The Menkes copper transporter is required for the activation of tyrosinase.
Menkes disease is an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A (MNK) gene. The MNK gene encodes a copper-transporting P-type ATPase, MNK, which is localized predominantly in the trans-Golgi 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 tyrosinase, a copper-dependent enzyme involved in melanogenesis that is synthesized within the secretory pathway. We demonstrate that recombinant tyrosinase expressed in immortalized Menkes fibroblast cell lines was inactive, whereas in normal fibroblasts known to express MNK protein there was substantial tyrosinase activity. Co-expression of the Menkes protein and tyrosinase from plasmid constructs in Menkes fibroblasts led to the activation of tyrosinase and melanogenesis. This MNK-dependent activation of tyrosinase was impaired by the chelation of copper in the medium of cells and after mutation of the invariant phosphorylation site at aspartic acid residue 1044 of MNK. Collectively, these findings suggest that the MNK protein transports copper into the secretory pathway of mammalian cells to activate copper-dependent enzymes and reveal a second copper transport role for MNK in mammalian cells. These findings describe a single cell-based system that allows both the copper transport and trafficking functions of MNK to be studied. This study also contributes to our understanding of the molecular basis of pigmentation in mammalian cells.
Petris
M J
MJ
Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, 221 Burwood Highway, Burwood 3125, Australia. petrism@missouri.edu
Strausak
D
D
Mercer
J F
JF
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000037:antagonists & inhibitors / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D004789:Enzyme Activation; D004790:Enzyme Induction; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D006801:Humans; D008543:Melanins / Q000096:biosynthesis; D007706:Menkes Kinky Hair Syndrome / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D010858:Pigmentation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D002614:Chelating Agents; D008543:Melanins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003300:Copper; D014442:Monophenol Monooxygenase; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases