Model for hepcidin-resistant Fpn internalization. Fpn is a dimer and both monomers must bind hepcidin for Jak2 to bind to Fpn. Once Jak2 is bound, it is phosphorylated and then phosphorylates Fpn, which is the signal for Fpn internalization. Missense mutants of Fpn can participate in Fpn dimer formation. An inability to bind hepcidin (FpnC326Y) in one monomer will prevent Jak2 binding to a WT mutant heterodimer, resulting in retention of Fpn on the cell surface. Mutant Fpn that can bind hepcidin (FpnN144H) may lead to Jak2 binding; however, Jak2 is not activated and will not phosphorylate the WT mutant heterodimer. This results in retention of Fpn on the cell surface and continued iron export.

Jak2 is required for hepcidin-mediated Fpn internalization. WT (2C4) and Jak2 deficient cells (γ2A) were transfected with a plasmid containing a CMV-regulated Fpn-GFP. Eighteen hours posttransfection, cells were incubated in the presence or absence of 1.0 μg/ml hepcidin for 4 or 24 h. Fpn-GFP localization was determined by epifluorescence microscopy. Cells were lysed and protein samples were analyzed by Western blot using rabbit anti-Fpn, mouse anti-tubulin, or rabbit anti-Jak2 followed by a peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG.

Hepcidin-mediated activation of Fpn bound Jak2 is highly cooperative. (A) HEK293T cells were transiently transfected with WT Fpn-FLAG and Fpn(Y302–303F)-GFP expressed under a CMV promoter. Cells were incubated in the presence or absence of 1 μg/ml hepcidin for 30 min, solubilized, and immunoprecipitated with rabbit anti-GFP or anti-FLAG antibodies as in Fig. 5. Immunoprecipitated samples were analyzed by Western blot using mouse anti-phosphotyrosine (1) or rabbit anti-Jak2 (2) followed by a peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG. A second immunoprecipitation was performed as in (A) and Jak2 phosphorylation assessed using mouse anti-phosphotyrosine followed by a peroxidase-conjugated goat anti-mouse IgG (panel 3). (B) HEK293T cells were transiently transfected with WT Fpn-FLAG and Fpn(N144H)-GFP expressed under a CMV promoter. Cells were incubated in the presence or absence of 1 μg/ml hepcidin for 30 min, solubilized, and immunoprecipitated with rabbit anti-GFP or anti-FLAG antibodies. Immunoprecipitated samples were analyzed by Western blots using mouse anti-phosphotyrosine (1) or rabbit anti-Jak2 (2) followed by a peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG. A second immunoprecipitation was performed as in (A) and Jak2 phosphorylation assessed using mouse anti-phosphotyrosine followed by a peroxidase-conjugated goat anti-mouse IgG (3).

Hepcidin binding to Fpn leads to the binding and activation of Jak2. (A) HEK293T cells were cotransfected with plasmids containing Fpn-GFP and DynaminK44A and incubated in the presence or absence of 1.0 μg/ml hepcidin for 30 min. Cells were placed at 0 °C and solubilized. Samples were immunoprecipitated with rabbit anti-Fpn antibodies as described in Methods. Immunoprecipitated samples were analyzed by Western blots probed using rabbit anti-Fpn (1) or rabbit anti-Jak2 (2) followed by a peroxidase-conjugated goat anti-rabbit IgG. Samples from the Fpn immunoprecipitation were immunoprecipitated for a second time with rabbit anti-Jak2 antibodies. Immunoprecipitated samples were analyzed by Western blot using mouse anti-phosphotyrosine followed by a peroxidase-conjugated goat anti-mouse IgG (3). (B) HEK293T cells were transfected with plasmids containing DMT1-EGFP-N1 and DynaminK44A. Cells were solubilized and immunoprecipitated with rabbit anti-GFP antibodies. Immunoprecipitated samples were analyzed by Western blot using rabbit anti-Jak2 or rabbit anti-GFP followed by a peroxidase-conjugated goat anti-rabbit IgG. (C) HEK293T cells were cotransfected with plasmids containing Fpn(Y302–303F)-GFP and Dynamin K44A. Cells were incubated in the presence or absence of 1 μg/ml hepcidin for 30 min, solubilized and immunoprecipitated with rabbit anti-Fpn antibodies. Immunoprecipitated samples were analyzed by Western blot using rabbit anti-Jak2 or rabbit anti-Fpn followed by a peroxidase-conjugated goat anti-rabbit IgG. (D) HEK293T cells were cotransfected with plasmids containing Fpn(N144H)-GFP and Dynamin K44A. Cells were incubated in the presence or absence of 1 μg/ml hepcidin for 30 min, solubilized, and immunoprecipitated with rabbit anti-Fpn antibodies. Immunoprecipitated samples were analyzed by Western blots using rabbit anti-Jak2 or rabbit anti-Fpn followed by a peroxidase-conjugated goat anti-rabbit IgG.

