A novel role for copper in Ras/mitogen-activated protein kinase signaling

Abstract

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.

Fig 1

Knockdown of Ctr1A in the prothoracic gland results in a large-fly phenotype. (A) Detection of Ctr1A in dissected prothoracic glands by indirect immunofluorescence assay. The genotype is shown above each image. The prothoracic gland driver used for these experiments, phm-Gal4::UAS-mCD8GFP, also expresses membrane-localized GFP under the control of the Phantom promoter. Knockdown of Ctr1A is indicated by arrows that show reduced plasma membrane staining of Ctr1A in the prothoracic gland. (B) Images of adult Drosophila. Flies carrying only the prothoracic gland Gal4 driver, Phantom-Gal4 (phm-Gal4), are smaller than flies with knockdown of Ctr1A in the prothoracic gland (phm-Gal4:UAS-Ctr1A^RNAi). The flies shown are both females. (C) Quantitative measurements of pupae. Comparisons of pupal length must be made within each sex, as female flies are larger than male flies. WT refers to the W¹¹¹⁸ stock. Genotypes are as follows. phm-Gal4/+, pupae possessing only the prothoracic gland Gal4 driver transgene; phm-Gal4:UAS-Ras^DN, pupae expressing a dominant negative allele of Ras in the prothoracic gland; phm-Gal4:UAS-Ctr1A^RNAi, pupae with knockdown of Ctr1A in the prothoracic gland. * indicates a P value of less than 0.01 when that genotype is compared to a WT fly of the same sex. n = 10 for all genotypes except phm-Gal4/+, where n = 8. The data shown here are for transgenic stocks with UAS-Ctr1A^RNAi on the second chromosome. Similar results were obtained with flies with a UAS-Ctr1A^RNAI transgene on the third chromosome (data not shown).

Fig 2

Knockdown of Ctr1A suppresses constitutively active Ras phenotypes in both the fly eye and wing. (A) Bright-field images of adult Drosophila wings. Expression of UAS-Ras^V12 using an apterous-Gal4 (ap-Gal4) driver, which drives expression in the dorsal compartment of the wing, is lethal, presumably because apterous is also expressed in portions of the central and peripheral parts of the nervous system, while expression of both the UAS-Ras^V12 and UAS-Ctr1A^RNAi transgenes yields viable adult flies with normal wings. Bright-field images of adult Drosophila wings. Shown on the far right are ×10 magnifications of the W¹¹¹⁸ female wing and the ap-Gal4:UAS-Ras^V12, Ctr1A^RNAi female wing. Note that the wings of some of the eclosed flies had an ectopic vein in the posterior portion of the marginal cell (arrow) in the ap-Gal4:UAS-Ras^V12, Ctr1A^RNAi wing. This phenotype is reminiscent of Ellipse mutants, a hyperactive allele of the epidermal growth factor receptor upstream of Ras, that leads to ectopic vein differentiation (3). Thus, some ectopic Ras activity is visible and not suppressed in flies expressing both the UAS-Ras^V12 and UAS-Ctr1A^RNAi transgenes in the wing. (B) SEM images of adult female Drosophila eyes, with the genotype shown above each image. The ey-Gal4 driver line expresses UAS promoter-driven transgenes in the eye. Expression of a constitutively active isoform of Ras^v14 results in a rough-eye phenotype (top far right image). Knockdown of Ctr1A in the eye can suppress the Ras^v14 phenotype (bottom images; compare bottom images to top far right and far left images).

Fig 3

Cu chelation or competition for Ctr1A-mediated Cu⁺ transport compromises Ras/MAPK signaling in Drosophila S2 cells. (A) Cu chelation downregulates Ras/MAPK signaling. Cells were not pretreated (−) or pretreated (+) with the Cu⁺-specific membrane-impermeant chelator BCS as indicated. Cells were left untreated (−) or treated (+) with insulin from 0 to 15 min, and total protein extracts were analyzed by immunoblotting for total Erk (Erk) and phospho-Erk (P-Erk) as shown. (B) Iron chelation with the membrane-impermeant Fe²⁺-specific chelator BPS was used as described for panel A. (C) Competition for Ctr1A-mediated transport by silver (Ag). Cells were not pretreated (−) or pretreated (+) with Ag and assayed as described for panels A and B.

