It is well known that insulin receptor substrates (IRS) act as a mediator for signal transduction of insulin, insulin-like growth factors, and several cytokines. To identify proteins that interact with IRS and modulate IRS-mediated signals, we performed yeast two-hybrid screening with IRS-1 as bait. Out of 109 cDNA-positive clones identified from a human placental cDNA library, two clones encoded 53BP2, p53-binding protein 2 (53BP2S), a short form splicing variant of the apoptosis-stimulating protein of p53 that possesses Src homology region 3 domain, and ankyrin repeats domain, and had been reported to interact with p53, Bcl-2, and NF-kappaB. Interaction of 53BP2S with IRS-1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays in COS-7 cells and 3T3-L1 adipocytes. The Src homology region 3 domain and ankyrin repeats domain of 53BP2S were responsible for its interaction with IRS-1, whereas the phosphotyrosine binding domain and a central domain (amino acid residues 750-861) of IRS-1 were required for its interaction with 53BP2S. In CHO-C400 cells, expression of 53BP2S reduced insulin-stimulated IRS-1 tyrosine phosphorylation with a concomitant enhancement of IRS-2 tyrosine phosphorylation. In addition, the amount of the phosphatidylinositol 3-kinase regulatory p85 subunit associated with tyrosine-phosphorylated proteins, and activation of Akt was enhanced by 53BP2S expression. Although 53BP2S also enhanced Akt activation in 3T3-L1 adipocytes, insulin-induced glucose transporter 4 translocation was markedly inhibited in accordance with reduction of insulin-induced AS160 phosphorylation. Together these data demonstrate that 53BP2S interacts and modulates the insulin signals mediated by IRSs.