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(22,26,43,44), and transcription factor subunits called STATs (signal transducers and activators of transcription) (8, 39). Phosphorylated STATs, which also contain Src homology 2 (SH2) domains, associate to form homo-and heterodimers (40, 51). These dimers, with or without additional cofactors, migrate to the nucleus, where they activate the transcription of IFN-responsive genes (5).IFN-␣ and -␤ induce formation of the transcription factor IFN-stimulated gene factor 3 (ISGF3) (4,20). The binding sites on DNA for ISGF3, called IFN-stimulated response elements (ISREs), are found near promoters of most IFN-␣/␤-responsive genes (7,19,31,33,34,48). The ISGF3 transcription factor is an oligomeric protein with three subunits: STAT1, STAT2, and a 48-kDa DNA-binding protein (9,10,38,46). STAT1 exists in two alternatively spliced forms of 91 kDa (STAT1␣) and 84 kDa (STAT1␤); either is capable of participating in ISGF3 formation (27). STAT2 is a 113-kDa protein having approximately 40% homology with STAT1␣ (10). Unlike IFN-␣/␤, IFN-␥ triggers the phosphorylation on tyrosine of STAT1 but not STAT2 (41), leading to formation of the gamma-activated transcription factor GAF, which binds to the gamma-activated sequences (GAS) (6,21,42). GAF is formed when STAT1␣ subunits dimerize through reciprocal SH2 domain-phosphotyrosine interactions (40). Phosphorylation of STAT1 and formation of STAT1 homodimers can also be activated by IFN-␣. Before the STAT proteins were recognized, this IFN-␣-activated factor was called AAF (6).Unphosphorylated STAT1␣ binds to a specific phosphotyrosine near the C terminus of the IFN-␥ receptor ␣ chain (12). Phosphorylation of this tyrosine upon binding of IFN-␥ to the receptor is an early step in the IFN-␥ signaling pathway, creating a binding site for the unphosphorylated transcription factor subunit in proximity to the receptor-bound tyrosine kinases JAK1 and JAK2 (26, 49). The IFN-␣ receptor may employ a similar mechanism. However, although phosphorylation of the IFN-␣ receptor has been observed (30), the site is not yet known.Genetic studies using mutant cell lines unresponsive to the IFNs (17,24,29,49) have established the functional importance of JAKs and STATs in the pathways. Two JAK family kinases, TYK2 (47) and JAK1 (26), as well as STAT1 (27) and the 48-kDa DNA-binding protein (17) are required for the response of most genes to IFN-␣. However, the situation is somewhat complex in that mutants lacking TYK2 retain a weak response to , probably mediated by formation of ISGF3 (15). Furthermore, some genes (IRF-1, for example) respond to IFN-␣/␤ independently of ISGF3, utilizing AAF to activate a GAS-like element (6, 13). The availability of mutant cell lines lacking the individual proteins of ISGF3 allows analysis of the function of each protein separately. Study of U3A cells, which lack STAT1␣ and -␤, has shown that STAT1␣ (but not STAT1␤) is required in the IFN-␥ pathway, that either STAT1␣ or STAT1␤ can function in the IFN-␣ pathway, and that Y-701 (the phosphorylation site in STAT1) and R...

Interferon a induction of transcription operates through interferon-stimulated-gene factor 3 (ISGF), a transcription factor two components ofwhich are members of the newly characterized Stat family of transcription factors.Interferon a induces tyrosine phosphorylation of Statl and Stat2 proteins that associate and, together with a 48-kDa protein, form ISGF3. Evidence is presented that a heterodimer of Statl and Stat2 is present in ISGF3 and that Statl and the 48-kDa protein make precise contact, while Stat2 makes general contact, with the interferon-stimulated response element, the binding site of the ISGF3.Cytokine attachment to cell surface receptors triggers gene activation in the cell nucleus (1). Many of these extracellular polypeptides cause their intracellular changes through the Jak-Stat pathway (2). The Jak proteins are protein-tyrosine kinases associated with cell surface receptors that are activated by receptor occupation. The Stat proteins serve the dual function of signal transduction and activation of transcription. The first polypeptide ligand recognized to use this pathway was interferon a (IFN-a), which leads to activation of a nuclear DNA-binding complex called interferon-stimulated-gene factor 3 (ISGF3) (3, 4), which upon purification proved to contain four protein species, 113, 91, 84, and 48 kDa in size (5). The first three of these were the proteins that yielded sequence information establishing the sequence similarity in the Stat family (6, 7); it was also demonstrated that the 113-, 91-, and 84-kDa proteins (renamed Stat2, Statla, and Statlf3, respectively) were activated by phosphorylation on a single similarly located tyrosine (8-10). (The 91-and 84-kDa proteins differ only in a 38-aa carboxyl-terminal extension in the 91-kDa protein.) The 48-kDa protein,

