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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...

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JC polyomavirus (JCV), which infects 90% of the human population, is detectable in human tumors. Its early protein, JCV T-antigen, transforms cells in vitro and is tumorigenic in experimental animals. Although T-antigen-mediated transformation involves genetic alterations of the affected cells, the mechanism underlying this genomic instability is not known. We show that JCV T-antigen inhibits homologous recombination DNA repair (HRR), which results in an accumulation of mutations. T-antigen does not operate directly but utilizes a cytosolic molecule, insulin receptor substrate 1 (IRS-1). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with artificial nuclear localization signal. Our observations define a new mechanism by which viral protein utilizes cytosolic molecule to inhibit faithful DNA repair, and suggest how polyomaviruses could compromise stability of the genome. (c) 2005 Wiley-Liss, Inc.

Glomerular visceral epithelial cells (podocytes) play a critical role in the pathogenesis of human immunodeficiency virus (HIV)-associated nephropathy.A key question concerns the mechanism(s) by which the HIV-1 genome alters the phenotype of the highly specialized, terminally differentiated podocytes. Here, using an in vitro system of conditionally immortalized differentiated human podocytes (CIDHPs), we document a pivotal role for the p66ShcA protein in HIV-1-induced reactive oxygen species generation and CIDHP apoptosis. CIDHP transfected with truncated HIV-1 construct (NL4-3) exhibit increased reactive oxygen species metabolism, DNA strand breaks, and a 5-fold increase in apoptosis, whereas the opposite was true for NL4-3/CIDHP co-transfected with mu-36p66ShcA (mu-36) dominant negative expression vector or isoform-specific p66-small interfering RNA. Phosphorylation at Ser-36 of the wild type p66ShcA protein, required for p66ShcA redox function and inhibition of the potent stress response regulator Foxo3a, was unchanged in mu-36/NL4-3/CIDHP but increased in NL4-3/CIDHP. Acute knockdown of Foxo3a by small interfering RNA induced a 50% increase in mu-36/NL4-3/CIDHP apoptosis, indicating that Foxo3a-dependent responses promote the survival phenotype in mu-36 cells. We conclude that inhibition of p66ShcA redox activity prevents generation of HIV-1 stress signals and activation of the CIDHP apoptosis program.Glomerular visceral epithelial cells or podocytes are highly specialized cells that play a pivotal role in the pathogenesis of focal segmental glomerular sclerosis (FSGS) and the collapsing variant of this entity, frequently encountered in HIVAN. The podocyte, strategically positioned along the glomerular basement membrane, is a critical component of the glomerular filtration barrier, functioning in tandem with its associated slit diaphragm to limit passage of albumin and plasma proteins to the urinary space (1, 2). Compelling evidence (3-7) supports a key role for HIV-1 gene products in the podocyte injury that leads to a breach in the integrity of the glomerular filtration barrier and the massive proteinuria that characterizes HIVAN. The absence of podocyte regeneration after cell injury or apoptosis is a major limitation to the development of innovative therapeutic strategies to arrest or prevent HIVAN and other glomerular diseases. Accordingly, interventions that increase the resistance of this terminally differentiated cell population to death signals offer a novel approach to preserve the integrity and permselectivity of the glomerular filtration barrier.Several lines of evidence support a dominant role for the p66ShcA protein in the intracellular pathways that convert oxidative stress to apoptosis (8, 9). The three overlapping Shc proteins, p66ShcA, p52ShcA, and p46ShcA, share a C-terminal Src homology 2 domain, central collagen homology region, and N-terminal phosphotyrosine binding domain. p46ShcA and p52ShcA are the product of alternative translation initiation sites within the same transcript, wherea...