An Wu

Functional Significance of Type 1 Insulin-like Growth Factor-mediated Nuclear Translocation of the Insulin Receptor Substrate-1 and β-Catenin

Previous work has shown that the transcriptional regulator β-catenin can translocate to the nuclei when cells are stimulated with the type 1 insulin-like growth factor (IGF-1). We show by immunocoprecipitation and by confocal microscopy that β-catenin binds to and co-localizes with the insulin receptor substrate-1 (IRS-1), a docking protein for both the insulin and the IGF-1 receptors. IRS-1 is required for IGF-1-mediated nuclear translocation of β-catenin, resulting in the activation of the β-catenin target genes. IGF-1-mediated nuclear translocation of β-catenin is facilitated by the nuclear translocation of IRS-1. Both IRS-1 and β-catenin are recruited to the cyclin D1 promoter, an established target for β-catenin, but only IRS-1 is recruited to the ribosomal DNA (rDNA) promoter. UBF proteins (known to interact with both IRS-1 and β-catenin) are also detectable in the cyclin D1 and rDNA promoters. These results indicate that IRS-1 (activated by the IGF-1 receptor) is one of several proteins that regulate the subcellular localization and activity of β-catenin. The ability of IRS-1 to localize to both RNA polymerase II (with β-catenin) and RNA polymerase I-regulated promoters suggest an explanation for the effect of IRS-1 on both cell growth in size and cell proliferation. This possibility is supported by the demonstration that enforced nuclear localization of IRS-1 causes nuclear translocation of β-catenin and transformation of normal mouse embryo fibroblasts (colony formation in soft agar).

Nuclear Translocation of Insulin Receptor Substrate-1 by the Simian Virus 40 T Antigen and the Activated Type 1 Insulin-like Growth Factor Receptor

32D cells are a murine hemopoietic cell line that undergoes apoptosis upon withdrawal of interleukin-3 (IL-3) from the medium. 32D cells have low levels of the type 1 insulin-like growth factor (IGF-I) receptor and do not express insulin receptor substrate-1 (IRS-1) or IRS-2. Ectopic expression of IRS-1 delays apoptosis but cannot rescue 32D cells from IL-3 dependence. In 32D/IRS-1 cells, IRS-1 is detectable, as expected, in the cytosol/membrane compartment. The SV40 large T antigen is a nuclear protein that, by itself, also fails to protect 32D cells from apoptosis. Co-expression of IRS-1 with the SV40 T antigen in 32D cells results in nuclear translocation of IRS-1 and survival after IL-3 withdrawal. Expression of a human IGF-I receptor in 32D/IRS-1 cells also results in nuclear translocation of IRS-1 and IL-3 independence. The phosphotyrosine-binding domain, but not the pleckstrin domain, is necessary for IRS-1 nuclear translocation. Nuclear translocation of IRS-1 was confirmed in mouse embryo fibroblasts. These results suggest possible new roles for nuclear IRS-1 in IGF-I-mediated growth and anti-apoptotic signaling.

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

The insulin receptor substrate-1 (IRS-1) is a docking protein of the insulin-like growth factor-1 (IGF-1) receptor and of the insulin receptor. IRS-1 sends a strong mitogenic, anti-apoptotic signal and plays an important role in cell transformation and cancer. IRS-1 translocates to nuclei of cells, where it increases the activity of the rDNA, c-myc and cyclin D1 promoters. We show, by chromatin immunoprecipitation, occupancy by IRS-1 of the same promoters. Both promoter activation and promoter occupancy are IGF-1-dependent. In cells that respond to IGF-1 but in which IRS-1 does not translocate to nuclei, promoter occupancy is absent and promoter activation is absent or much reduced. Transcriptional activation of c-myc and cyclin D1 promoters by nuclear IRS-1 does not occur with a mutant, inactive IRS-1 protein (deletion of the phosphotyrosine-binding domain, PTB) and does not require PI3-kinase activity. Taken together, these results indicate a novel mechanism by which nuclear IRS-1 activates cell cycle genes.

