Protein tyrosine phosphatases are regulated by mononuclear iron dicitrate

Abstract

The involvement of macrophages (Mvarphis) as host, accessory, and effector cells in the development of infectious diseases, together with their central role in iron homeostasis, place these immune cells as key players in the interface between iron and infection. Having previously shown that the functional expression of NRAMP-1 results in increased protein phosphorylation mediated in part by an iron-dependent inhibition of Mvarphi protein-tyrosine phosphatase (PTP) activity, we sought to study the mechanism(s) underlying this specific event. Herein we have identified the mononuclear dicitrate iron complex [Fe(cit)(2)H(4-x)]((1+x)-) as the species responsible for the specific inhibition of Mvarphi PTP activity. By using biochemical and computational approaches, we show that [Fe(cit)(2)](5-) targets the catalytic pocket of the PTP SHP-1, competitively inhibiting its interaction with an incoming phosphosubstrate. In vitro and in vivo inhibition of PTP activity by iron-citrate results in protein hyperphosphorylation and enhanced MAPK signaling in response to LPS stimulation. We propose that iron-citrate-mediated PTP inhibition represents a novel and biologically relevant regulatory mechanism of signal transduction.

FIGURE 1.

Iron-dependent PTP inhibition is specific to Fe-cit. Mϕ PTP activity was determined following incubation of (A) 10 μg of B10R total protein lysates (B) or GST-purified recombinant PTPs for 10 min at RT with Fe-cit, FeSO₄ or FeCl₃ to a final concentration of 500 μm. PTP activity was evaluated by pNPP hydrolysis and absorbance reading at 405 nm. Statistically significant differences (**) were considered when p < 0.01.

FIGURE 2.

Effect of iron-to-citrate ratio on PTP inhibition. Three Fe-cit solutions were prepared modifying the iron-to-citrate ratios to obtain 1:1, 1:4, and 1:10 Fe:cit solutions. A, dose response curve of B10R Mϕ protein extracts incubated with 50–500 μm 1:1, 1:2, B, 1:4 and 1:10 Fe:cit or C, 0.1–5 mm citric acid was performed. PTP activity was evaluated by pNPP hydrolysis and absorbance reading at 405 nm. Statistically significant differences (**) were considered when p < 0.01.

FIGURE 3.

Specific inhibition of PTPs by mononuclear iron dicitrate. Electrospray mass spectra of (A) 1:1, (B) 1:4, and (C) 1:10 Fe:cit solutions.

FIGURE 4.

Fe-cit-dependent PTP inhibition is independent of Fe-catalyzed ROS. A, B10R Mϕ protein lysates were incubated for 10 min at room temperature with 500 μm 1:10 Fe:cit, in the presence or absence of 5 mm dithiothreitol, 50 units/ml superoxide dismutase (SOD), and/or 50 units/ml catalase (CAT). PTP activity was evaluated by pNPP hydrolysis and absorbance reading at 405 nm. Statistically significant differences (*) were considered when p < 0.05. B, 50 μg of total B10R protein lysate were subjected to in-gel PTP assay. During the final renaturation step, gels were incubated with 500 μm 1:10 Fe:cit (right panel) or left untreated (left panel). Samples are loaded in duplicates. Bands of dephosphorylation represent active PTPs. Arrows represent PTP targeted by Fe-cit.

FIGURE 5.

Computational docking of SHP-1 and Fe-cit complexes. Docking solutions of SHP-1 (PDB code: 1GWZ) and the mononuclear iron dicitrate complex C7 [Fe(cit)₂]⁵⁻ and with the dinuclear dicitrate iron complex C2 [Fe2(cit)2(H₂O)₂]²⁻. A, shown is a surface representation of SHP-1 and the docked C7 (green base color) and C2 (purple base color) molecules in stick representation. For reference the phosphotyrosine-containing substrate analog (PDB code: 1FPR) is also shown (transparent blue base color) illustrating that C7 and the phosphotyrosine occupy the same binding site. B, detailed view of the SHP-1 active site, highlighting the catalytically important residues Cys-455 and Arg-461, and other residues lining the wall of the pocket, as well as the inhibitory Fe-cit molecule C7.

FIGURE 6.

Fe-cit inhibits PTP activity in Mϕs and modulates protein phosphorylation. A, B10R Mϕs were incubated with 500 μm 1:10 and 1:1 Fe:cit, FeCl₃, or FeSo₄ in serum-free DMEM for 5–60 min. PTP activity was evaluated by pNPP hydrolysis and absorbance reading at 405 nm. Data represent the mean ± S.E. of five independent experiments performed in duplicate. Statistically significant differences (**) were considered when p < 0.01. B, 4G10 anti-phosphotyrosine Ab was used to evaluate the phosphotyrosine content of 1:10 Fe:cit-treated Mϕs. Loading control was performed using anti-β-actin Ab. C, Mϕs were pretreated with 500 μm 1:10 Fe:cit in serum-free medium for 5–60 min or left untreated. Next, cells were stimulated with 100 ng/ml LPS and SAPK/JNK and ERK1/2 phosphorylation was evaluated by Western blot. Shown are the loading controls using anti-JNK and anti-ERK1/2 antibodies. Data from densitometric analyses of three independent experiments are presented as the mean ± S.E. of integrated densitometric values (IDV).