Fig 4

Ctr1 function in Ras/MAPK signaling is dependent upon Cu⁺ transport activity. (A) MEFs wild type (Ctr1^+/+) or null (Ctr1^−/−) for Ctr1 were treated with insulin, and phospho-Erk was analyzed over time by SDS-PAGE and immunoblotting. Total Erk1/2 was evaluated as a loading control. (B) Ctr1^−/− cells stably expressing either wild-type human Ctr1 (Ctr1) or a transport-defective mutant form of human Ctr1 (Ctr1^M150A) were analyzed for insulin-stimulated Ras/MAPK activity in the phosphorylation of Erk. The Ctr1^M150A transport-defective cell line is Cu deficient, as indicated by the increased levels of CCS, compared to those of the Ctr1 wild-type cell line. Total Erk1/2 was assessed as a loading control. Note that the same film exposures for Ctr1 wild-type and Ctr1^M150A samples were used in examining protein levels; the dotted line indicates the removal of additional, superfluous controls loaded between wild-type and Ctr1^M150A sample lanes.

Fig 5

In Ctr1^−/− cells, Ras/MAPK pathway signal transduction is intact through Mek1/2 activation. Ctr1^+/+ and Ctr1^−/− cells were serum starved for 16 h and subsequently stimulated with FGF for the times indicated, and the phosphorylated and total levels of B-Raf, Mek1/2, Erk1/2, and Akt1 were assessed by SDS-PAGE and immunoblotting of the total protein extracts.

Fig 6

Mek1 is affinity purified by Cu-chelated resins. (A) GSH resin, either alone or preincubated and loaded with Cu, was incubated with total protein extracts from wild-type MEFs. The levels of Mek1, GAPDH, and Erk1/2 were assayed from the input proteins, GSH resin affinity-purified proteins, and Cu-charged GSH resin-purified proteins by SDS-PAGE and immunoblotting. (B) Pentadentate-chelated beads complexed with no metal, zinc, or Cu were incubated with lysates from Ctr1^+/+ cells, and Mek1 and the KSR1 scaffold protein were analyzed by immunoblotting as for panel A. (C) Purified recombinant rat Mek1 was added to pentadentate beads that were uncharged (metal free) or charged with zinc (Zn) or Cu, and affinity-purified Mek1 was analyzed by SDS-PAGE and immunoblotting.

Fig 7

Recombinant Mek1 metal-binding characteristics. (A) Dialysis experiments under the indicated equilibrium conditions showing a Cu/Mek1 ratio of 2 to 3, depending on competitive or noncompetitive conditions. Competition experiments decreased the ratio by unity, suggesting the presence of a low-affinity site. (B) Demonstration of saturation binding by equilibrium dialysis with increasing CuCl₂ concentrations in the dialysate using an independent set of purified rat Mek1. Mek1 was used at concentrations of 4 to 6 μM. (C) Determination of the Cu²⁺ dissociation constant, K_D, of Mek1 using the probe PAR showing overall spectral changes of the Cu-PAR complex on Mek1 titration. The inset shows the decrease at 500 nm relative to Mek1 additions for a [Cu-PAR]_total of 3.9 μM and a [PAR]_total of 9.3 μM. (D) Apparent K_Ds at pH 7.4 derived from competition titration using Cu²⁺-PAR. The asterisk indicates Mek1 dialyzed against a Cu²⁺-His complex overnight and then dialyzed against 0.5 M NaCl and 100 mM EDTA.

Fig 8

Mek1 kinase activity and association with Erk are stimulated by Cu. (A) Recombinant, GST-tagged human kinase-dead Erk2 and recombinant, GST-tagged human Mek1 were incubated with increasing amounts of CuSO₄ with or without TTM or Mek inhibitor 1. Mek1 phosphorylation of Erk2 was assessed by Western blotting with Erk1/2 phosphospecific antibody (n = 3). (B) Erk kinase activity in not enhanced by the addition of Cu. Recombinant GST-hErk2 and recombinant MBP were incubated with increasing amounts of CuSO₄. Erk2 phosphorylation of MBP was assessed by Western blotting with MBP phosphospecific antibody (n = 2). (C) Coimmunoprecipitation of Mek1 and Erk1/2 in Ctr1^+/+ and Ctr1^−/− MEFs. Mek1 immunoprecipitation (IP) from Ctr1^+/+ and Ctr1^−/− and binding of Erk1/2 were assessed by Western blotting with Mek1 and Erk1/2 antibodies. RalB immunoprecipitation was used as a negative control. Cu deficiency was assessed by immunoblot analysis of CCS protein levels in whole-cell extract (WCE). Immunoblot analyses of total Mek1, Erk1/2, and β-tubulin served as loading controls (n = 3).

Fig 9

Ras/MAPK signaling from mice with Ctr1-deficient hearts recapitulates cell culture observations. Heart lysates from two Ctr1 wild-type (Ctr1^flox/flox) animals were analyzed by immunoblotting for Mek1, Mek2, phospho-Mek1/1, Erk, phospho-Erk, CCS, and tubulin as a loading control. C indicates an individual Ctr1^flox/flox mouse, and M indicates an individual Ctr1^hrt/hrt mouse analyzed.