Characterization of the Interaction between the Interferon-Induced Protein P56 and the Int6 Protein Encoded by a Locus of Insertion of the Mouse Mammary Tumor Virus

Abstract

For determining cellular functions of the interferon-inducible human cytoplasmic protein P56, we undertook a Saccharomyces cerevisiae two-hybrid screen that identified Int6 as a P56-interacting protein. That the interaction also occurs in human cells was confirmed by coimmunoprecipitation and the observed cytoplasmic displacement of nuclear Int6 upon coexpression of P56. Because Int6 has been claimed to be both a cytoplasmic and a nuclear protein, we investigated the structural basis of this discrepancy. By mutational analyses, we showed that the Int6 protein contains a bipartite nuclear localization signal and a nuclear export signal at the far end of the amino terminus. The 20 amino-terminal residues of Int6, when they were attached to a different nuclear protein, were sufficient to translocate that protein to the cytoplasm. Within this region, replacement of any of the three leucine residues with alanine destroyed the function of the export signal. The specific domain of P56 that is required for its interaction with Int6 was mapped using the yeast two-hybrid assay and a mammalian coimmunoprecipitation assay. Both assays demonstrated that the C-terminal region of P56 containing three specific tetratricopeptide motifs is required for this interaction. In contrast, removal of an internal domain of P56 enhanced the interaction, as quantified by the two-hybrid assay.

Identification and expression analysis of two IFN-inducible genes in crucian carp (Carassius auratus L.)

“…tertiary structure of the helix and impair the contacts (Guo and Sen, 2000). Previous reports demonstrated that the last three specific TPR motifs in the C-terminal region are required for interaction of IFI56 with the translation initiation factor eIF-3 (Guo and Sen, 2000;Guo et al, 2000a), which is critical for human IFI56 inhibiting cellular protein synthesis, possibly including viral protein synthesis.…”

“…Previous reports demonstrated that the last three specific TPR motifs in the C-terminal region are required for interaction of IFI56 with the translation initiation factor eIF-3 (Guo and Sen, 2000;Guo et al, 2000a), which is critical for human IFI56 inhibiting cellular protein synthesis, possibly including viral protein synthesis. In the present study, all 10 TPR motifs were found in CaIFI58 as compared to human IFI58.…”

“…These genes encode structurally related proteins, which contain multiple tetratricopeptide repeat (TPR) domains that are a loosely conserved 34-amino acid residue repeat unit involved in specific protein -protein interactions (Lamb et al, 1995;Das et al, 1998). Recently, one cellular function of IFI56 has been revealed by the discovery that IFI56 binds to the large multimeric complex of eIF-3 via p48 and blocks its function in initiating protein synthesis, which is presumed to mediate inhibiting viral replication and cell growth (Guo and Sen, 2000;Guo et al, 2000a), and also possibly mediate cell apoptosis (Grandvaux et al, 2002).…”

Int6 Expression Can Predict Survival in Early-Stage Non–Small Cell Lung Cancer Patients

“…In fission yeasts, Int6 deletion mutants accumulate ubiquitinated proteins or misfolded proteins after treatment with canavanine, indicating a proteasome malfunction (9). In addition, it has been shown that Int6 binds the IFN-induced protein p56 during the course of viral infection (13,24). Based on these and other observations, it has been suggested that Int6 may act particularly in cellular stress conditions, which cause protein misfolding during translation or ubiquitination (15).…”

“…The constitutive photomorphogenesis 9 signalosome is predominantly nuclear (10), eIF3 is cytoplasmic, and the proteasome can be distributed in both cell compartments (11,12). Because Int6 contains a bipartite nuclear localization sequence and a putative NH 2 -terminal nuclear export signal (13), it can shuttle between the nucleus and the cytoplasm and interact with these three complexes (14). All of these observations suggest that Int6 could exert a regulatory activity in both protein translation and degradation and possibly act on specific transcripts (15).…”