Interferon-inducible Myc/STAT-interacting protein Nmi associates with IFP 35 into a high molecular mass complex and inhibits proteasome-mediated degradation of IFP 35.

Nmi is an interferon (IFN)-inducible protein homologous to IFN-inducible protein IFP 35. The homology consists of a novel Nmi/IFP 35 domain (NID) of 90-92 amino acids that is repeated in tandem in each protein and mediates Nmi-Nmi protein interactions and subcellular localization. In a yeast two-hybrid screen with a fragment of Nmi protein containing both NIDs, we identified an interaction between Nmi and IFP 35. Deletion derivatives of the proteins indicate that both NIDs are required for the interaction between Nmi and IFP 35. In mammalian cells, Nmi and IFP 35 co-immunoprecipitate and co-localize in large cytoplasmic speckles. Nmi and IFP 35 proteins associate into a high molecular mass complex of 300-400 kDa as determined by native gel electrophoresis and gel filtration. The association of Nmi and IFP 35 into a complex can be demonstrated in multiple cell lines and is not dependent on treatment with IFN. Short term and long term cultures of transfected HEK293 cells suggest that Nmi and IFP 35 proteins stabilize each other through complex formation. IFP 35 appears to be more labile because Nmi was stable in the absence of IFP 35, whereas IFP 35 was degraded in the absence of Nmi. A deletion analysis revealed that Nmi must interact with IFP 35 to prevent its degradation and that the amino terminus of Nmi is required, but not sufficient, for this function. Inhibition of the proteasome, but not other proteases, led to increased levels of IFP 35. Thus, we have shown that Nmi and IFP 35 associate into a protein complex, that IFP 35 is degraded in a proteasome-mediated process, and that a novel function of Nmi is to prevent IFP 35 degradation. The stabilization of IFP 35 by Nmi may serve to amplify the physiologic effects of IFNs.

Interferon-alpha induces nmi-IFP35 heterodimeric complex formation that is affected by the phosphorylation of IFP35.

Nmi and IFP35 are interferon (IFN)-induced proteins. In cells treated with IFN-gamma, Nmi enhances the association of transcription co-activator CBP/p300 with signal transducer and activator of transcription proteins, and IFP35 forms a high molecular weight cytosolic complex of unknown constituents. Here we show that Nmi and IFP35 co-immunoprecipitate with an anti-keratin 19 antibody, which is due to cross-reaction of the antibody with Nmi, and suggests an Nmi-IFP35 physical association. In support of this, Nmi and IFP35 co-immunoprecipitate using anti-Nmi and anti-IFP35 antibodies, manifest enhanced colocalization as determined by immunofluorescence staining of IFN-treated cells, and form heterodimers as determined by chemical cross-linking. Nmi and IFP35 are primarily cytosolic proteins, and their interaction is increased after IFN-alpha treatment of cells as early as 1 h after exposure. Sucrose gradient sedimentation and size fractionation showed a shift of Nmi-IFP35 heterodimers toward a heavier fraction (100-200 kDa) in IFN-alpha-treated cells. This dynamic complex formation is reversed by pretreatment with okadaic acid. Two-dimensional gel analysis indicates that the IFN-induced complex formation correlates with IFP35 dephosphorylation. Our data demonstrate Nmi-IFP35 cytosolic localization and heterodimerization, and an IFN-alpha-regulated molecular event in which Nmi and IFP35 participate, reversibly and by a dephosphorylation dependent fashion, in a 100-200-kDa molecular complex formation.

RGS1 is expressed in monocytes and acts as a GTPase-activating protein for G-protein-coupled chemoattractant receptors.

The leukocyte response to chemoattractants is transduced by the interaction of transmembrane receptors with GTP-binding regulatory proteins (G-proteins). RGS1 is a member of a protein family constituting a newly appreciated and large group of proteins that act as deactivators of G-protein signaling pathways by accelerating the GTPase activity of G-protein alpha subunits. We demonstrate here that RGS1 is expressed in human monocytes; by immunofluorescence and subcellular fractionation RGS1 was localized to the plasma membrane. By using a mixture of RGS1 and plasma membranes, we were able to demonstrate GAP activity of RGS1 on receptor-activated G-proteins; RGS1 did not affect ligand-stimulated GDP-GTP exchange. We found that RGS1 desensitizes a variety of chemotactic receptors including receptors for N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and C5a. Interaction of RGS proteins and ligand-induced G-protein signaling can be demonstrated by determining GTPase activity using purified RGS proteins and plasma membranes.

A cytoplasmic structure resembling large protein aggregates induced by interferons.

IFP 35 is an interferon (IFN)-regulated leucine zipper protein, expression of which is observed in a variety of cell types including monocytes/macrophages, epithelial cells and fibroblasts. Using immunofluorescence studies, we demonstrate that IFP 35 is found in characteristic punctate cytoplasmic structures after IFN treatment. Co-localization experiments using double immunofluorescence and confocal laser scanning microscopy failed to show association of IFP 35 with known organelles (mitochondria, peroxisomes, endoplasmic reticulum, lysosomes, endosomes, Golgi complex), ribosomes, or actin filaments. Subcellular fractionation to separate membrane-associated from cytoplasmic proteins demonstrated that IFP 35 localizes to the cytoplasm. Separation of postnuclear supernatant from HeLa cells by gel filtration revealed that IFP 35 eluted at a molecular mass of 200-440 kD, suggesting that IFP 35 is part of protein complexes. Electron microscopic studies showed cytoplasmic clusters of a few aggregates of IFP 35 in IFN-treated cells which were neither associated with nor surrounded by a membrane. A combination of immunoprecipitation and immunofluorescence studies of cells transfected with a hemagglutinin epitope-tagged IFP 35 expression construct demonstrated complex formation and co-localization of endogenous and transfected IFP 35. Taken together, our studies demonstrate that IFP 35 associates with unique cytoplasmic structures that are distinct from known organelles and resemble large protein aggregates.