with Zip5 and Zip10, belongs to a branch of Zip transporters characterized by the presence of a prion-like ectodomain that has been shown to be relevant in the interactome of Zip6 and its function during cell proliferation and development (7,31,32). Whether this specific role of Zip6 on activation might relay on these specific molecular determinants is an attractive hypothesis that requires further studies.…”

“…We also studied the role of the zinc-dependent kinase GSK-3b, which has been found to bind to Zip6 (7,23). During T cell activation, GSK-3b is inactivated by phosphorylation, allowing NFAT retention in the cell nucleus (24,25).…”

“…At the molecular level, besides being a structural component of many proteins, zinc has also been shown to be a second messenger. Thus, zinc movements influence signaling cascades by regulating key enzymes such as AKT and GSK-3 (6,7), as well as transcription programs by acting on transcription factors such as MTF-1 and NF-кB (8,9). In this dynamic scenario, several transporters and zinc binding proteins work in a coordinated way to tightly regulate cytosolic zinc concentrations.…”