Transformation by the simian virus 40 T antigen is regulated by IGF-I receptor and IRS-1 signaling

Previous work has shown that the Simian Virus 40 T antigen (T antigen) cannot transform mouse embryo fibroblasts (MEFs) that do not express the type 1 insulin-like growth factor receptor (IGF-IR). We have now investigated the mechanism(s) by which the transforming activity of T antigen is affected by IGF-IR signaling. We demonstrate that transformation by T antigen of MEFs and several other cell lines requires an insulin receptor substrate-1 (IRS-1) phosphorylated on tyrosines. If IRS-1 is not expressed, or is serine phosphorylated or otherwise inactive, T antigen fails to transform cells in culture. For instance, while T antigen cannot transform 32D myeloid cells (that do not express IRS-1), its transforming activity is restored by the expression of a wild-type IRS-1, but not of an IRS-1 mutated at the PI3K binding sites. The importance of IRS-1 activation of PI3K in T-antigen transformation is supported by the finding that a constitutively activated p110 subunit of PI3K, a target of IRS-1, overcomes the inability of T antigen to transform MEFs with a serine phosphorylated IRS-1. Taken together, these results indicate that the IRS-1/PI3K signaling is one of the mechanisms regulating transformation by the SV40 T antigen. We propose that the requirement for a tyrosyl-phosphorylated IRS-1 provides a mechanism to explain the failure of T antigen to transform MEFs with deleted IGF-IR genes.

Nuclear translocation of insulin receptor substrate‐1 by the insulin receptor in mouse embryo fibroblasts

Translocation of the insulin receptor substrate‐1 (IRS‐1) to the nuclei has been reported to occur in cells stimulated by insulin‐like growth factor‐1 (IGF‐I) or expressing certain viral and cellular oncogenes. We show here that insulin can also induce nuclear translocation of IRS‐1 in mouse embryo fibroblasts (MEF), that do not express the type 1 insulin‐like growth factor receptor (IGF‐IR). Only the A isoform of the insulin receptor (IR) can induce IRS‐1 nuclear translocation, which is significant when the receptor is over‐expressed. At physiological receptor levels, translocation occurs only in a fraction of cells, and only at high concentrations of ligand.

Regulation of insulin receptor substrate‐1 expression levels by caveolin‐1

The insulin receptor substrate‐1 (IRS‐1), a docking protein of the type 1 insulin‐like growth factor receptor (IGF‐IR) plays a significant role in cell proliferation and differentiation. The expression of IRS‐1 is down‐regulated in mouse embryo fibroblasts (MEFs) with a deletion of caveolin‐1 (cav1) genes (KO cells). Levels of IRS‐1 mRNA are not affected. Re‐introduction of cav1 into KO cells rescues IRS‐1 expression. Stabilization of protein levels is reciprocal and a strict correlation between IRS‐1 and cav1 levels was confirmed in five cell lines, and in mouse tissues. IRS‐1 binds through its phosphotyrosine binding (PTB) domain to tyrosine 14 (Y14) of cav1, the residue phosphorylated by IGF‐1 stimulation and by v‐src. The down‐regulation of IRS‐1 in cav−/− cells occurs via the proteasome pathway. These results indicate a novel mechanism for the regulation of IRS‐1 expression levels, an important finding in view of IRS‐1 role in cell proliferation and transformation. J. Cell. Physiol. 217: 281–289, 2008.

Insulin receptor substrate-1 regulates the transformed phenotype of BT-20 human mammary cancer cells.

Although originating from a human breast cancer, BT-20 cells do not form colonies in soft agar. BT-20 cells do not express insulin receptor substrate-1 (IRS-1), which is known to promote both normal and abnormal growth and to inhibit differentiation. Stable expression of IRS-1 confers to BT-20 cells the ability to form colonies in soft agar. BT-20 cells form tumors in xenografts in mice, but the size of tumors is twice as large when the cells express IRS-1. The increased transformed phenotype is characterized by occupancy of the rDNA and cyclin D1 promoters by IRS-1 and the activation of the cyclin D1, c-myc, and rDNA promoters. In addition, the retinoblastoma protein, which is detectable in the rDNA promoter of quiescent BT-20/IRS-1 cells, is replaced by IRS-1 after insulin-like growth factor-I stimulation. Our results indicate that in BT-20 human mammary cancer cells, expression of IRS-1 activates promoters involved in cell growth and cell proliferation, resulting in a more transformed phenotype. Targeting of IRS-1 could be effective in inhibiting the proliferation of mammary cancer cells.

The role of insulin receptor substrate-1 in the oncogenicity of simian virus 40 T antigen.

The Simian Virus 40 large T antigen (Tag) and the insulin receptor substrate-1 (IRS-1) interact with each other. Tag is a nuclear protein even in the absence of IRS-1, but IRS-1 is required for occupancy and activation by Tag of the ribosomal DNA (rDNA), cyclin D1 and c-myc promoters. Our results indicate that IRS-1 plays a significant role in Tag activation of cell cycle progression genes.