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.

Subcellular localization of interferon-inducible Myc/stat-interacting protein Nmi is regulated by a novel IFP 35 homologous domain.

Nmi was initially identified through a yeast two-hybrid interaction with N-Myc but it also interacts with c-Myc, Max, Fos, and several other transcription factors, including signal transducer and activator of transcription (Stat) proteins. Nmi is an interferon (IFN)-inducible protein with 25% amino acid identity to the IFN-inducible protein IFP 35. We have found that this homology consists of a novel domain of approximately 90-92 amino acids (aa) that is repeated in tandem in each protein. This region, termed Nmi/IFP 35 domain (NID), is important for subcellular localization of Nmi. Full-length Nmi protein or deletion constructs containing a single NID are localized to the cytoplasm, but amino-terminal Nmi fragments of up to 92 aa containing neither NID are nuclear. Fusion of the amino-terminal end of Nmi to pyruvate kinase, an exclusively cytoplasmic protein, results in a cytoplasmic fusion protein, suggesting that the amino-terminal end of Nmi does not contain a classic nuclear localization signal (NLS). Fusion of the amino-terminal end of Nmi to green fluorescent protein (GFP), which is normally found in both nuclear and cytoplasmic compartments, does not alter GFP distribution, whereas fusion of a single NID to GFP targets the fusion to the cytoplasm. Fusion of a nuclear localization signal (NLS) to full-length Nmi or NID repeats targets the hybrid to the nucleus, suggesting that a strong NLS is dominant to the cytoplasmic localization function of NID. NID may mediate cytoplasmic localization of the full-length Nmi protein through NID-NID protein interactions as demonstrated by yeast two-hybrid assay, immunoprecipitation, and the presence of Nmi in a high molecular weight protein complex. These results suggest that Nmi is composed of a modular structure with an amino-terminal domain that when separated from the rest of the protein is nuclear. The carboxy-terminal two thirds of the protein is composed of two NID that mediate cytoplasmic localization of the full-length protein.

Interferon-induced upregulation and cytoplasmic localization of Myc-interacting protein Nmi.

Nmi interacts with c-Myc, N-Myc, Max, and fos, as demonstrated by yeast two-hybrid and coimmunoprecipitation assays. Nmi is partially homologous to IFP 35, an interferon (IFN)-inducible protein. In this study, we show that basal expression of Nmi is upregulated by IFN in multiple tumor-derived cell lines. Treatment with IFN results in an increased amount of cytoplasmic Nmi distributed in a punctate granular pattern. We also demonstrate that Nmi is expressed in various fetal and adult tissues. As Nmi does not contain a known DNA-binding motif, it has the potential to form inactive heterodimers with its putative DNA-binding partners. Our studies suggest that Nmi may modulate its binding partners in an IFN-inducible manner.