Related Experiment Videos
Interferon-alpha induces nmi-IFP35 heterodimeric complex formation that is affected by the phosphorylation of IFP35
X Zhou1, J Liao, A Meyerdierks
1Palo Alto Veterans Affairs Medical Center and Stanford University, Palo Alto, California 94304, USA.
Abstract:
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.
Insights
Interferon (IFN)-induced Nmi and IFP35 proteins form a cytosolic heterodimer complex. This interaction is regulated by IFN-alpha and IFP35 dephosphorylation, forming a 100-200 kDa complex.
Area of Science:
- Cellular and Molecular Biology
- Immunology
- Protein Interactions
Background:
- Nmi and IFP35 are interferon-induced proteins.
- Nmi interacts with transcription co-activators, while IFP35 forms cytosolic complexes.
- Previous studies suggested potential interactions but lacked direct evidence.
Purpose of the Study:
- To investigate the physical association and complex formation between Nmi and IFP35.
- To elucidate the role of interferon signaling in regulating Nmi-IFP35 interactions.
- To characterize the molecular properties of the Nmi-IFP35 complex.
Main Methods:
- Co-immunoprecipitation using specific antibodies.
- Immunofluorescence staining for colocalization analysis.
- Chemical cross-linking to confirm heterodimerization.
- Sucrose gradient sedimentation and size fractionation.
- Two-dimensional gel electrophoresis.
Main Results:
- Nmi and IFP35 were shown to physically associate and form heterodimers.
- Interferon-alpha treatment significantly increased Nmi-IFP35 interaction and complex formation (100-200 kDa).
- Complex formation was dependent on IFP35 dephosphorylation and reversible by okadaic acid.
Conclusions:
- Nmi and IFP35 form a cytosolic heterodimer complex.
- Interferon-alpha dynamically regulates this complex formation via IFP35 dephosphorylation.
- This interaction represents a novel interferon-regulated molecular event.