Two hepcidin binding sites on the Fpn dimer are required for Jak2 binding. (A) Schematic of experimental design. Cells were transfected with plasmids expressing Fpn-FLAG and mutant Fpn-GFP. Cells were incubated in the presence or absence of hepcidin and then immunoprecipitate with either anti-FLAG or anti-GFP antibodies. The immunoprecipitates were then analyzed for Fpn, phospho-Fpn, Jak2, and phospho-Jak2. (B) HEK293T cells were transiently transfected with plasmids containing WT Fpn-FLAG and Fpn(C326Y)-GFP. Cells were incubated in the presence of 1 μg/ml hepcidin for 30 min, solubilized, and immunoprecipitated with rabbit anti-GFP or anti-FLAG antibodies. The immunoprecipitates were analyzed by Western blot using anti-FLAG or anti-GFP antibodies. (C) Immunoprecipitated samples were analyzed by Western blot using mouse anti-phosphotyrosine (1) or rabbit anti-Jak2 followed by a peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (2). A second immunoprecipitation was performed with rabbit anti-Jak2 and the immunoprecipitation samples were analyzed by Western blot using mouse anti-phosphotyrosine followed by a peroxidase-conjugated goat anti-mouse IgG (3).

Silencing of Jak2 in human cells prevents hepcidin-mediated Fpn internalization. (A) HEK293TFpn-GFP cells were transfected with either nonspecific siRNA (N.S.) or human Jak2 specific siRNA oligonucleotide pools using OligofectAMINE. Forty-eight hours later cells were induced to express Fpn-GFP, and after 18 h cells were incubated in the presence or absence of hepcidin. Fpn-GFP localization was examined by epifluorescence microscopy. Silencing was assessed by Western blot with tubulin as a loading control. (B) HEK293TFpn-GFP cells were silenced as in (A). Forty-eight hours later, cells were transfected with pCMV-mouse Jak2 and induced to express Fpn-GFP. After 18 h cells were incubated in the presence or absence of hepcidin and Fpn-GFP localization was examined by epifluorescence microscopy 1 hr later. The data were quantified by determining the percent of cells with internalized Fpn-GFP in 2 data sets of >100 cells each, and error bars indicate standard deviations.

Silencing Jak2 in macrophages specifically inhibits hepcidin-mediated Fpn internalization. (A) Mouse bone marrow macrophages were transfected with either nonspecific (N.S.) or mouse Jak2 specific siRNA pools using OligofectAMINE. Forty-eight hours later, cells were incubated with FAC (10 μM Fe) to induce the expression of Fpn. After a further 24 h the cells were incubated in the presence or absence of hepcidin and Fpn localization was examined by immunofluorescence microscopy. Silencing efficiency was assessed by Western blot. (B) Mouse bone marrow macrophages were incubated with FAC (10 μM Fe) to induce the expression of Fpn and after 24 h cells were incubated in the presence or absence of 1 μg/ml hepcidin for 30 min. Cells were placed at 0 °C, solubilized, and samples immunoprecipitated with rabbit anti-Fpn antibodies. Immunoprecipitated samples were analyzed by Western blots probing for Jak2 or Fpn. (C) Cells as in (A) were incubated in the presence or absence of BCS (250 μM) for 18 h and levels were analyzed by Western blot.