Association of Insulin Receptor Substrate 1 with Simian Virus 40 Large T Antigen

Abstract

Mouse embryo cells expressing a wild-type number of insulin-like growth factor I receptors (IGF-IR) (W cells) can be transformed either by simian virus 40 large T antigen (SV40 T) or by overexpressed insulin receptor substrate 1 (IRS-1), singly transfected. Neither SV40 T antigen nor IRS-1, individually, can transform mouse embryo cells with a targeted disruption of the IGF-IR genes (R ؊ cells). However, cotransfection of SV40 T antigen and IRS-1 does transform R؊ cells. In this study, using different antibodies and different cell lines, we found that SV40 T antigen and IRS-1 are coprecipitated from cell lysates in a specific fashion, regardless of whether the lysates are immunoprecipitated with an antibody to SV40 T antigen or an antibody to IRS-1. The same antibody to SV40 T antigen, however, fails to coprecipitate another substrate of IGF-IR, the transforming protein Shc, and two other signal-transducing molecules, Grb2 and Sos. Finally, an SV40 T antigen lacking the amino-terminal 250 amino acids fails to coprecipitate IRS-1 and also fails to transform R ؊ cells overexpressing mouse IRS-1. These experiments indicate that IRS-1 associates with SV40 T antigen and that this association plays a critical role in the combined ability of these proteins to transform R ؊ cells. This finding is discussed in light of the crucial role of the IGF-IR in the establishment and maintenance of the transformed phenotype.The insulin-like growth factor I (IGF-I) receptor (IGF-IR) (56) plays a crucial role in the establishment and maintenance of the transformed phenotype. Antibodies to IGF-IR (1, 18), antisense expression plasmids to either IGF-I (55), IGF-IR (4, 40, 41, 48), or IGF-II (6), and dominant negative mutants of IGF-IR (25, 37) can all reverse the transformed phenotype and/or inhibit tumorigenesis. Conversely, overexpression of the wild-type (but not a mutant) IGF-IR induces transformation (7,19,25,28,46), while overexpression of IGF-II in transgenic mice increases the incidence of certain malignancies (42).Recently, we have generated from mouse embryos homozygous for a targeted disruption of the IGF-IR genes and from their wild-type littermates (2, 26) cell lines of 3T3-like fibroblasts, designated, respectively, R Ϫ (receptor minus) and W (wild-type) cells (46,47). R Ϫ cells grow in 10% serum (albeit more slowly than W cells) but do not grow at all in serum-free medium supplemented with the growth factors that sustain the growth of W cells, and of other 3T3-like cells, such as plateletderived growth factor (PDGF), epidermal growth factor, IGF-I, IGF-II, insulin at supraphysiological concentrations, and fibroblast growth factors (7, 46). In addition, R Ϫ cells are refractory to transformation by simian virus 40 (SV40) T antigen (47) or by other oncoproteins, such as an activated HaRas (46, 52), singly or in combination. The growth deficits of R Ϫ cells, including their resistance to transformation, are abrogated by the stable transfection of a plasmid expressing a wild-type (but not a mutant) human IGF-IR cDNA (7,9,25...

Activation of the IGF-IR system contributes to malignant growth of human and mouse medulloblastomas

“…Considering that both T-positive and T-negative cells have similar levels of the IGF-IR and P13-K proteins, and both grossly overexpress IRS-1 (Figure 3), it is feasible that the IGF-IR signaling pathway and JCV Tantigen may cooperate by amplifying the signal towards transformation in this cellular system. There are several reports that support this notion: JCV Tantigen has high homology to SV40 large T-antigen (Gallia et al, 1998); both JCV T-antigen and SV40 Tantigen have transforming properties (Gallia et al, 1998); SV40 T-antigen is not able to transform cells in the absence of IGF-IR (Sell et al, 1993); cells expressing functional IGF-IR can be transformed either by ectopic expression of SV40 T-antigen or by IRS-1 overexpression (Fei et al, 1995); IRS-1 coprecipitates with the SV40 T-antigen (Fei et al, 1995), and SV40 T-antigen enhances expression from the IGF-1 promoter (Porcu et al, 1994). Whether similar interactions exist between the IGF-IR system and the JCV T-antigen, and whether these interaction/s contribute to the development and progression of medulloblastoma remains to be determined.…”

“…Conversely, reactivation of IRS-1 expression in cells from the external granular layer (Kulik et al, 1997;Reiss et al, 2000). Additionally, the IRS-1 protein has transforming properties but only when overexpressed in cells with a functional IGF-IR (Fei et al, 1995). In addition, the IRS-1 protein has been found to co-precipitate with the SV40 T antigen.…”

“…In addition, the IRS-1 protein has been found to co-precipitate with the SV40 T antigen. Simultaneous ectopic expression of both IRS-1 and SV40 T is able to sustain the transformed phenotype of the cell, even in the absence of the IGF-IR (Fei et al, 1995). We have selected two mouse medulloblastoma cell lines (Bs-1b and Bs-1c) that are JCV T-antigen negative and two cell lines (BcB8 and BsB13) that are JCV T-antigen positive (Krynska et al, 2000).…”

Adenovirus 5 E1A is responsible for increased expression of insulin receptor substrate 4 in established adenovirus 5-transformed cell lines and interacts with IRS components activating the PI3 kinase/Akt signalling pathway

“…Another viral oncoprotein, SV40T antigen, interacts directly with IRS-1 (Fei et al, 1995) relocalizing it to the nucleus. Transformation by SV40T requires IRS-1 to be phosphorylated on tyrosine residues to allow the activation of PI3 kinase, such that the absence or inactivation of IRS-1 renders cells refractory to transformation (DeAngelis et al, 2006).…”

Deactivation of Akt by a small molecule inhibitor targeting pleckstrin homology domain and facilitating Akt ubiquitination

“…S3). To test if SC66 could inhibit the Akt signaling pathway, HEK293T cells, which were shown to contain a high level of PIP3 (19), were treated with different amounts of SC66, and the whole-cell lysates were examined for the phosphorylation level of Akt and its known target proteins (Fig. 1B).…”