The insulin receptor substrate-1 (IRS-1) is one of the major substrates of both the insulin and IGF-I receptors and is generally localized in the cytosol/membrane fraction of the cell. We show here that a substantial fraction of IRS-1 is translocated to the nucleus in mouse embryo fibroblasts (MEF) expressing the simian virus 40 T antigen. Nuclear translocation of IRS-1 occurs also in MEF stimulated with IGF-I or in MEF expressing the oncogene v-src. Nuclear translocation of IRS-1 can be demonstrated by confocal microscopy, immunohistochemistry, or subcellular fractionation. An antibody to IRS-1 immunoprecipitates from nuclear fractions (but not from cytosolic fractions) the upstream binding factor, which is a key regulator of RNA polymerase I activity and ribosomal RNA (rRNA) synthesis. In agreement with this finding, in 32D murine hemopoietic cells, nuclear translocation of IRS-1 correlates with a markedly increased rRNA synthesis. Our experiments suggest that nuclear IRS-1 may play a specialized role in rRNA synthesis and/or processing.The insulin receptor substrate (IRS) 1 proteins are a family of docking proteins, which include IRS-1-4, Gab-1, and p62 dok (1). IRS-1 was the first to be identified as a docking protein for both the insulin and the IGF-I receptors. It transmits a signal from both receptors by interacting with a number of partners, including phosphatidylinositol 3-kinase, SHP2, Grb2, Crk, and others (2, 3). Tyrosine kinase activity of the receptors and phosphorylation of IRS-1 are essential steps in signal transduction. Of the downstream signals generated by IRS-1, the best studied is the phosphatidylinositol 3-kinase signaling pathway, which plays a major role in a number of biological responses to growth factors (1, 4). IRS-1 interacts directly with both the insulin and the IGF-I receptors, and the domains required for their interaction have also been identified (5). Because of its direct interaction with the receptors, its size, and its downstream signaling, it has been generally assumed that IRS-1 is an exclusively cytosolic (or plasma membrane) protein (6, 7). However, IRS-1 is known to interact with the SV40 T antigen (8, 9) and nucleolin (10). T antigen and nucleolin are predominantly nuclear proteins, although minor fractions of either protein can be found in the cytosol (11-13). It has been therefore tacitly assumed that IRS-1, anchored to the receptor, was interacting with the minor cytosolic fractions of T antigen and nucleolin. There is evidence, however, that signal-transduction molecules can translocate to the nucleus. They include mitogen-activated protein kinase (14), p70 S6K /TOR (15, 16), the STAT proteins (17, 18), Akt (19), ␤-catenin (20), the epidermal growth factor receptor (21), phosphatases (22), IRS-3 (23), and a cleaved ErbB-4 receptor (24). Indeed, Jans and Hassan (25) have summarized in a review the evidence that growth factors and their receptors can accumulate in the nuclei of cells. IGF-I, IGF-IR, and IRS-1 are not mentioned in that review, but insulin is (26).T...

The insulin receptor substrate 1 (IRS-1) can translocate to the nuclei and nucleoli of several types of cells. Nuclear translocation can be induced by an activated insulin-like growth factor 1 receptor (IGF-IR), and by certain oncogenes, such as the Simian virus 40 T antigen and v-src. We have asked whether IRS-2 could also translocate to the nuclei. In addition, we have studied the effects of functional mutations in the IGF-IR on nuclear translocation of IRS proteins. IRS-2 translocates to the nuclei of mouse embryo fibroblasts expressing the IGF-IR, but, at variance with IRS-1, does not translocate in cells expressing the Simian virus 40 T antigen. Mutations in the tyrosine kinase domain of the IGF-IR abrogate translocation of the IRS proteins. Other mutations in the IGF-IR, which do not interfere with its mitogenicity but inhibit its transforming capacity, result in a decrease in translocation, especially to the nucleoli. Nuclear IRS-1 and IRS-2 interact with the upstream binding factor, which is a key regulator of RNA polymerase I activity and, therefore, rRNA synthesis. In 32D cells, wild-type, but not mutant, IRS-1 causes a significant activation of the ribosomal DNA promoter. The interaction of nuclear IRS proteins with upstream binding factor 1 constitutes the first direct link of these proteins with the ribosomal DNA transcription machinery.

Insulin receptor substrate 1 (IRS-1) is a major signaling molecule activated by the insulin and insulin-like growth factor I receptors. Recent data obtained in different cell models suggested that in addition to its conventional role as a cytoplasmic signal transducer, IRS-1 has a function in the nuclear compartment. However, the role of nuclear IRS-1 in breast cancer has never been addressed. Here we report that in estrogen receptor a (ERa)-positive MCF-7 cells, (1) a fraction of IRS-1 was translocated to the nucleus upon 17-b-estradiol (E2) treatment; (2) E2-dependent nuclear translocation of IRS-1 was blocked with the antiestrogen ICI 182,780; (3) nuclear IRS-1 colocalized and co-precipitated with ERa; (4) the IRS-1:ERa complex was recruited to the E2-sensitive pS2 gene promoter. Notably, IRS-1 interaction with the pS2 promoter did not occur in ERa-negative MDA-MB-231 cells, but was observed in MDA-MB-231 cells retransfected with ERa. Transcription reporter assays with E2-sensitive promoters suggested that the presence of IRS-1 inhibits ERa activity at estrogen-responsive elementcontaining DNA. In summary, our data suggested that nuclear IRS-1 interacts with ERa and that this interaction might influence ERa transcriptional activity.