The loop connecting metal-binding domains 3 and 4 of ATP7B is a target of a kinase-mediated phosphorylation

Mee Y. Bartee, Martina Ralle, Svetlana Lutsenko

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Cu-ATPase ATP7B (Wilson's disease protein) transports copper into the trans-Golgi network for biosynthetic incorporation into ceruloplasmin and sequesters excess copper to endocytic vesicles for further export out of the cell. The activity and intracellular location of ATP7B are regulated by copper levels; the trafficking of ATP7B between cellular compartments is coupled to changes in the level of protein phosphorylation. Neither the nature of the kinase(s) phosphorylating ATP7B nor the location of phosphorylation sites is known. We demonstrate that the membrane-bound ATP7B is phosphorylated by an ATPdependent, GTP-independent kinase that can be either soluble or membrane-associated. Mg2+ or Mn2+ is necessary for kinase activity. We further show that the recombinant N-terminal domain of ATP7B (N-ATP7B) is a specific target for a kinase-mediated phosphorylation in vitro and in cells. Although exogenous addition of copper is not required for kinase activity, copper binding to N-ATP7B markedly alters the exposure of loops connecting the metal-binding subdomains (MBDs) to proteolysis and facilitates phosphorylation by 25-30%. MBD1-2 and MBD4-5 linkers become protected, while MBD2-3 and MBD3-4 regions remain exposed. A significant, 5-fold increase in the level of phosphorylation is also observed for the ATP7B variant that lacks the 29 kDa N-terminal fragment (mostly likely comprised of MBD1-3). Analysis of phosphorylated peptides by two-dimensional gel electrophoresis and mass spectrometry points to the loop connecting MBD3 and MBD4 as a region of phosphorylation. Altogether, the results suggest a mechanism in which kinase-mediated phosphorylation of ATP7B is controlled by a conformational state of N-ATP7B.

Original languageEnglish (US)
Pages (from-to)5573-5581
Number of pages9
JournalBiochemistry
Volume48
Issue number24
DOIs
StatePublished - Jun 23 2009

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Phosphorylation
Phosphotransferases
Metals
Copper
Proteolysis
Membranes
trans-Golgi Network
Transport Vesicles
Ceruloplasmin
Electrophoresis, Gel, Two-Dimensional
Protein Transport
Guanosine Triphosphate
Electrophoresis
Mass spectrometry
Adenosine Triphosphatases
Mass Spectrometry
Gels
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

The loop connecting metal-binding domains 3 and 4 of ATP7B is a target of a kinase-mediated phosphorylation. / Bartee, Mee Y.; Ralle, Martina; Lutsenko, Svetlana.

In: Biochemistry, Vol. 48, No. 24, 23.06.2009, p. 5573-5581.

Research output: Contribution to journalArticle

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abstract = "Cu-ATPase ATP7B (Wilson's disease protein) transports copper into the trans-Golgi network for biosynthetic incorporation into ceruloplasmin and sequesters excess copper to endocytic vesicles for further export out of the cell. The activity and intracellular location of ATP7B are regulated by copper levels; the trafficking of ATP7B between cellular compartments is coupled to changes in the level of protein phosphorylation. Neither the nature of the kinase(s) phosphorylating ATP7B nor the location of phosphorylation sites is known. We demonstrate that the membrane-bound ATP7B is phosphorylated by an ATPdependent, GTP-independent kinase that can be either soluble or membrane-associated. Mg2+ or Mn2+ is necessary for kinase activity. We further show that the recombinant N-terminal domain of ATP7B (N-ATP7B) is a specific target for a kinase-mediated phosphorylation in vitro and in cells. Although exogenous addition of copper is not required for kinase activity, copper binding to N-ATP7B markedly alters the exposure of loops connecting the metal-binding subdomains (MBDs) to proteolysis and facilitates phosphorylation by 25-30{\%}. MBD1-2 and MBD4-5 linkers become protected, while MBD2-3 and MBD3-4 regions remain exposed. A significant, 5-fold increase in the level of phosphorylation is also observed for the ATP7B variant that lacks the 29 kDa N-terminal fragment (mostly likely comprised of MBD1-3). Analysis of phosphorylated peptides by two-dimensional gel electrophoresis and mass spectrometry points to the loop connecting MBD3 and MBD4 as a region of phosphorylation. Altogether, the results suggest a mechanism in which kinase-mediated phosphorylation of ATP7B is controlled by a conformational state of N-ATP7B.",
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