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D J Hilton

Publications and source records attributed to D J Hilton.

At least 37 records · Page 2Linked to original sources
PubMed2000

Suppressor of cytokine signaling-3 preferentially binds to the SHP-2-binding site on the shared cytokine receptor subunit gp130.

Suppressor of cytokine signaling-3 (SOCS-3) is one member of a family of intracellular inhibitors of signaling pathways initiated by cytokines that use, among others, the common receptor subunit gp130. The SH2 domain of SOCS-3 has been shown to be essential for this inhibitory activity, and we have used a quantitative binding analysis of SOCS-3 to synthetic phosphopeptides to map the potential sites of interaction of SOCS-3 with different components of the gp130 signaling pathway. The only high-affinity ligand found corresponded to the region of gp130 centered around phosphotyrosine-757 (pY757), previously shown to be a docking site for the tyrosine phosphatase SHP-2. By contrast, phosphopeptides corresponding to other regions within gp130, Janus kinase, or signal transducer and activator of transcription proteins bound to SOCS-3 with weak or undetectable affinity. The significance of pY757 in gp130 as a biologically relevant SOCS-3 docking site was investigated by using transfected 293T fibroblasts. Although SOCS-3 inhibited signaling in cells transfected with a chimeric receptor containing the wild-type gp130 intracellular domain, inhibition was considerably impaired for a receptor carrying a Y-->F point mutation at residue 757. Taken together, these data suggest that the mechanism by which SOCS-3 inhibits the gp130 signaling pathway depends on recruitment to the phosphorylated gp130 receptor, and that some of the negative regulatory roles previously attributed to the phosphatase SHP-2 might in fact be caused by the action of SOCS-3.

Amino Acid Sequence↗
PubMed2000

Ligand-specific utilization of the extracellular membrane-proximal region of the gp130-related signalling receptors.

The receptor gp130 is used by the interleukin-6 (IL-6)-type cytokines, which include IL-6 and leukaemia-inhibitory factor (LIF). To investigate the role of the three extracellular membrane-proximal fibronectin-type-III-like (FNIII) modules of gp130 and the related receptor for granulocyte colony-stimulating factor (G-CSFR) in cytokine signal transduction we have transfected into murine myeloid M1-UR21 cells the chimaera (GR-FNIII)gp130, which contains the membrane-proximal FNIII modules of the G-CSFR on a gp130 backbone, and its complement, the chimaera (gp130-FNIII)GR. Whereas the binding affinities of (125)I-labelled IL-6 to (GR-FNIII)gp130, or of (125)I-Tyr1,3-G-CSF to (gp130-FNIII)GR, were similar to wild-type gp130 and wild-type G-CSFR, respectively, (125)I-LIF failed to bind with high affinity to (GR-FNIII)gp130. In assays measuring differentiation the (gp130-FNIII)GR cells were fully responsive to G-CSF, whereas the (GR-FNIII)gp130 cells responded fully to the agonistic anti-gp130 monoclonal antibody (mAb) B-S12, but not to IL-6 or LIF. Neutralizing mAbs that recognize the membrane-proximal FNIII modules of gp130 or the G-CSFR differentially interfered with signalling by B-S12, LIF and G-CSF. The data suggest that B-S12 and G-CSF induce the correct orientation or conformation for signalling by the wild-type and chimaeric homodimeric receptors, that the membrane-proximal region of gp130 is important for the correct formation of the signalling IL-6-IL-6 receptor-gp130 complex and that this region is also involved in LIF-dependent receptor heterodimerization and signalling.

Animals↗
PubMed2000

Adaptor protein SKAP55R is associated with myeloid differentiation and growth arrest.

Activation of the SRC family of protein tyrosine kinases is an important component of intracellular signaling in hematopoiesis, but their critical substrates are less well understood. In this report, we describe the cloning and functional characterization of murine SKAP55R (mSKAP55R), an SRC family kinase substrate. Expression of mSKAP55R was examined by Northern blot. Phosphorylation of mSKAP55R was examined by transient transfection of COS cells. For overexpression studies, mSKAP55R was cloned into a bicistronic murine stem cell virus-based retrovirus. Transduced cells (FDC-P1 cell line and murine bone marrow) were FACS isolated by expression of the selectable marker green fluorescent protein.mSKAP55R showed 90% amino acid identity to the recently published human SKAP55R. mSKAP55R contained a central pleckstrin homology domain, a C-terminal SH3 domain, and a putative SRC kinase consensus substrate DEIY(260). mSKAP55R was expressed in all hematopoietic lineages, with relative mRNA levels greatest in cells of the myeloid and erythroid lineages. Induced myeloid differentiation of M1 and HL-60 cell lines was associated with an eight-fold increase in mSKAP55R mRNA. Transient expression of mSKAP55R in COS cells demonstrated that tyrosine 260 was the predominant site of phosphorylation by FYN kinase. Furthermore, this phosphotyrosine was essential for coimmunoprecipitation of FYN with mSKAP55R. Enforced expression of mSKAP55R inhibited in vitro growth of the myeloid FDC-P1 cell line and primary hematopoietic progenitors. In contrast, a tyrosine 260 mutant mSKAP55R had no effect on in vitro growth. These studies implicate mSKAP55R in the processes of myeloid differentiation and growth arrest.

