Biochemical and Biophysical Research Communications
Protein kinase-dependent phosphorylation of the Menkes copper P-type ATPase
Section snippets
Materials and methods
Cell culture. Chinese hamster ovary cells (CHO-K1) stably transfected with the wild-type (wtMNK) and the mutant construct (with six putative metal-binding sites –CxxC– mutated to –SxxS–, mMBS) of the Menkes protein cDNA were described earlier [5]. Empty vector transfected CHO-K1 cells (EV) were used as a control in these studies. The growth conditions for the transfected cells were identical. The cells were cultured as a monolayer in Eagle’s basal medium with Earle’s salts (BME) supplemented
In vitro phosphorylation
We demonstrated earlier that, as a part of its catalytic cycle, MNK could undergo copper-dependent transient acyl-phosphorylation at the invariant aspartate residue in the 1044DKTG motif, which is in accordance with the catalytic mechanism for P-type ATPases [11]. Characteristically, the reaction is completed within 20–40 s and the resulting acyl-phosphate is sensitive to hydroxylamine [11]. In this study we used isolated membrane vesicles containing the Golgi membranes, the endoplasmic
Discussion
Protein kinase-dependent phosphorylation is important in regulating both the catalytic activity and subcellular localisation of various ATPases, including the Na/K-ATPase, Ca-ATPases, and CFTR [7], [8], [9]. Copper-dependent phosphorylation of the liver-specific copper P-type ATPase, WND, has been reported [10]. It was suggested that phosphorylation may be linked to trafficking of WND, although no causal relationship was demonstrated [10]. Our current study provides the first evidence that MNK
Acknowledgements
This work was supported by the NH&MRC, J.N. Peter’s Bequest, K.M. Brutton Award, and the Wellcome Trust.
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Copper homeostasis in chronic kidney disease and its crosstalk with ferroptosis
2024, Pharmacological ResearchRegulation of Copper Transporters in Human Cells
2012, Current Topics in MembranesCitation Excerpt :The cytosolic copper chaperone Atox1 was shown to transfer copper to the regulatory MBDs of the Cu-ATPases and regulate their occupancy (Walker et al., 2004). Current data indicate that copper binding to N-terminal domain of both ATP7A and ATP7B is necessary for their copper-responsive phosphorylation (Vanderwerf et al., 2001; Voskoboinik et al., 2003). Upon binding, copper induces rearrangements of the loops connecting MBDs in the N-terminal domain of ATP7B and also facilitates phosphorylation by a kinase, suggesting that conformational changes due to copper binding might be important in the exposure of additional phosphorylation sites (Bartee et al., 2009).
Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase
2009, International Journal of Biochemistry and Cell BiologyCellular multitasking: The dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance
2008, Archives of Biochemistry and BiophysicsCitation Excerpt :At the protein level, ATP7A and ATP7B are 50–60% identical. Both Cu-ATPases are phosphorylated at Ser residue(s) by a yet to be characterized kinase; the extent of a kinase-mediated phosphorylation is regulated by copper ([38,39], see Localization and trafficking of human Cu-ATPases for details). The membrane portion of ATP7A and ATP7B has eight transmembrane segments (TMS) with the N- and C-termini of the protein both oriented towards the cytosol [40].
Trafficking of the copper-ATPases, ATP7A and ATP7B: Role in copper homeostasis
2007, Archives of Biochemistry and BiophysicsCitation Excerpt :A scan of the PROSITE database using ScanProsite and Proscan [145,146] for signaling motifs within the ATP7B N-terminal 63 amino acids revealed a putative casein kinase II site (TARE), which may represent one of several targets since Vanderwerf et al. (2001) [144] found that an inhibitor of casein kinase II partially prevented the copper-responsive phosphorylation. A similar phenomenon of basal versus copper-stimulated phosphorylation was observed for ATP7A [147]. In addition, these authors determined that serine residues within three major phosphopeptides were the targets of the basal phosphorylation, while one of these three phosphopeptides and one novel phosphopeptide were phosphorylated in response to elevated copper levels.
- 1
Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Vic. 8006, Australia.
- 2
These authors contributed equally to this work.