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. 2021 Dec;297(6):101314.
doi: 10.1016/j.jbc.2021.101314. Epub 2021 Oct 27.

V体育官网 - The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation

Michael Grasso  1 , Gavin J Bond  2 , Ye-Jin Kim  3 , Stefanie Boyd  4 , Maria Matson Dzebo  5 , Sebastian Valenzuela  5 , Tiffany Tsang  6 , Natalie A Schibrowsky  1 , Katherine B Alwan  7 , Ninian J Blackburn  7 , George M Burslem  8 , Pernilla Wittung-Stafshede  5 , Duane D Winkler  4 , Ronen Marmorstein  9 , Donita C Brady  10
Affiliations

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation

"VSports app下载" Michael Grasso et al. J Biol Chem. 2021 Dec.

Abstract

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers VSports手机版. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS. .

Keywords: copper chaperone; copper homeostasis; metalloallostery; mitogen-activated protein kinase; protein kinase; signal transduction V体育安卓版. .

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Conflict of interest statement

Conflict of interest D. C. B. holds ownership in Merlon Inc. D. C VSports最新版本. B. is an inventor on the patent application 20150017261 entitled “Methods of treating and preventing cancer by disrupting the binding of copper in the MAP kinase pathway”. All other authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
CCS directly interacts with MEK1 to facilitate Cu binding and kinase activity.A–C, single-cycle SPR experiments of HIS-tagged PARVALBUMIN, ATOX1, or CCS binding to MEK1 in which increasing concentrations of (A), PARAVALBUMIN (2.5–40 μM, black line), (B), ATOX1 (2.5–40 μM, blue line), or (C), CCS (5–40 μM, red line was injected onto immobilized MEK1 and the The 1:1 binding model fits [B] black lines). n = 3 independent experiments. D, electrophoretic mobility shift assays (EMSAs) of increasing concentrations of apo-CCS or Cu reconstituted CCS (Cu-CCS) with increasing concentrations of MEK1. E–G, size exclusion chromatography runs of (E), Cu-CCS (blue line), (F), MEK1 (black line), or (G), Cu-CCS and MEK1 (red line) and protein detection in indicated fractions detected by Coomassie Brilliant Blue. H, scatter dot plot with bar at mean luminescence units ±SEM from ELISA detection of recombinant phosphorylated (P)-ERK2K54R from MEK1WT incubated with increasing concentrations of Cu reconstituted ATOX1 (Cu-ATOX1) or CCS (Cu-CCS). n = 2 technical replicates. The results were compared using a two-way ANOVA followed by a Tukey's multi-comparisons test. ∗p = 0.0185. I, size exclusion chromatography runs of Cu-CCS and MEK1 mixed 1:1 (blue line) or Cu-CCS and MEK1 mixed 1:1 in presence of 5:1 ratio of bathocuproinedisulfonic acid (BCS) to protein (red line) and CCS or MEK1 positive fractions analyzed by inductively-coupled plasma mass spectrometry (ICP-MS) detected by Coomassie Brilliant Blue. J, scatter dot plot with bar at mean ratio of Cu detected by ICP-MS to protein ±SEM of CCS, as isolated, MEK1, as isolated, Cu and CCS mixed 1:1, CCS positive fractions 14 to 18 or MEK1 positive fractions 20 to 22 from Cu-CCS and MEK1 mixed 1:1, or CCS positive fractions 14 to 18 or MEK1 positive fractions 20 to 22 from Cu-CCS and MEK1 mixed 1:1 in presence of BCS. n = 1 to 4 independent experiments. The results were compared using one-way ANOVA followed by a Tukey's multi-comparisons test. ∗∗∗p = 0.0003, ∗∗p = 0.0028. CCS, Cu chaperone for superoxide dismutase; Cu, copper; ICP-MS, inductively-coupled plasma mass spectrometry; SPR, surface plasmon resonance.
Figure 2
Figure 2
CCS is the necessary Cu chaperone for MEK1.A, immunoblot detection of biotinylated MEK1, ATP7A, SOD1, MYC-BirA, MYC-BirA-ATOX1, or MYC-BirA-CCS from strepavadin pulldowns and whole cell extracts from HEK-HT cells stably expressing MYC-BirA, MYC-BirA-ATOX1, or MYC-BirA-CCS treated with media with (+) or without (−) biotin for 48 h. n = 2 biologically independent experiments. B, immunoblot detection of biotinylated MEK1 or MYC-BirA-CCS from strepavadin pulldowns from HEK-HT cells stably expressing MYC-BirA-CCS treated with media with (+) or without (−) biotin for 48 h in the presence of vehicle, 500 μM bathocuproinedisulfonic acid (BCS), or 1 μM CuCl2. n = 2 biologically independent experiments. C and D, immunoblot detection of phosphorylated (P)-ERK1/2, total (T)-ERK1/2, P-MEK1/2, T-MEK1/2, CCS, or β-ACTIN from HEK-HT cells stably expressing doxycycline inducible shRNA against control (−) or CCS (#1 or #2) treated with doxycycline for 24 h followed by (B), vehicle (VEH) or 1 μM CuCl2 for 20 min or (C), VEH or 0.1 ng/ml EGF for 15 min. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n = 3 biologically independent experiments. E, immunoblot detection of P-ERK1/2, T-ERK1/2, P-MEK1/2, T-MEK1/2, SOD1, or β-ACTIN from HEK-HT cells stably expressing doxycycline inducible shRNA against control (−) or SOD1 (+) treated with doxycycline for 48 h n = 2 biologically independent experiments. F, immunoblot detection of P-ERK1/2, T-ERK1/2, P-MEK1/2, T-MEK1/2, MYC, or β-ACTIN from HEK-HT cells stably expressing doxycycline inducible shRNA against control (−) or CCS (#2) reconstituted with MYC-CCSWT(WT), MYC-CCS Domain 1 Mutant (D1), MYC-CCS Domain 3 Mutant (D3), or MYC-CCS Domain 1 + 3 Mutant (D1+D3) treated with doxycycline for 24 h. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n = 3 biologically independent experiments. CCS, Cu chaperone for superoxide dismutase; Cu, copper; EGF, epidermal growth factor.
Figure 3
Figure 3
Treatment with small molecular inhibitor of CCS blunts MAPK pathway activation at the level of MEK1/2.A, immunoblot detection of phosphorylated (P)-ERK1/2, total (T)-ERK1/2, or β-ACTIN from HEK-HT cells treated with vehicle (0) or increasing concentrations of DCAC50 for 24 h. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n = 3 biologically independent experiments. B and C, immunoblot detection of P-ERK1/2, T-ERK1/2, P-MEK1/2, T-MEK1/2, or β-ACTIN from HEK-HT cells treated with vehicle (−) or 10 μM DCAC50 for 24 h followed stimulation with (B), 1 μM CuCl2 or (C), 0.1 ng/ml EGF for indicated time. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n = 3 biologically independent experiments. D, model of CCS-mediated Cu activation of MEK1/2. CCS, Cu chaperone for superoxide dismutase; Cu, copper; EGF, epidermal growth factor; MAPK, mitogen-associated protein kinase; VEH, vehicle.
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