Alpha interferon (IFN-alpha)-induced transcriptional activation requires the induction of a complex of DNA-binding proteins, including tyrosine-phosphorylated Stat1 and Stat2, and of p48, a protein which is not phosphorylated on tyrosine and which comes from a separate family of DNA-binding proteins. The isolation and characterization of U6A cells, which lack Stat2, have allowed the introduction of normal and mutant forms of Stat2 so that various functions of the Stat2 protein can be examined. As reported earlier, Stat1, which is the second target of tyrosine phosphorylation in IFN-alpha-treated cells, is not phosphorylated in the absence of Stat2. We show that all mutations that block Stat2 phosphorylation also block Stat1 phosphorylation. These include not only the mutations of Y-690 and SH2 domain residues that are involved in tyrosine phosphorylation but also short deletions at the amino terminus of the protein. Two mutants of Stat2 that are not phosphorylated on tyrosine can act as dominant negative proteins in suppressing wild-type Stat2 phosphorylation, most likely by competition at the receptor-kinase interaction site(s). We also show that the COOH-terminal 50 amino acids are required for transcriptional activation in response to IFN-alpha. Mutants lacking these amino acids can be phosphorylated, form IFN-stimulated gene factor 3, and translocate to the nucleus but cannot stimulate IFN-alpha-dependent transcription. Seven acidic residues are present in the deleted COOH-terminal residues, but 24 acidic residues still remain in the 100 carboxy-terminal amino acids after deletion. Thus, transcriptional activation is unlikely to depend on acidic amino acids alone.

The present studies have examined the effects of ionizing radiation on control of the early growth response 1 (EGRI) gene. Exposure of human HL-525 cells to x-rays was associated with increases in EGRI mRNA levels. Nuclear run-on assays showed that this effect is related at least in part to activation ofEGRI gene transcription. Sequences responsive to ionizing radiation-induced signals were determined by deletion analysis of the EGRI promoter. The results demonstrate

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The cellular response to ionizing radiation includes induction of the early growth response 1 gene (EGRI). The present work has examined the involvement of reactive oxygen intermediates (ROTs) in this response. Exposure of human HL-525 cells, an HL-60 subclone deficient in protein kinase C-mediated signaling, to both ionizing radiation and H202 was associated with increases in EGR-1 transcripts. These increases in EGR-1 expression were inhibited by the antioxidant N-acetyl-L-cysteine (NAC). Nuclear run-on assays demonstrate that NAC inhibits the activation of EGRI transcription by these agents. Previous studies have shown that induction ofEGRI by x-rays is conferred by serum response or CC(A/T)6GG (CArG) elements. The present studies demonstrate similar findings with H202 and the finding that activation of the EGRI promoter region containing CArG elements is abrogated by NAC. Moreover, we show that NAC inhibits the ability of a single CArG box to confer x-ray and H202 inducibility when linked to a heterologous promoter. Taken together, these fmdings indicate that ROIs induce EGRI transcription by activation of CArG elements.

We recently described the identification of a non-peptidyl fungal metabolite (L-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T., Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974 -977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver. In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.Insulin elicits a diverse array of biological responses by binding to its specific receptor (1). The insulin receptor (IR) 1 is a heterotetrameric protein consisting of two extracellular ␣ subunits and two transmembrane ␤ subunits. The binding of the ligand to the ␣ subunit of IR not only concentrates insulin at its site of action, but also induces conformational changes in the receptor, which in turn stimulates the tyrosine kinase activity intrinsic to the ␤ subunit of the IR. Extensive studies have indicated that the ability of the receptor to autophosphorylate and phosphorylate intracellular substrates is essential for its mediation of the complex cellular responses of insulin (2-5).Insulin receptors trans-phosphorylate several immediate substrates (on Tyr residues), including insulin receptor substrate (IRS) proteins 1-4, Shc, and Gab 1, each of which provide specific docking sites for other signaling proteins containing Src homology 2 domains (6). These events lead to the activation of downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-kinase). Numerous studies have adduced that PI 3-kinase is required for the metabolic effec...