c-Src kinase is involved in the tyrosine phosphorylation and activity of SLC11A1 in differentiating macrophages
Fig 6
The proline-rich motif of SLC11A1 is required for association with c-Src kinase.
(A) Schematic representation of the P130A, P231A and PRM-deletion constructs of SLC11A1. (B) U937 cells were transfected with constructed pCB6 vectors expressing c-Myc-tagged wild-type SLC11A1, P231A, P130A or PRM-deletion mutant together with c-Src expression vector. Transfected cells were treated with PMA for 48 hrs and cell lysates were immunoprecipitated with anti-c-Myc antibody (9E10). The bound proteins were probed with a rabbit polyclonal antibody against c-Src. Expression of c-Myc-tagged wild-type SLC11A1, P231A, P130A or PRM-deletion mutant in transfected cells were also detected by Western blot analysis. (C) In vitro binding of c-Src to the PXXP motifs of SLC11A1. The SLC11A1 PRM peptide I (GEVCHLYYPKVPRTVLWLTI), PRM peptide II (ISSPTSPTSPGPRQAPPRE T), the negative control peptide (IPDTKPGTFSLRK LWAFTGPGFLM) and the positive control peptide (Src substrate Sam68), which were described in Materials and Methods, were coupled to activated CH-Sepharose 4B. Binding of these peptides to purified c-Src was detected by immunoblotting with anti-Src antibody. A representative Western blot is shown. (D) Peptide competition binding assay was performed. Purified c-Src was added 1, 2 or 5 μg of negative peptide, peptide I or peptide II before incubating with the CH-Sepharose 4B coupled with the positive control peptide. The illustrated showing the inhibitory effects of different peptides on c-Src binding to the positive peptide.