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.
Zhou
X
X
Palo Alto Veterans Affairs Medical Center and Stanford University, Palo Alto, California 94304, USA.
Liao
J
J
Meyerdierks
A
A
Feng
L
L
Naumovski
L
L
Bottger
E C
EC
Omary
M B
MB
D000906:Antibodies; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents; D019281:Dimerization; D005455:Fluorescent Antibody Technique; D006801:Humans; D051796:Inhibitor of Differentiation Proteins; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D007700:Kinetics; D016350:Leucine Zippers; D009687:Nuclear Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; C085378:IFI35 protein, human; D051796:Inhibitor of Differentiation Proteins; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D047908:Intracellular Signaling Peptides and Proteins; C099927:NMI protein, human; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D007371:Interferon-gamma