The SOCS proteins are induced by several cytokines and are involved in negative feedback loops. Here we demonstrate that in 3T3-L1 adipocytes, insulin, a hormone whose receptor does not belong to the cytokine receptor family, induces SOCS-3 expression but not CIS or SOCS-2. Using transfection of COS-7 cells, we show that insulin induction of SOCS-3 is enhanced upon Stat5B expression. Moreover, Stat5B from insulin-stimulated cells binds directly to a Stat element present in the SOCS-3 promoter. Once induced, SOCS-3 inhibits insulin activation of Stat5B without modifying the insulin receptor tyrosine kinase activity. Two pieces of evidence suggest that this negative regulation likely results from competition between SOCS-3 and Stat5B binding to the same insulin receptor motif. First, using a yeast two-hybrid system, we show that SOCS-3 binds to the insulin receptor at phosphotyrosine 960, which is precisely where Stat5B binds. Second, using confocal microscopy, we show that insulin induces translocation of SOCS-3 from an intracellular compartment to the cell membrane, leading to colocalization of SOCS-3 with the insulin receptor. This colocalization is dependent upon phosphorylation of insulin receptor tyrosine 960. Indeed, in cells expressing an insulin receptor mutant in which tyrosine 960 has been mutated to phenylalanine, insulin does not modify the cellular localization of SOCS-3. We have thus revealed an insulin target gene of which the expression is potentiated upon Stat5B activation. By inhibiting insulin-stimulated Stat5B, SOCS-3 appears to function as a negative regulator of insulin signaling.

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Microbe-macrophage interactions play a central role in the pathogenesis of many infections. The ability of some bacterial pathogens to induce macrophage apoptosis has been suggested to contribute to their ability to elude innate immune responses and successfully colonize the host. Here, we provide evidence that activation of liver X receptors (LXRs) and retinoid X receptors (RXRs) inhibits apoptotic responses of macrophages to macrophage colony-stimulating factor (M-CSF) withdrawal and several inducers of apoptosis. In addition, combined activation of LXR and RXR protected macrophages from apoptosis caused by infection with Bacillus anthracis, Escherichia coli, and Salmonella typhimurium. Expression-profiling studies demonstrated that LXR and RXR agonists induced the expression of antiapoptotic regulators, including AIM͞ CT2, Bcl-XL, and Birc1a. Conversely, LXR and RXR agonists inhibited expression of proapoptotic regulators and effectors, including caspases 1, 4͞11, 7, and 12; Fas ligand; and Dnase1l3. The combination of LXR and RXR agonists was more effective than either agonist alone at inhibiting apoptosis in response to various inducers of apoptosis, and it acted synergistically to induce expression of AIM͞CT2. Inhibition of AIM͞CT2 expression in response to LXR͞RXR agonists partially reversed their antiapoptotic effects. These findings reveal unexpected roles of LXRs and RXRs in the control of macrophage survival and raise the possibility that LXR͞RXR agonists may be exploited to enhance innate immunity to bacterial pathogens that induce apoptotic programs as a strategy for evading host responses.oxysterol ͉ transcription M acrophages serve essential functions as regulators of immunity and homeostasis (1, 2). As participants in native immunity, macrophages phagocytose and kill invading microorganisms and elaborate signaling molecules that amplify acute inflammatory responses. Macrophages also contribute to acquired immune responses by means of specialized functions that include antigen presentation and regulation of T cell responses. Thus, regulation of macrophage differentiation and survival is critical to the overall control of the magnitude, duration, and characteristics of immune responses. Programmed cell death, or apoptosis, of lymphocyte and myeloid cells is regulated tightly through cell death receptor and mitochondrial pathways to limit amplification of immune responses and facilitate resolution of inflammation (3). Apoptosis and survival pathways are also targeted by pathogens as a means of either escaping immune surveillance or establishing residence within host cells (4). The inhibition of macrophage apoptosis may offer a strategy for augmenting innate immunity to highly virulent bacterial pathogens, such as Bacillus anthracis, Yersinia pestis, Salmonella spp., and Shigella flexneri, which have evolved various ways to kill host macrophages. The execution of all forms of programmed cell death involves the proteolytic activation of a cascade of intracellular cysteine proteases, known as caspases....

We compared the interaction between the insulin receptor (IR) and the IR substrate (IRS) proteins IRS-1 and IRS-2) using the yeast two-hybrid system. Both IRS proteins interact specifically with the cytoplasmic portion of the IR and the related insulin-like growth factor-I receptor, and these interactions require receptor tyrosine kinase activity. Alignment of IRS-1 and IRS-2 revealed two conserved domains at the NH2 terminus, called IH1PH and IH2PTB, which resemble a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, respectively. The IH2PTB binds to the phosphorylated NPXY motif (Tyr-960) in the activated insulin receptor, providing a specific mechanism for the interaction between the receptor and IRS-1. Although the IH2PTB of IRS-2 also interacts with the NPEY motif of the insulin receptor, it is not essential for the interaction between the insulin receptor and IRS-2 in the yeast two-hybrid system. IRS-2 contains another interaction domain between residues 591 and 786, which is absent in IRS-1. This IRS-2-specific domain is independent of the IH2PTB and does not require the NPEY motif; however, it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop (Tyr-1146, Tyr-1150, and Tyr-1151). Importantly, this novel domain mediates the association between IRS-2 and insulin receptor lacking the NPXY motif and may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances IRS-2 phosphorylation.

