Ferroportin is a multi-pass membrane protein that serves as an iron exporter in many vertebrate cell types. Ferroportin-mediated iron export is controlled by the hormone hepcidin, which binds ferroportin, causing its internalization and degradation. Mutations in ferroportin cause a form of the iron overload disease hereditary hemochromatosis. Relatively little is known about ferroportin's properties or the mechanism by which mutations cause disease. Here we expressed and purified human ferroportin to characterize its biochemical/biophysical properties in solution and conducted cell biological studies in mammalian cells. We show that purified, detergent-solubilized ferroportin was a well-folded monomer that bound hepcidin. In cell membranes, the N-and C-termini were both cytosolic, implying an even number of transmembrane regions, and ferroportin was mainly localized to the plasma membrane. Hepcidin addition resulted in a redistribution of ferroportin to intracellular compartments that labeled with early endosomal and lysosomal, but not Golgi, markers and that trafficked along microtubules. An analysis of 16 disease-related ferroportin mutants revealed that all formed well-folded monomers that localized to the plasma membrane, but some were resistant to hepcidin-induced internalization. The characterizations reported here form a basis upon which models for ferroportin's role in regulating iron homeostasis in health and disease can be interpreted.

Human VHR (vaccinia H1 related phosphatase) is a member of the dual-specificity phosphatases (DSPs) that often act on bisphosphorylated protein substrates. Unlike most DSPs, VHR displays a strong preference for dephosphorylating phosphotyrosine residues over phosphothreonine residues. Here we describe the 2.75 A crystal structure of the C124S inactive VHR mutant in complex with a bisphosphorylated peptide corresponding to the MAP kinase activation lip. This structure and subsequent biochemical studies revealed the basis for the strong preference for hydrolyzing phosphotyrosine within bisphosphorylated substrates containing -pTXpY-. In the structure, the two phospho residues are oriented into distinct pockets; the phosphotyrosine is bound in the exposed yet deep active site cleft while the phosphothreonine is loosely tethered into a nearby basic pocket containing Arg(158). As this structure is the first substrate-enzyme complex reported for the DSP family of enzymes, these results provide the first glimpse into how DSPs bind their protein substrates.

The dual specificity mitogen-activated protein kinase phosphatase MKP3 has been shown to down-regulate mitogenic signaling through dephosphorylation of extracellular signal-regulated kinase (ERK). Camps et al. Studies have shown that these MAP kinase phosphatases (MKPs) display distinct in vivo substrate preferences for the various MAP kinases (8 -11). Of the proposed MKPs, one of the most selective enzymes is MKP3 (9) (also named rVH6 (12) and Pyst1 (8)). The NH 2 -terminal "noncatalytic" region of MKP3 was shown to display tight binding to its in vivo substrate, ERK (13). This NH 2 -terminal domain is distinct from the common catalytic core that resides at the COOH-terminal half of the protein. Not only does the NH 2 -terminal domain bind ERK protein independently, but in the context of the full-length protein, ERK binding activates the phosphatase activity by at least 35-fold (14). ERK phosphorylation was not required for either binding or activation. The physical basis for this dramatic activation has not been established. Recently, the x-ray structure for the catalytic domain of Pyst1 was solved. The overall structure was remarkably similar to the previously published x-ray structure of the DSP VHR (15) and that predicted from modeling studies based on the VHR structure (16). The full-length VHR structure (15) represents the minimal catalytic domain shared with larger DSPs such as MKP3, MKP1, PAC1, which share 30% sequence identity to VHR within the catalytic domain but contain a second domain that is predicted to extend from the amino terminus. VHR has served as the archetypal DSP in detailed biochemical

No abstract

No abstract