Insulin Receptor Substrate 1 Translocation to the Nucleus by the Human JC Virus T-antigen

Abstract

Insulin receptor substrate 1 (IRS-1) is the major signaling molecule for the insulin and insulin-like growth factor I receptors, which transduces both metabolic and growth-promoting signals, and has transforming properties when overexpressed in the cells. Here we show that IRS-1 is translocated to the nucleus in the presence of the early viral protein-T-antigen of the human polyomavirus JC. Nuclear IRS-1 was detected in T-antigenpositive cell lines and in T-antigen-positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JC virus T-antigen binding was localized within the N-terminal portion of IRS-1 molecule, and the binding was independent from IRS-1 tyrosine phosphorylation and was strongly inhibited by IRS-1 serine phosphorylation. In addition, competition for the IRS-1-T-antigen binding by a dominant negative mutant of IRS-1 inhibited growth and survival of JC virus T-antigen-transformed cells in anchorageindependent culture conditions. Based on these findings, we propose a novel role for the IRS-1-T-antigen complex in controlling cellular equilibrium during viral infection. It may involve uncoupling of IRS-1 from its surface receptor and translocation of its function to the nucleus. Insulin receptor substrate 1 (IRS-1)1 is a 160-kDa cytosolic protein implicated in insulin and IGF-I signal transduction. IRS-1 plays an essential role in IGF-I-mediated cell proliferation (1, 2), and has transforming properties when overexpressed in different cell types (3, 4). The structure of IRS-1 reveals two conserved regions within the N-terminal portion of the protein (5, 6). The first one is called PH for its similarity to a pleckstrin homology domain (7), and the second shows similarity to a putative phosphotyrosine-binding (PTB) domain present in Shc and other proteins (6). The PTB domain recognizes phosphorylated tyrosine within NPXY motifs, providing a mechanism to couple IRS-1 with the Tyr 950 in the juxtamembrane region of the IGF-IR (8). PH domains contain a positively charged binding pocket that may mediate interaction with phospholipids (9) and with proteins containing acidic motifs (10). Following activation, over 20 phosphorylation sites on the IRS-1 docking molecule can recruit a variety of proteins equipped with Src homology domains (11). Independent from its tyrosine phosphorylation, IRS-1 interacts with ␣ v ␤ 3 (12) and ␣ 5 ␤ 1 (13, 14) integrins, with typical nuclear proteins such as the SV40 large T-antigen (3) and nucleolin (10) and is constitutively phosphorylated in v-Src transformed cells (15). Transforming properties of IRS-1 were suspected for quite some time even before the first convincing evidence was furnished by utilizing R Ϫ cells (3T3-like fibroblasts derived from mice with targeted disruption of IGF-IR gene) (3, 4). Although R Ϫ cells are remarkably resistant to transformation (15, 16), co-expression of IRS-1 and SV40 T-antigen induced R Ϫ transformation, a phenotype efficiently reversed by antisense IRS-1 mRNA (4). Importantly, overexpressio...

Nuclear insulin receptor substrate 1 interacts with estrogen receptor α at ERE promoters

“…Lower panel: The abundance of pS2 and 36B4 mRNAs in COS-7 cells transfected with different plasmids was detected by RT-PCR, as described in Materials and methods Nuclear IRS-1/ERa interactions C Morelli et al tion of IRS-1 is blocked with ICI and does not occur in ERa-negative cells; (3) nuclear IRS-1 interacts with ERa; (4) nuclear IRS-1 is corecruited with ERa to the ERE-containing pS2 promoter; and (5) the presence of IRS-1 decreases ERa transcription at ERE promoters. Nuclear localization of IRS-1 has recently been described in different cellular systems (Lassak et al, 2002;Prisco et al, 2002;Sun et al, 2003;Tu et al, 2002;Sciacca et al, 2003). The mechanism by which IRS-1 enters cell nucleus is still not clear.…”

“…In addition to its conventional role as a cytoplasmic signaling molecule, IRS-1 appears to function in the nuclear compartment. Several rigorously controlled studies demonstrated that nuclear IRS-1 can be found in cells transformed by oncogenic proteins, for example, T antigens of the JCV (Lassak et al, 2002) and SV40 viruses, and v-src (Tu et al, 2002). Nuclear translocation of IRS-1 has also been described in mouse embryo fibroblasts stimulated with IGF-I (Tu et al, 2002;Sun et al, 2003), 32D murine cells (Sciacca et al, 2003), osteoblasts (Seol and Kim, 2003), and hepatocytes (Boylan and Gruppuso, 2002).…”

“…The nuclear localization of IRS-1 requires specific IRS-1 domains, but these requirements appear to be different depending on the experimental system. For instance, in JCV T-antigenexpressing cells, nuclear localization of IRS-1 depends on its pleckstrin homology domain (Lassak et al, 2002), while in IGF-I-stimulated cells, the phosphotyrosine binding domain is required .…”

Nuclear insulin receptor substrate-1 activates promoters of cell cycle progression genes

“…IRS-1 is translocated to the nuclei in cells that express the SV40T antigen or its human equivalent (Lassak et al, 2002). However, T antigen cannot transform MEFs unless IRS-1 is tyrosyl phosphorylated (DeAngelis et al, 2006).…”

“…More recently, IRS-1 has been found in nuclei and nucleoli of cells, either after stimulation with IGF-1 (Tu et al, 2002), or in association with the T antigens of SV40 or its human equivalent (Lassak et al, 2002;Prisco et al, 2002). Nuclear IRS-1 binds the upstream binding factor 1 (UBF1), one of the proteins that regulate RNA polymerase I activity (Tu et al, 2002).…”