The Menkes copper transporter is required for the activation of tyrosinase

Menkes disease is an X-linked recessive copper deficiency disorder caused b y mutations in the ATP7A (MNK) gene. The MNK gene encodes a copper-transpor ting P-type ATPase, MNK, which is localized predominantly in the trans-Golg i network (TGN). The MNK protein relocates to the plasma membrane in cells exposed to elevated copper where it functions in copper efflux, A role for MNK at the TGN in mammalian cells has not been demonstrated. In this study, we investigated whether the MNK protein is required for the activity of ty rosinase, a copper-dependent enzyme involved in melanogenesis that is synth esized within the secretory pathway. We demonstrate that recombinant tyrosi nase expressed in immortalized Menkes fibroblast cell lines was inactive, w hereas in normal fibroblasts known to express MNK protein there was substan tial tyrosinase activity. Go-expression of the Menkes protein and tyrosinas e from plasmid constructs in Menkes fibroblasts led to the activation of ty rosinase and melanogenesis. This MNK-dependent activation of tyrosinase was impaired by the chelation of copper in the medium of cells and after mutat ion of the invariant phosphorylation site at aspartic acid residue 1044 of MNK, Collectively, these findings suggest that the MNK protein transports c opper into the secretory pathway of mammalian cells to activate copper-depe ndent enzymes and reveal a second copper transport role for MNK in mammalia n cells, These findings describe a single cell-based system that allows bot h the copper transport and trafficking functions of MNK to be studied, This study also contributes to our understanding of the molecular basis of pigm entation in mammalian cells.