Protein kinase-dependent phosphorylation of the Menkes copper P-type ATPase

doi:10.1016/S0006-291X(03)00329-2

Biochemical and Biophysical Research Communications

Volume 303, Issue 1, 28 March 2003, Pages 337-342

https://doi.org/10.1016/S0006-291X(03)00329-2 Get rights and content

Abstract

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a key regulator of copper homeostasis in humans. It has a dual role in supplying copper to essential cuproenzymes in the trans-Golgi network (TGN) and effluxing copper from the cell. These functions are achieved through copper-regulated trafficking of MNK between the TGN and the plasma membrane. However, the exact mechanism(s) which regulate the localisation and biochemical functions of MNK are still unknown. Here we investigated copper-dependent phosphorylation of MNK by a putative protein kinase(s). We found that in the presence of elevated copper there was a substantial increase in phosphorylation of the wild-type MNK in vivo. The majority of copper-dependent phosphorylation was on serine residues in two phosphopeptides. In contrast, there was no up-regulation of phosphorylation of a non-trafficking MNK mutant with mutated cytosolic copper-binding sites. Our findings suggest a potentially important role of kinase-dependent phosphorylation in the regulation of function of the MNK protein.

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.

References (18)

J. Camakaris et al.
Biochem. Biophys. Res. Commun.
(1999)
I. Voskoboinik et al.
Adv. Protein Chem.
(2002)
D. Strausak et al.
J. Biol. Chem.
(1999)
I. Voskoboinik et al.
J. Biol. Chem.
(1999)
X.B. Chang et al.
J. Biol. Chem.
(1993)
S.M. Vanderwerf et al.
J. Biol. Chem.
(2001)
I. Voskoboinik et al.
J. Biol. Chem.
(2001)
K.I. Mitchelhill et al.
J. Biol. Chem.
(1997)
E.A. Ostrakhovitch et al.
Arch. Biochem. Biophys.
(2002)

There are more references available in the full text version of this article.

Cited by (26)

Copper homeostasis in chronic kidney disease and its crosstalk with ferroptosis
2024, Pharmacological Research
Chronic kidney disease (CKD) has become a global public health problem with high morbidity and mortality. Renal fibrosis can lead to end-stage renal disease (ESRD). However, there is still no effective treatment to prevent or delay the progression of CKD into ESRD. Therefore, exploring the pathogenesis of CKD is essential for preventing and treating CKD. There are a variety of trace elements in the human body that interact with each other within a complex regulatory network. Iron and copper are both vital trace elements in the body. They are critical for maintaining bodily functions, and the dysregulation of their metabolism can cause many diseases, including kidney disease. Ferroptosis is a new form of cell death characterized by iron accumulation and lipid peroxidation. Studies have shown that ferroptosis is closely related to kidney disease. However, the role of abnormal copper metabolism in kidney disease and its relationship with ferroptosis remains unclear. Here, our current knowledge regarding copper metabolism, its regulatory mechanism, and the role of abnormal copper metabolism in kidney diseases is summarized. In addition, we discuss the relationship between abnormal copper metabolism and ferroptosis to explore the possible pathogenesis and provide a potential therapeutic target for CKD.
Regulation of Copper Transporters in Human Cells
2012, Current Topics in Membranes
Citation 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).
Copper is essential for normal growth and development of human organisms. The role of copper as a cofactor of important metabolic enzymes, such as cytochrome c oxidase, superoxide dismutase, lysyl oxidase, dopamine-β-hydroxylase, and many others, has been well established. In recent years, new regulatory roles of copper have emerged. Accumulating evidence points to the involvement of copper in lipid metabolism, antimicrobial defense, neuronal activity, resistance of tumor cells to platinum-based chemotherapeutic drugs, kinase-mediated signal transduction, and other essential cellular processes. For many of these processes, the precise mechanism of copper action remains to be established. Nevertheless, it is increasingly clear that many regulatory and signaling events are associated with changes in the intracellular localization and abundance of copper transporters, as well as distinct compartmentalization of copper itself. In this review, we discuss current data on regulation of the localization and abundance of copper transporters in response to metabolic and signaling events in human cells. Regulation by kinase-mediated phosphorylation will be addressed along with the emerging area of the redox-driven control of copper transport. We highlight mechanistic questions that await further testing.
Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase
2009, International Journal of Biochemistry and Cell Biology
The Menkes copper-translocating P-type ATPase (ATP7A) is a critical copper transport protein functioning in systemic copper absorption and supply of copper to cuproenzymes in the secretory pathway. Mutations in ATP7A can lead to the usually lethal Menkes disease. ATP7A function is regulated by copper-responsive trafficking between the trans-Golgi Network and the plasma membrane. We have previously reported basal and copper-responsive kinase phosphorylation of ATP7A but the specific phosphorylation sites had not been identified. As copper stimulates both trafficking and phosphorylation of ATP7A we aimed to identify all the specific phosphosites and to determine whether trafficking and phosphorylation are linked. We identified twenty in vivo phosphorylation sites in the human ATP7A and eight in hamster, all clustered within the N- and C-terminal cytosolic domains. Eight sites were copper-responsive and hence candidates for regulating copper-responsive trafficking or catalytic activity. Mutagenesis of the copper-responsive phosphorylation site Serine-1469 resulted in mislocalization of ATP7A in the presence of added copper in both polarized (Madin Darby canine kidney) and non-polarized (Chinese Hamster Ovary) cells, strongly suggesting that phosphorylation of specific serine residues is required for copper-responsive ATP7A trafficking to the plasma membrane. A constitutively phosphorylated site, Serine-1432, when mutated to alanine also resulted in mislocalization in the presence of added copper in polarized Madin Darby kidney cells. These studies demonstrate that phosphorylation of specific serine residues in ATP7A regulates its sub-cellular localization and hence function and will facilitate identification of the kinases and signaling pathways involved in regulating this pivotal copper transporter.
Cellular multitasking: The dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance
2008, Archives of Biochemistry and Biophysics
Citation 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].
The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.
Cyclic AMP-dependent protein kinase controls energy interconversion during the catalytic cycle of the yeast copper-ATPase
2008, FEBS Letters
The pathogenesis of human Menkes and Wilson diseases depends on alterations in copper transport. Some reports suggest that intracellular traffic of copper might be regulated by kinase-mediated phosphorylation. However, there is no evidence showing the influence of kinase-related processes in coupled ATP hydrolysis/copper transport cycles. Here, we show that cyclic AMP-dependent protein kinase (PKA) regulates Ccc2p, the yeast Cu(I)-ATPase, with PKA-mediated phosphorylation of a conserved serine (Ser²⁵⁸) being crucial for catalysis. Long-range intramolecular communication between Ser²⁵⁸ and Asp⁶²⁷ (at the catalytic site) modulates the key pumping event: the conversion of the high-energy to the low-energy phosphorylated intermediate associated with copper release.
Trafficking of the copper-ATPases, ATP7A and ATP7B: Role in copper homeostasis
2007, Archives of Biochemistry and Biophysics
Citation 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.
Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.

View all citing articles on Scopus

¹: Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Vic. 8006, Australia.

²: These authors contributed equally to this work.

View full text

Protein kinase-dependent phosphorylation of the Menkes copper P-type ATPase

Abstract

Section snippets

Materials and methods

In vitro phosphorylation

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

Adv. Protein Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.