Protein kinase CK 2 triggers cytosolic zinc signaling pathways by phosphorylation of zinc channel ZIP 7

The transition element zinc, which has recently been identified as an intracellular second messenger, has been implicated in various signaling pathways, including those leading to cell proliferation. Zinc channels of the ZIP protein family (Solute Carrier Family 39A, SLC39A) transiently increase the cytosolic free zinc (Zn 2+ ) concentration in response to extracellular signals. Here, we show that phosphorylation of evolutionarily conserved residues in zinc transporter ZIP7 is associated with the gated release of Zn 2+ from intracellular stores, leading to activation of tyrosine kinases and the phosphorylation of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Through pharmacological manipulation, proximity assay, and mutagenesis, we identified CK2 as the kinase responsible for ZIP7 activation. Together, the present results show that eukaryotic transition element channels can be activated post-translationally by phosphorylation eliciting a cell signaling cascade. Our study links the regulated release of zinc from intracellular stores to phosphorylation of kinases involved in proliferative responses and cell migration, suggesting a functional role for ZIP7 and zinc signals for these events which are characteristic of cancerous cells. Furthermore, the interaction of ZIP7 with CK2, a kinase that is antiapoptotoc and promotes cell division, highlights the potential for ZIP7 as a target for anticancer drug development. Introduction The essential role of zinc 1 is demonstrated by the deleterious effects of zinc deficiency 2 and by the link between zinc regulatory dysfunction and the pathophysiology of various disease states, including neurodegeneration 3 , inflammation 4 , diabetes 5 , cancer 6 , and others 7 . Recent data reinforces zinc’s essential role, confirming its widespread involvement in development, immunity, reproduction, endocrinology, and neurotransmission 3, 4, 8-10 . Zinc acts as a cofactor for an estimated 3,000 human proteins 11 , representing 10% of the genome, and has a well-established role in regulation of gene expression through metal-responsive transcription-factor-1 (MTF1) 12, 13 . However, the true extent of zinc’s participation in cellular processes is only now emerging and suggests a complexity and importance comparable to that attributed to calcium. This is emphasized by zinc’s role as a second messenger 14 involved in a regulating various pathways 3, 8, 9 , often by inhibiting protein tyrosine phosphatases (PTPs) 15 . Transient increases in the cytosolic free zinc ion (Zn 2+ ) can occur through the release of zinc from zinc-binding proteins driven by changes in cellular redox potential 16 , and by flux of Zn 2+ into the cytosol 6, 14, 17 through zinc channels. The concentration of cytosolic Zn 2+ must be tightly controlled to prevent cell death due to zinc insufficiency or toxicity 1 . This requirement, together with the fact that zinc cannot traverse biological membranes unaided, is addressed through two families of proteins dedicated to mediating zinc movement across membranes. These are the ZnT (Official name: Solute Carrier Family 30A, SLC30A) family of zinc transporters that remove zinc from the cytosol into organelles or out of the cell and the ZIP (Official name: Solute carrier Family 39A, SLC39A) family of zinc channels that increase cytosolic zinc by mediating influx across the plasma membrane or from organelles. There are a total of 10 ZnT and 14 ZIP gene loci in humans allowing for fine-tuning of zinc Science Signaling 7 February 2012 Vol 5 Issue 210 ra11 distribution among tissues and intracellular compartments 18, 19 . A study of the prokaryotic homologue, ZIPB, which translocates zinc in a non-saturable fashion, indicates that the ZIP proteins function as Zn 2+ ion channels 20 . However, the mechanisms whereby zinc transport is regulated have been unclear. Zinc channel ZIP7, which resides in the membrane of the endoplasmic reticulum (ER), the Golgi, or both 19, 2123 , has orthologs in many species, including Drosophila (CATSUP) and Arabidopsis (IAR1) 19, 24 , that are also found in the ER 25 . At least in some cells, human ZIP7 21 is essential for zinc release from the ER, a process that results in activation of downstream signaling pathways 6 that promote cell proliferation 26 . Activation of these pathways may occur through phosphatase inhibition 15 , thereby preventing de-activation of phosphorylated tyrosine kinases. The apparent role of ZIP7 as a gatekeeper of cytosolic zinc release from the ER 9 , highlights the involvement of zinc in rapid signaling, in addition to, and contrasting with, its role in mediating transcriptional responses 13, 27, 28 . Studies identifying ZIP7 as essential for zinc release from the ER employed Tamoxifen-resistant MCF-7 breast cancer cells (TamR), which natively exhibit increased intracellular zinc 6 and ZIP7, but not other ZIP transporters 29 , compared to wild-type MCF-7 cells, and thus provide an excellent model for investigating the role of ZIP7 in cellular zinc homeostasis. TamR cells are more aggressive than wildtype MCF-7 cells; they show increased proliferation and increased invasiveness 30 , both of which are driven by hyperactivation of the EGFR (epidermal growth factor receptor) and IGF1-R (insulin-like growth factor 1 receptor) signaling pathways and thereby of downstream effectors such as AKT and MAPK (mitogen-activated protein kinase) 31, 32 . However, the ubiquitous expression of ZIP7 among organisms and cell types 21 suggests that it mediates a fundamental role of Zn 2+ flux from intracellular stores. We hypothesised that exposure of cells to exogenous stimuli, such as application of extracellular zinc, results in ZIP7-dependant release of zinc from the ER without a prior rise in cytosolic Zn 2+ concentration. Here, we investigated the molecular mechanism underlying gated zinc release by ZIP7 and showed that extracellular stimulation activates ZIP7 through phosphorylation at specific residues by the protein kinase CK2 (formerly casein kinase 2). Protein kinase CK2 is a ubiquitously expressed tetrameric threonine/serine kinase composed of two catalytic α subunits and two regulatory β subunits 33 . Although CK2 has numerous cellular targets and has not been previously thought to be regulated, it has been particularly implicated in cell survival and proliferation, participating in such processes as apoptosis and mitosis 34 . CK2 shuttles between the cytosol and nuclei of cells in order to support apoptosis or mitosis, respectively. ZIP7 phosphorylation by CK2 results in ZIP7-mediated zinc release from the ER and the subsequent activation of multiple downstream pathways that enhance cell proliferation and migration.