Members of the suppressor of cytokine signaling (SOCS) family are potentially key physiological negative regulators of interleukin-6 (IL-6) signaling. To examine whether SOCS3 is involved in regulating this signaling, we have used conditional gene targeting to generate mice lacking Socs3 in the liver or in macrophages. We show that Socs3 deficiency results in prolonged activation of signal transducer and activator of transcription 1 (STAT1) and STAT3 after IL-6 stimulation but normal activation of STAT1 after stimulation with interferon-gamma (IFN-gamma). Conversely, IL-6-induced STAT activation is normal in Socs1-deficient cells, whereas STAT1 activation induced by IFN-gamma is prolonged. Microarray analysis shows that the pattern of gene expression induced by IL-6 in Socs3-deficient livers mimics that induced by IFN-gamma. Our data indicate that SOCS3 and SOCS1 have reciprocal functions in IL-6 and IFN-gamma regulation and imply that SOCS3 has a role in preventing IFN-gamma-like responses in cells stimulated by IL-6.

ABSTRACT-/-mice has revealed that SOCS1 plays a key role in the negative regulation of interferon-γ signaling and in T cell differentiation. Socs2 -/-mice are 30%-40% larger than wild-type mice, demonstrating that SOCS2 is a critical regulator of postnatal growth. Additionally, the study of embryos lacking socs3 has revealed that SOCS3 is an important regulator of fetal liver hematopoiesis. The biological role of other SOCS proteins remains to be determined.

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SOCS-6 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS-1 to SOCS-7 and CIS) which each contain a central SH2 domain and a carboxyl-terminal SOCS box. SOCS-1, SOCS-2, SOCS-3, and CIS act to negatively regulate cytokine-induced signaling pathways; however, the actions of SOCS-4, SOCS-5, SOCS-6, and SOCS-7 remain less clear. Here we have used both biochemical and genetic approaches to examine the action of SOCS-6. We found that SOCS-6 and SOCS-7 are expressed ubiquitously in murine tissues. Like other SOCS family members, SOCS-6 binds to elongins B and C through its SOCS box, suggesting that it might act as an E3 ubiquitin ligase that targets proteins bound to its SH2 domain for ubiquitination and proteasomal degradation. We investigated the binding specificity of the SOCS-6 and SOCS-7 SH2 domains and found that they preferentially bound to phosphopeptides containing a valine in the phosphotyrosine (pY) ؉1 position and a hydrophobic residue in the pY ؉2 and pY ؉3 positions. In addition, these SH2 domains interacted with a protein complex consisting of insulin receptor substrate 4 (IRS-4), IRS-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase. To investigate the physiological role of SOCS-6, we generated mice lacking the SOCS-6 gene. SOCS-6 ؊/؊ mice were born in a normal Mendelian ratio, were fertile, developed normally, and did not exhibit defects in hematopoiesis or glucose homeostasis. However, both male and female SOCS-6 ؊/؊ mice weighed approximately 10% less than wild-type littermates.

