Communication between the N- and C-termini is required for Cu-stimulated Ser/Thr phosphorylation of Cu-(I)ATPase (ATP7B) [Cell Biology]

February 9th, 2015 by Braiterman, L. T., Gupta, A., Chaerkady, R., Cole, R. N., Hubbard, A. L.

The Wilson Disease protein ATP7B exhibits Cu-dependent trafficking. In high Cu, ATP7B exits the trans-Golgi network (TGN) and moves to the apical domain of hepatocytes, where it facilitates elimination of excess Cu through the bile. Cu levels also affect ATP7B phosphorylation. ATP7B is basally phosphorylated in low Cu and becomes more phosphorylated (″hyperphosphorylated″) in elevated Cu. The functional significance of hyperphosphorylation remains unclear. We showed that hyperphosphorylation occurs even when ATP7B is restricted to the TGN. We performed comprehensive phosphoproteomics of ATP7B in low versus high Cu, which revealed 24 Ser/Thr residues in ATP7B could be phosphorylated, and only four of which were Cu-responsive. Most of the phosphorylated sites were found in the N- and C-terminal cytoplasmic domains. Using truncation and mutagenesis, we showed that inactivation or elimination of all 6 N-terminal metal binding domains (MBDs) did not block Cu-dependent, reversible, apical trafficking but did block hyperphosphorylation in hepatic cells. We showed that 9 of 15 of Ser/Thr in the C-terminal domain were phosphorylated. Inactivation of 13 C-terminal phosphorylation sites reduced basal phosphorylation and eliminated hyperphosphorylation, suggesting that Cu binding at the N-terminus propagates to the ATP7B C-terminal region. C-terminal mutants with either inactivating or phosphomimetic substitutions showed little effect upon Cu-stimulated trafficking, indicating that trafficking does not depend on phosphorylation at these sites. Thus, our studies revealed Cu-dependent conformational changes in the N-terminal region lead to hyperphosphorylation at C-terminal sites, which seem not to affect trafficking and may instead fine-tune Cu sequestration.