The role of metal binding and phosphorylation domains in the regulation of cisplatin-induced trafficking of ATP7B

Abstract

The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 μM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.

Copper efflux transporters ATP7A and ATP7B: Novel biomarkers for platinum drug resistance and targets for therapy

“…A variant in which the CxxC motifs in all 6 MBDs are converted to SxxS could localize to the TGN but fails to traffic when exposed to either Cu or cisplatin. Deletion of either the first 5 MBDs or all 6 MBDs renders the loss of TGN localization . Thus, CxxC motifs in MBD5 and MBD6 are required for platinum‐induced trafficking, while MBD1–4 for binding platinum drugs.…”

Human copper transporter ATP7B (Wilson disease protein) forms stable dimers in vitro and in cells

“…Mutations in ATP7B cause Wilson disease, a hepatoneurologic disorder with a significant variability in the time of onset, rate of progression, and specific manifestations (3). In addition, certain ATP7B polymorphisms have been seen more frequently in patients with Alzheimer disease (4), and overexpression of ATP7B has been observed in cancers (5). Only a small fraction of disease-causing mutants, as well as polymorphisms, have been characterized so far.…”

Effects of Metal Ions (Mg2+, Ca2+, Cd2+ and Zn2+) on Adenosine Triphosphate Hydrolysis

“…Magnesium and cadmium ions appear to increase the rate constant, but the catalytic effect is relative lower than the calcium ions, zinc ions and TAME. Metal ions also influenced the amount of phosphoramidate intermediate, greater percentages being observed in reactions with magnesium, calcium, cadmium and zinc ions 52 .…”