The recovery of transfected IRP1 was analysed by immunoblotting with the FLAG antibody (lower panel). Earlier experiments with PMA-treated HL-60 cells have suggested that IRP1 and IRP2 are targets of phosphorylation, most probably by PKC (protein kinase C) [5]. phosphorylation assays of synthetic oligopeptides with PKC provided indirect evidence that the Rabbit Polyclonal to SNAP25 conserved Ser-138 and Ser-711 of IRP1 are, probably, physiologically relevant phosphorylation sites. The interest in a potential regulatory role for Ser-138 phosphorylation has been spurred by results obtained with phosphomimetic IRP1 mutants expressed in an aconitase-deficient yeast strain (aco1). Wild-type IRP1 complements aerobic growth of aco1 cells; however, S138D (Ser-711Asp) or S138E substitutions Hydroxocobalamin (Vitamin B12a) fail to do so, due to defects in the [4Fe-4S] cluster assembly pathway [6,7]. We have demonstrated that IRP1S138E expressed in mammalian cells not only fails to assemble the [4Fe-4S] cluster but, furthermore, undergoes iron-dependent degradation [8] in a fashion akin to the regulation of IRP2. The potential role of Ser-711 phosphorylation in IRP1 regulation has not received much attention so far, despite the fact that the oligopeptides containing Ser-711 appeared to be better substrates for PKC (and in transfected cells. The phosphomimetic S711E substitution is associated with a severe impairment of both aconitase and IRE-binding activities of IRP1, thus suggesting that Ser-711 is a critical site for regulation. MATERIALS AND METHODS Materials Haemin, PMA, bovine liver rhodanese, citrate, DL-isocitrate, BL21 (DE3) and recombinant human IRP1 was purified by affinity chromatography on Ni-NTA (Ni2+-nitrilotriacetic acid) beads, followed by a second purification step with anion-exchange chromatography on a Resource Q FPLC column (Pharmacia, Baie d’Urf, Quebec, Canada) [8]. Iron-sulphur cluster reconstitution The iron-sulphur cluster of human recombinant IRP1 (wild-type and mutants) was reconstituted by three different approaches: 5?g of purified protein was incubated for Hydroxocobalamin (Vitamin B12a) 25?min at 25?C either with 10?mM Hydroxocobalamin (Vitamin B12a) cysteine/HCl (pH?7.4) and 100?M ferrous sulphate [10], or with 100?mM DTT (dithiothreitol), 1?mM thiosulphate, 400?M ferric citrate and 10?M bovine liver rhodanese (enzyme catalysing the transfer of sulphur between thiosulphate and a thiophilic anion) [11], or with 70?mM DTT, 800?M sodium sulphide and 350?M ferric citrate [12]. Aconitase assay Aconitase activity was determined by the reduction of NADP+ at 340?nm in a coupled reaction with isocitrate dehydrogenase [10], with 200?M citrate or experiments with oligopeptides suggested that Ser-138 and Ser-711 are potential phosphorylation sites [5]. To map the phosphorylation site(s) of IRP1 phosphorylation (Figure 1C). Taken together, these results suggest that PMA triggers IRP1 phosphorylation at Ser-711. Open in a separate window Figure 1 Ser-711 is the site of IRP1 phosphorylation in cells treated with PMA(A) HEK-293 cells were transiently transfected with constructs encoding FLAG-tagged wild-type (wt) or mutant (S138A, S711A or S138A/S711A) IRP1. Parent and transfected cells were metabolically labelled with [32P]orthophosphate. After 30?min, 0.2?M PMA (dissolved in DMSO) or solvent alone were added in the radioactive medium, and the treatment was continued for 90?min. To assess the phosphorylation status of transfected wild-type IRP1, IRP1S138A, IRP1S711A and IRP1S138A/S711A, cytoplasmic extracts (1000?g) were subjected to quantitative immunoprecipitation (IP) with a FLAG antibody (8.8?g). The immunoprecipitated material was analysed by SDS/PAGE (8% polyacrylamide). Hydroxocobalamin (Vitamin B12a) Phosphorylated proteins were visualized by autoradiography (upper panel) and the recovery of transfected IRP1 was analysed by immunoblotting (IB) with 1:1000 diluted FLAG antibody (lower panel). (B) The indicated amounts of NSYG[pS]RRGND and NSYGSRRGND peptides were spotted on a Hydroxocobalamin (Vitamin B12a) nitrocellulose filter and analysed by IB with 1:1000 diluted 711[pS], a phospho-specific antibody raised against NSYG[pS]RRGND. (C) HEK-293 cells were transiently transfected with a construct encoding FLAG-tagged wild-type IRP1 and treated with 0.2?M PMA or DMSO alone for 90?min. To assess phosphorylation of IRP1 at Ser-711, cytoplasmic extracts (1000?g) were subjected to quantitative IP with the FLAG antibody (8.8?g) and subsequently analysed by IB with 1:1000?diluted 711[pS] antibody (upper panel). The recovery of transfected IRP1 was analysed by immunoblotting with the FLAG antibody (lower panel). The asterisks denote non-specific bands. Purified recombinant IRP1S711E displays minimal aconitase and IRE-binding activities Having established that Ser-711 is a biologically relevant phosphorylation site, we utilized recombinant wild-type and mutated versions of human IRP1, including the phosphorylation-deficient IRP1S711A and the phosphomimetic IRP1S711E mutants, to investigate further the role of this residue in IRP1 function. The proteins were tagged with an N-terminal His6 epitope, expressed in and purified by affinity and ion exchange chromatography. The purity of the preparations was very high, as evaluated by SDS/PAGE and staining with Coomassie Brilliant Blue (Figure 2A)..