Negative feedback is a mechanism commonly employed in biological processes as a means of maintaining homeostasis. We have investigated the roles of suppressor of cytokine signaling (SOCS) proteins in regulating the kinetics of negative feedback in response to cytokine signaling. In mouse livers and bone marrow-derived macrophages, both interferon-␥ (IFN␥) and interleukin-6 (IL-6) rapidly induced the tyrosine phosphorylation of signal transducer and activator of transcription-1 (STAT1) and STAT3. STAT3 tyrosine phosphorylation was bi-phasic in response to continuous IL-6 signaling. In macrophages lacking Socs3, however, continuous IL-6 signaling induced uniformly high levels of STAT3 tyrosine phosphorylation, and early IL-6-inducible genes were inappropriately expressed at intermediate time points. SOCS3 therefore imposes bi-phasic kinetics upon IL-6 signaling. Compared with Socs3 mRNA, Socs1 mRNA was induced relatively slowly, and SOCS1 simply attenuated the duration of IFN␥ signaling. Surprisingly, heightened Socs1 mRNA expression but minimal STAT1 tyrosine phosphorylation was observed after prolonged stimulation with IFN␥, indicating that STAT1 may not play a large role in inducing Socs1 mRNA during steady-state IFN␥ signaling. We also demonstrate that both SOCS1 and SOCS3 can desensitize primary bone marrowderived macrophages to IFN␥ and IL-6 signaling, respectively. Consistent with the kinetics with which Socs1 and Socs3 mRNAs were induced, SOCS3 desensitized cells to IL-6 rapidly, whereas SOCS1-mediated desensitization to IFN␥ occurred at later time points. The kinetics with which SOCS proteins are induced by cytokine may therefore be a parameter that is "hard-wired" into specific cytokine signaling pathways as a means of tailoring the kinetics with which cells become desensitized. Interleukin-6 (IL-6)2 and interferon-␥ (IFN␥) are cytokines with key roles in regulating the immune response. Signaling by IL-6 and IFN␥ begins at the surface of the cell where the cytokines associate with their respective receptor complexes (1). The IFN␥-receptor complex consists of the ligand-binding IFN␥-receptor ␣ subunit and the signal-transducing IFN␥-receptor ␤ subunit, whereas the IL-6-receptor complex consists of the ligand-binding IL-6-receptor ␣ subunit and the signal-transducing glycoprotein-130 subunit (1). Associated with the receptor subunits are the Janus kinases (JAKs), which become activated upon receptor dimerization, and phosphorylate signal transducers and activators of transcription (STAT) transcription factors (2, 3). Both IFN␥ and IL-6 can activate JAK1 (4 -6) and JAK2 (7-10), and subsequently STAT1 (11, 12) and STAT3 (6,13,14). The phosphorylated STATs (P-STATs) then dimerize and are transported to the nucleus, where they bind to promoter sequences, and activate the transcription of a suite of genes, among which are those encoding the suppressor of cytokine signaling-1 (SOCS1) and SOCS3 proteins (15, 16). STAT3 activates the transcription of Socs3 mRNA by associating with the 5Ј promoter region of the Soc...

To identify new potential substrates for the MAP kinase signal-integrating kinases (Mnks), we employed a proteomic approach. The Mnks are targeted to the translational machinery through their interaction with the cap-binding initiation factor complex. We tested whether proteins retained on cap resin were substrates for the Mnks in vitro, and identified one such protein as PSF (the PTB (polypyrimidine tract-binding protein)-associated splicing factor). Mnks phosphorylate PSF at two sites in vitro, and our data show that PSF is an Mnk substrate in vivo. We also demonstrate that PSF, together with its partner, p54 nrb , binds RNAs that contain AU-rich elements (AREs), such as those for proinflammatory cytokines (e.g. tumor necrosis factor ␣ (TNF␣)). Indeed, PSF associates specifically with the TNF␣ mRNA in living cells. PSF is phosphorylated at two sites by the Mnks. Our data show that Mnk-mediated phosphorylation increases the binding of PSF to the TNF␣ mRNA in living cells. These findings identify a novel Mnk substrate. They also suggest that the Mnk-catalyzed phosphorylation of PSF may regulate the fate of specific mRNAs by modulating their binding to PSF⅐p54 nrb .Polypyrimidine tract-binding protein (PTB) 3 -associated splicing factor (PSF) and p54 nrb are highly homologous DNA/ RNA-binding proteins that form a multifunctional heterodimer implicated in nuclear processes such as transcription, nuclear RNA processing, nuclear retention of edited RNA, DNA relaxation, and tumorigenesis (reviewed in Ref. 1). These proteins also cooperate in the inhibition of human immunodeficiency virus type 1 mRNA expression (2). Moreover, PSF is also been reported to repress gene expression through its association with nuclear hormone receptors (3) or through binding to insulin-like growth factor-1 response elements (4). PSF and p54 nrb are both phosphoproteins. Phosphorylation may be involved in the relocalization of PSF during apoptosis (5) and in regulating the binding properties of p54 nrb during mitosis (6). All eukaryotic cytoplasmic mRNAs have a 5Ј-terminal cap structure that contains 7-methyl-GTP (m 7 GTP) and promotes their efficient translation (7,8). The cap is bound by eukaryotic translation initiation factor eIF4E, which also binds to the scaffold eIF4G and through this with other translational factors to recruit the 40 S ribosomal subunit to the mRNA. eIF4G also binds the poly(A)-binding protein (PABP), which interacts with the 3Ј-end of the mRNA, thus circularizing it (reviewed in Ref. 9). eIF4E is phosphorylated in vitro and in vivo by the MAP kinase-signal integrating (or MAP kinase-interacting) kinases (Mnks) (10 -13). There are two Mnk genes in humans, each of which generates two different polypeptides as a consequence of alternative splicing (14, 15). The longer Mnk1 isoform, Mnk1a, is switched on by signaling through the ERK and p38 MAP kinase pathways, whereas Mnk2a (the longer Mnk2 isoform), in contrast, shows high basal activity (11,16,17). We recently showed that Mnks play an important role in the control ...

Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity.

Crk proteins are Src homology (SH) 2/SH3-containing adapter proteins that can mediate the formation of signaling complexes. We show that engaging the B cell antigen receptor (BCR) on the RAMOS B cell line caused both Crk-L and Crk II to associate with several tyrosinephosphorylated proteins. We identified two of these phosphoproteins as Cas and Cbl and showed that both bound to the Crk SH2 domain after BCR engagement. BCR ligation also increased the amount of Crk proteins in the particulate fraction of the cells and induced the formation of Crk⅐Cas and Crk⅐Cbl complexes in the particulate fraction. We propose that tyrosine phosphorylation of membrane-associated Cas and Cbl creates binding sites for the Crk SH2 domain and recruits Crk complexes to cellular membranes. Thus, Crk proteins may participate in BCR signaling by using their SH2 domains to direct the interactions and subcellular localization of proteins that bind to their SH3 domains. In RAMOS cells, we found that the SH3 domains of Crk-L and Crk II bound C3G. Since C3G activates Rap, a negative regulator of the Ras pathway, Crk proteins may participate in regulation of Ras signaling by the BCR.Signaling by the B cell antigen receptor (BCR) 1 plays an important role in both the establishment of immunologic tolerance and the generation of antibody (Ab) responses to foreign antigens. Immature B cells that bind self-antigens while still in the bone marrow are eliminated by apoptosis (1). In contrast, antigen binding by the BCR on newly formed mature B cells results in either activation, anergy, or apoptosis depending on the nature of the antigen and whether or not the B cell receives a co-stimulatory signal through other receptors such as CD40 (2, 3). In the presence of appropriate co-stimulatory signals and cytokines, BCR signaling promotes mature B cells to enter the cell cycle, proliferate, and differentiate into antibody-secreting cells (4).To understand how BCR engagement regulates B cell survival and activation, it is necessary to elucidate the signaling pathways used by the BCR. Cross-linking of the BCR by multivalent antigens or by anti-immunoglobulin (Ig) Abs results in activation of several Src family tyrosine kinases as well as the Syk and Btk tyrosine kinases (5-7). These kinases then activate the signaling pathways that are controlled by phospholipase C-␥, phosphatidylinositol (PtdIns) 3-kinase, and Ras (2). It is likely that the BCR also activates other signal transduction pathways that mediate the diverse effects of BCR engagement on B cells.A common feature of many receptor signaling pathways is that the components of the pathway are physically separated in resting cells but are then assembled into signaling complexes after the receptor is engaged. In many cases, the assembly of these complexes is necessary to bring enzymes close to their substrates. This strategy promotes efficient signaling in receptor-activated cells while ensuring low basal levels of signaling in resting cells. Adapter proteins that contain SH2 and SH3 protein interacti...