Protein kinase-dependent phosphorylation of the Menkes copper P-type ATPase I. Voskoboinik, R. Fernando, N. Veldhuis, K.M. Hannan, 1 N. Marmy-Conus, 1 R.B. Pearson, 1,2 and J. Camakaris * ,2 Department of Genetics, The University of Melbourne, Melbourne, Vic. 3010, Australia Received 6 February 2003 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. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Copper; Menkes protein; Protein kinase; Phosphorylation Copper is an essential trace element, whose unique properties make it indispensable for the active sites of enzymes involved in redox reactions such as cytochrome c oxidase, lysyl oxidase, and superoxide dismutase. However, excess copper is highly toxic to biological systems through the same redox mechanism that makes it essential to the cell. As a result, a complex mechanism has evolved to allow the delivery of copper to target proteins localised in various compartments and to de- toxify the excess amounts of copper by scavenging and efflux from the cell [1]. Two copper transporting P-type ATPases have been shown to play a key role in regulating copper homeo- stasis in mammals: the Menkes protein (ATP7A; MNK) and the 60% homologous Wilson protein (ATP7B; WND) [2]. The MNK protein, which is expressed in most tissues, except the liver where WND is expressed, has several roles in copper homeostasis: it is essential for systemic copper absorption, it delivers copper to cup- roenzymes in the trans-Golgi network (TGN) compart- ment, and facilitates copper efflux when at the plasma membrane (PM) [1]. To perform these tasks in different subcellular compartments, MNK has evolved the ability to traffic from the TGN to the PM and back [3]. There is constitutive recycling between these two compartments [4] and, importantly, copper-regulated relocalisation of MNK to the PM [3]. Several motifs have been identified and characterised as essential for copper-dependent trafficking of MNK. Among these are the N-terminal metal-binding sites (CysXXCys), which play important regulatory roles in copper-induced trafficking and also in catalysis [5,6]. Current information on MNK trafficking suggests that there may be several, potentially interrelated, mecha- nisms controlling the steady-state localisation and cop- per-induced trafficking of MNK [2]. Overall it appears Biochemical and Biophysical Research Communications 303 (2003) 337–342 www.elsevier.com/locate/ybbrc BBRC * Corresponding author. Fax: +613-8344-5139. E-mail address: j.camakaris@unimelb.edu.au (J. Camakaris). 1 Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Vic. 8006, Australia. 2 These authors contributed equally to this work. 0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00329-2