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...