The Human Protein HSPC021 Interacts with Int-6 and Is Associated with Eukaryotic Translation Initiation Factor 3

Myriam Ferro,Christelle Morris-Desbois,Pierre Jalinot,Stéphane Réty,Jérôme Garin

doi:10.1074/jbc.m104966200

Abstract

The Int-6 protein has been shown to be a subunit of eukaryotic translation initiation factor 3 (eIF3) and to play a role in the control of cell growth. By immunoprecipitation experiments and mass spectrometry analyses, we identified a human protein previously known as HSPC021 that is associated with Int-6. Exposure of Jurkat cells to the phosphatase inhibitor H(2)O(2) triggers a marked phosphorylation on tyrosine of HSPC021. Several experiments were performed to evaluate whether this protein is associated with eIF3. It was observed that HSPC021 coelutes with Int-6 and eIF3 in gel filtration, coimmunoprecipitates with eIF3, and is incorporated into eIF3 both in rabbit reticulocyte lysates and in COS7 cells. A direct protein-protein interaction occurs between HSPC021 and Int-6, but the analysis of different mutants of HSPC021 indicated that a larger region of the protein is necessary for incorporation into eIF3 as compared with binding to Int-6. Taken together, our results establish that HSPC021 is tightly associated with the mammalian translation initiation factor eIF3. Analysis of the primary sequence of HSPC021 from different species revealed the presence of a tetratricopeptide repeat, a proteasome-COP9 (constitutive photomorphogenesis 9) signalosome-initiation factor 3 domain along with a Pumilio FBF repeat. These protein motifs are also present in subunits of eIF3, of the lid of the 26 S proteasome, and of the COP9 signalosome.

Highlights

The Int-6 protein has been identified by three independent approaches
Studies in mice infected by the mouse mammary tumor virus have shown that the int-6 gene is a common site of integration of the provirus in preneoplastic and neoplastic mammary lesions [1]
A similar effect was observed with the S. pombe homolog of eukaryotic translation initiation factor 3 (eIF3)-p66, Moe1, which interacts with Int-6

Summary

Introduction

The Int-6 protein has been identified by three independent approaches. Studies in mice infected by the mouse mammary tumor virus have shown that the int-6 gene is a common site of integration of the provirus in preneoplastic and neoplastic mammary lesions [1]. By immunoprecipitation experiments and mass spectrometry analyses, we identified a human protein previously known as HSPC021 that is associated with Int-6. Immunoblot analysis of these extracts with antibodies recognizing phosphotyrosine proteins showed that H2O2 induced appearance of multiple bands in both cell lines (Fig. 1, compare lanes 2 and 4 with lanes 1 and 3, respectively).

Results

Conclusion