The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a key regulator of copper homeostasis in humans. It has a dual role in supplying copper to essential cuproenzymes in the trans-Golgi network (TGN) and effluxing copper from the cell. These functions are achieved through copper-regulated trafficking of MNK between the TGN and the plasma membrane. However, the exact mechanism(s) which regulate the localisation and biochemical functions of NINK are still unknown. Here we investigated copper-dependent phosphorylation of NINK by a putative protein kinase(s). We found that in the presence of elevated copper there was a substantial increase in phosphorylation of the wild-type NINK in vivo. The majority of copper-dependent phosphorylation was on serine residues in two phosphopeptides. In contrast, there was no up-regulation of phosphorylation of a non-trafficking NINK mutant with mutated cytosolic copper-binding sites. Our findings suggest a potentially important role of kinase-dependent phosphorylation in the regulation of function of the NINK protein. (C) 2003 Elsevier Science (USA). All rights reserved.