Defining the molecular mechanisms that coordinately regulate proliferation and differentiation is a central issue in development. Here, we describe a mechanism in which induction of the Ets repressor METS/PE1 links terminal differentiation to cell cycle arrest. Using macrophages as a model, we provide evidence that METS/PE1 blocks Ras-dependent proliferation without inhibiting Ras-dependent expression of cell type-specific genes by selectively replacing Ets activators on the promoters of cell cycle control genes. Antiproliferative effects of METS require its interaction with DP103, a DEAD box-containing protein that assembles a novel corepressor complex. Functional interactions between the METS/DP103 complex and E2F/ pRB family proteins are also necessary for inhibition of cellular proliferation, suggesting a combinatorial code that directs permanent cell cycle exit during terminal differentiation.

Using the yeast two-hybrid system, a genetic assay for studying protein-protein interactions, we have examined and compared the interaction of the insulin-like growth factor-I receptor (IGF-IR) and the insulin receptor (IR) with their two known substrates p52Shc and the insulin receptor substrate-1 (IRS-1). We also mapped the specific domains of the IGF-IR and p52Shc participating in these interactions. Our findings can be summarized as follows: (i) the tyrosine kinase activity of the IGF-IR is essential for the interaction with p52Shc and IRS-1, (ii) p52Shc and IRS-1 bind to the IGF-IR in the NPEYjuxtamembrane motif, (iii) contrary to p52Shc, IRS-1 binds also to the major autophosphorylation sites (Tyr-1131, -1135, and -1136) of the IGF-IR, and (iv) the aminoterminal domain of p52Shc is required for its association with the IR and the IGF-IR. We propose that (i) the IGF-IR and the IR share at least in part the same molecular mechanism underlying their interplay with their two substrates, p52Shc and IRS-1, and (ii) IRS-1 interacts with the IGF-IR in a fashion that is different from that used by p52Shc. Finally, our data highlight the crucial role of the juxtamembrane domain in signaling by both the IR and the IGF-IR.

The diverse biological actions of insulin and insulin-like growth factor I (IGF-I) are initiated by binding of the polypeptides to their respective cell surface tyrosine kinase receptors. These activated receptors phosphorylate a series of endogenous substrates on tyrosine, amongst which the insulin receptor substrate (IRS) proteins are the best characterized. Their phosphotyrosine-containing motifs become binding sites for Src homology 2 (SH2) domains on proteins such as SH2 domain-containing protein-tyrosine-phosphatase (SHP)-2/Syp, growth factor receptor bound-2 protien, (Grb-2), and phosphatidyl inositol 3 kinase (PI3 kinase), which participate in activation of specific signaling cascades. However, the IRS molecules are not only platforms for signaling molecules, they also orchestrate the generation of signal specificity, integration of signals induced by several extracellular stimuli, and signal termination and modulation. An extensive review is beyond the scope of the present article, which will be centered on our own contribution and reflect our biases.

In addition to the pleckstrin homology domain and the phosphotyrosine binding domain in insulin receptor substrate (IRS)-1 and IRS-2, a region between amino acids 591 and 786 in IRS-2 (IRS-2-(591-786)) binds to the insulin receptor. Based on peptide competition studies, this region interacts with the phosphorylated regulatory loop of the insulin receptor; we designate this region the kinase regulatory loop binding (KRLB) domain. Two tyrosine residues in the KRLB domain at positions 624 and 628 are crucial for this interaction. Phosphorylation of tyrosine residues in the KRLB domain by the insulin receptor inhibits the binding to the receptor. These results reveal a novel mechanism regulating the interaction of the insulin receptor and IRS-2 that may distinguish the signal of IRS-2 from IRS-1. The insulin receptor (IR)1 mediates tyrosine phosphorylation of several cellular substrates, including IRS-1, IRS-2, and Gab-1 (1-3). These IRS (insulin receptor substrate) proteins provide an interface between the activated insulin receptor and various signaling proteins. IRS proteins are composed of a COOH terminus containing multiple tyrosine phosphorylation sites in various amino acid sequence motifs that bind to the Src homology-2 domain in certain enzymes and adapter molecules (4 -7). In addition to the phosphorylation sites, IRS proteins contain other domains to engage activated membrane receptors. At the extreme NH 2 terminus, the IRS proteins contain a pleckstrin homology (PH) domain (IH1 PH ). The IH1 PH is essential in IRS-1 for the interaction with a physiological level of insulin receptor (8); this domain plays a similarly important role in IRS-2 and Gab-1. In addition to the PH domain, IRS-1 and IRS-2 contain a phosphotyrosine binding (PTB) domain (IH2 PTB ), which binds to the phosphorylated NPEY motif in the cytoplasmic region of the receptors of insulin, insulin-like growth factor-I, and interleukin-4 (9 -15).A third region between residues 591 and 786 in IRS-2 engages the activated insulin receptor (13, 16). Using a yeast two-hybrid analysis, Tyr 624 and Tyr 628 in IRS-2 were found to contribute significantly to this interaction. The amino acid sequence in the region does not reveal a known protein-protein interaction domain, such as the PH domain, PTB domain, Src homology 2 domain, Src homology 3 domain, or WW domain (17-21). However, since it interacts with the phosphorylated regulatory loop of the insulin receptor ␤-subunit, we propose to designate it as the kinase regulatory loop binding (KRLB) domain. The binding of the KRLB domain of IRS-2 to the insulin receptor is independent of tyrosine phosphorylation sites in the COOH terminus and the NPEY motif in the juxtamembrane region (13). Since IRS-1 does not contain a KRLB domain (13, 16), this domain may contribute to a unique signaling potential of IRS-2. EXPERIMENTAL PROCEDURESYeast Strains and Plasmids-The yeast strain L40 (MAT a, trp1, leu2, his3, LYS2::lexA-His3, URA3::lexA-lacZ) and the yeast expression plasmids pBTM116 were obtained from A. Vojte...