A Novel Role for Copper in Ras/Mitogen-Activated Protein Kinase Signaling

Abstract

c Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. In vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer. Copper (Cu) is a metal ion that functions as a redox-active cofactor for a broad range of biochemical reactions, including mitochondrial oxidative phosphorylation, protection from reactive oxygen species, connective tissue maturation, iron absorption, neuropeptide biogenesis, and other processes (28, 43). Numerous studies point to the essentiality of Cu for normal growth and development, while aberrant Cu accumulation in tissues, as manifested in Wilson's disease patients, results in significant pathologies (33,35,42,47,60,61). However, the precise roles Cu plays and the mechanistic processes by which Cu drives cellular proliferation and growth are not well understood.The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is an evolutionarily conserved pathway involved in the control of many fundamental biological processes, including cell proliferation, apoptosis, survival, differentiation, motility, and metabolism (26,30). Aberrant Ras/MAPK signaling has significant consequences; loss of function of several components of the Ras/MAPK signaling cascade results in lethality, whereas gain-offunction mutations in many of the Ras/MAPK signaling components underlie cancer (2,12,26,55).Here we identify the Ctr1 high-affinity Cu ϩ transporter, conserved from yeast to humans, as being important for stimulation of the MAPK Erk in response to extracellular growth factor-mediated activation of the Ras signaling pathway. Moreover, genetic, physiological, and biochemical experiments point to a direct role for Cu in the ability of the MAPK kinase Mek1 to phosphorylate Erk in fruit flies, cultured cells, and mice. These studies suggest that the MAPK signaling pathway is a key cellular proliferation pathway that is stimulated by Cu and may be a direct target of potent cancer chemotherapeutics that function via Cu chelation. MATERIALS AND METHODS Drosophila melanogaster stocks and crosses. Phantom Gal4, UAS mCD8::GFP/TM6, Tb flies were from Michael O'Connor, University of Minnesota (44). The UAS-Ctr1ARNA...

Comparison of 18F-Fluoroazomycin-Arabinofuranoside and 64Cu-Diacetyl-Bis(N4-Methylthiosemicarbazone) in Preclinical Models of Cancer

“…However, the intracellular localization of Ctr1, found in PC-3, although unable to promote the redistribution of circulating copper, may promote its sequestration in intracellular compartments and facilitate the dissociation of the 64 Cu-ATSM complex. Finally, whereas the study of copper transport distribution does not explain the differences in tracer uptake, it does highlight a need for greater understanding of the role of copper in cell proliferation and of the involvement of transporters in the resistance and in the toxicity of platinum derivatives (36,37). 64 Cu-ATSM at 2 h, and 64 Cu-ATSM at 24 h after injection in the same mouse.…”

Copper signaling in the brain and beyond

“…Foundational studies by Thiele and co-workers in fly and mouse cell models led to the discovery that copper is required for MEK1/2 activity (81). Biochemical characterization demonstrated that copper binds directly to purified MEK1/2, facilitating phosphorylation of recombinant ERK1/2 in vitro; conversely, copper chelation inhibits ERK1/2 phosphorylation in vitro (81). Elegant work by Brady et al showed the physiological importance of this copper-MAPK connection in a cancer setting.…”

Chemotherapy triggers HIF-1–dependent glutathione synthesis and copper chelation that induces the breast cancer stem cell phenotype

“…Recently, it has been reported that copper is a required cofactor for the kinase activity of MEK1 (34,35). We hypothesized that glutathione inhibits MEK1 activity through chelating copper.…”