Involvement of Protein Kinase D in Expression and Trafficking of ATP7B (Copper ATPase)

Abstract

ATP7B is a P-type

Communication between the N and C Termini Is Required for Copper-stimulated Ser/Thr Phosphorylation of Cu(I)-ATPase (ATP7B)

“…Previous studies showed that Ser-1121 in the flexible loop of the nucleotide binding domain was phosphorylated in response to copper (11,12); our mass spectroscopy analysis confirmed this finding. Because copper has been shown to alter interdomain interactions that may be functionally relevant for trafficking (35), we used site-directed mutagenesis to inactivate Ser-1121 as well as the closely associated site Ser-1116 located in the nucleotide (N) domain (13 S/TϾA/GϩN).…”

“…An ATP7B mutant with all four of these Ser changed to Ala exhibited defective TGN exit, suggesting that phosphorylation at these sites is required for TGN exit (12). The conformation of a different Ser cluster (Ser-340 and Ser-341), located in the loop between MBD3 and MBD4 also appears to be important for TGN exit in HEK293TRex cells (13).…”

“…Previous studies showed that copper-dependent TGN exit of ATP7B correlated with its hyperphosphorylation (9). ATP7B phosphorylation also occurs during its biosynthesis; these modifications have been referred to as "basal" phosphorylation and seem to affect protein stability, lifetime, and/or quality control (9,(12)(13)(14). Mass spectrometry (MS) analysis of recombinant ATP7B isolated from Sf9 cells revealed that the loop between metal binding domain 3 (MBD3) and MBD4 as well as 10 other N-terminal (N-term) peptides were phosphorylated following in vitro radiolabeling in the presence of mammalian cell extract (15).…”

Distinctive Features of Catalytic and Transport Mechanisms in Mammalian Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases

“…We now find that even though a copper binding requirement for the E2 to the E1 transition is demonstrated by copper site mutations (33), experiments analogous to those with SERCA, in which Ca 2ϩ is first chelated with EGTA and then Ca 2ϩ is added to produce activation, cannot be easily done with ATP7A/B. In fact copper chelation with BCS produces inactivation due to removal of copper from the NMBD in addition to the TMBS, and very low phosphoenzyme formation is then obtained after the addition of copper and ATP.…”

“…alkali labile) corresponds to aspartyl phosphate intermediate. Alkali labile phosphorylation of ATP7B does not occur after mutations at the transmembrane copper binding site (33) and is, therefore, linked specifically to enzyme activation (E2 to E1 transition) by occupancy of the cation transport site. It is noteworthy that ATP7A undergoes aspartate phosphorylation with kinetics analogous to ATP7B, but a lower level of protein kinase assisted phosphorylation (34).…”