Copper Specifically Regulates Intracellular Phosphorylation of the Wilson's Disease Protein, a Human Copper-transporting ATPase

Abstract

Copper is a trace element essential for normal cell homeostasis. The major physiological role of copper is to serve as a cofactor to a number of key metabolic enzymes. In humans, genetic defects of copper distribution, such as Wilson's disease, lead to severe pathologies, including neurodegeneration, liver lesions, and behavior abnormalities. Here, we demonstrate that, in addition to its role as a cofactor, copper can regulate important posttranslational events such as protein phosphorylation. Specifically, in human cells copper modulates phosphorylation of a key copper transporter, the Wilson's disease protein (WNDP). Copper-induced phosphorylation of WNDP is rapid, specific, and reversible and correlates with the intracellular location of this copper transporter. WNDP is found to have at least two phosphorylation sites, a basal phosphorylation site and a site modified in response to increased copper concentration. Comparative analysis of WNDP, the WNDP pineal isoform, and WNDP C-terminal truncation mutants revealed that the basal phosphorylation site is located in the C-terminal Ser All living organisms require copper for growth and development. Copper is an integral cofactor for numerous enzymes that play key roles in various cell processes, including oxidative metabolism, neurotransmitter synthesis, free radical detoxification, and iron uptake (1-3). Either copper deficiency or copper accumulation is deleterious to cells, and the intracellular concentration of cooper is tightly controlled. Two homologous proteins, products of the ATP7A (Menkes disease) and ATP7B (Wilson's disease) genes, are essential for transmembrane transport of copper in mammalian cells (4 -6). Mutations in these genes cause severe pathologies in humans associated either with inborn copper deficiency (Menkes disease) or with vast accumulation of copper in tissues (Wilson's disease). Both Menkes and Wilson's disease proteins (WNDP) 1 are coppertransporting P-type ATPases that utilize energy of ATP hydrolysis to transport copper from the cytosol across cell membranes (7-9). Recent studies indicate that copper plays an active role in regulation of its own metabolism, modulating gene transcription, copper uptake, and protein trafficking (10 -13). Although the ability of copper to regulate these cellular events is firmly established, the precise molecular mechanisms through which copper acts are poorly understood. In this study we demonstrate that in human cells changes in copper concentration are "translated" into alterations of the phosphorylation levels for at least one protein target, WNDP. Phosphorylation of WNDP in response to copper is likely to represent a regulatory event, because it is rapid, specific, and reversible and has association with another cellular process, the subcellular redistribution of WNDP. We conclude that protein phosphorylation could be one of the molecular mechanisms by which copper regulates its own metabolism. EXPERIMENTAL PROCEDURES Treatment of Cells with Various Metals-Human hepatoma (HepG2)and hepatocarci...

Molecular Events Initiating Exit of a Copper-transporting ATPase ATP7B from the Trans-Golgi Network

“…In low copper, ATP7B has a basal level of phosphorylation. Copper elevation results in a hyperphosphorylation of ATP7B, and this event is coupled to trafficking of ATP7B from the TGN to vesicles, whereas dephosphorylation is associated with the return of ATP7B from vesicles to the TGN (16). The basal and copper-induced phosphorylation sites are different and involve different kinases (16).…”

Posttranslational regulation of copper transporters

“…This hyperphosphorylation of ATP7B appears dependent on copper and on the presence of an intact amino terminus [125,126]. In contrast, copper depletion results in dephosphorylation of ATP7B and concomitant retrograde trafficking of ATP7B to the TGN [125].…”

“…This hyperphosphorylation of ATP7B appears dependent on copper and on the presence of an intact amino terminus [125,126]. In contrast, copper depletion results in dephosphorylation of ATP7B and concomitant retrograde trafficking of ATP7B to the TGN [125]. The kinase responsible for this phosphorylation event is currently unknown, but it is tempting to speculate that coupling of protein phosphorylation to copper binding at the aminoterminal domains of copper-transporting P-type ATPases comprises the mechanism by which the amino terminus regulates protein function and localization in a copperdependent way.…”

“…Phosphorylation of ATP7B, in a manner distinct from the transient phosphorylation of the invariant D1027 during the catalytic cycle, has been described and correlated with localization of ATP7B to the periphery of the cell [125]. This hyperphosphorylation of ATP7B appears dependent on copper and on the presence of an intact amino terminus [125,126].…”

“…Alternatively, it is also possible that copper-initiated signal transduction pathways exist and result in phosphorylation of ATP7A or ATP7B. Furthermore, a basal phosphorylation site that is independent of copper was characterized, and involves serine residues in the carboxy-terminal Ser796-Tyr1384 region of ATP7B [125,126]. The function of basal phosphorylation is currently unknown, but it may be involved in interactions with proteins either to retain ATP7B in the TGN or to modulate copper transport activity.…”