Amino Acid Sequence↗
PubMed2000

SOCS: physiological suppressors of cytokine signaling.

Cytokines regulate cellular behavior by interacting with receptors on the plasma membrane of target cells and activating intracellular signal transduction cascades such as the JAK-STAT pathway. Suppressors of cytokine signaling (SOCS) proteins negatively regulate cytokine signaling. The SOCS family consists of eight proteins: SOCS1-SOCS7 and CIS, each of which contains a central Src-homology 2 (SH2) domain and a C-terminal SOCS box. The expression of CIS, SOCS1, SOCS2 and SOCS3 is induced in response to stimulation by a wide variety of cytokines, and overexpression of these proteins in cell lines results in inhibition of cytokine signaling. Thus, SOCS proteins appear to form part of a classical negative feedback loop. The analysis of mice lacking SOCS1 has revealed that it is critical in the negative regulation of IFN(gamma) signaling and in the differentiation of T cells. Additionally, the analysis of mouse embryos lacking SOCS3 suggests that SOCS3 negatively regulates fetal liver erythropoiesis, probably through its ability to modulate erythropoietin (Epo) signaling. Thus, the use of gene targeting has confirmed that SOCS proteins regulate cytokine signaling in a physiological setting.

Animals↗
PubMed1999

STAT5b mediates the GH-induced expression of SOCS-2 and SOCS-3 mRNA in the liver.

Suppressor of cytokine signalling (SOCS) proteins act as part of a classical negative feedback loop regulating cytokine signal transduction. Expression of SOCS proteins is induced in response to cytokines and down-regulates the cytokine signal by inhibiting the JAK/STAT pathway. Growth hormone (GH) was previously shown to induce strong transient expression of SOCS-3 and to a lesser extent CIS, SOCS-1 and SOCS-2 in mouse liver (Adams, T.E., Hansen, J.A., Starr, R., Nicola, N.A., Hilton, D.J., Billestrup, N., 1998. Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signalling. J. Biol. Chem. 273, 1285-1287.). In this work we have compared GH-induced SOCS gene expression in wild-type and STAT5b-deficient mice, and show that STAT5b is required for the induction of SOCS-2 and SOCS-3 in liver. In contrast, the absence of STAT5b has no effect on the GH-induced expression of CIS and SOCS-2 mRNA in the mammary gland. Suprisingly, there is no activation of SOCS-3 expression in mammary glands of wild-type and STAT5b mutant mice following GH administration. These results highlight both tissue- and factor-specific differences in the regulation of SOCS gene expression by STAT5a/b.

Animals↗
PubMed1999

SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine.

Mice lacking suppressor of cytokine signaling-1 (SOCS1) develop a complex fatal neonatal disease. In this study, SOCS1-/- mice were shown to exhibit excessive responses typical of those induced by interferon gamma (IFNgamma), were hyperresponsive to viral infection, and yielded macrophages with an enhanced IFNgamma-dependent capacity to kill L. major parasites. The complex disease in SOCS1-/- mice was prevented by administration of anti-IFNgamma antibodies and did not occur in SOCS1-/- mice also lacking the IFNgamma gene. Although IFNgamma is essential for resistance to a variety of infections, the potential toxic action of IFNgamma, particularly in neonatal mice, appears to require regulation. Our data indicate that SOCS1 is a key modulator of IFNgamma action, allowing the protective effects of this cytokine to occur without the risk of associated pathological responses.

Alphavirus Infections↗
PubMed1999

Suckling defect in mice lacking the soluble haemopoietin receptor NR6.

Cytokines control a variety of cellular responses including proliferation, differentiation, survival and functional activation, via binding to specific receptors expressed on the surface of target cells [1]. The cytokine receptors of the haemopoietin family are defined by the presence of a conserved 200 amino acid extracellular domain known as the haemopoietin domain [2]. We report here the isolation of NR6, a haemopoietin receptor that, like the p40 subunit of interleukin-12 (IL-12) [3] and the EBI3 gene induced by Epstein-Barr virus infection in lymphocytes [4], contains a typical haemopoietin domain but lacks transmembrane and cytoplasmic domains. Although in situ hybridisation revealed NR6 expression at multiple sites in the developing embryo, mice lacking NR6 did not display obvious abnormalities and were born in the expected numbers. Neonatal NR6(-/-) mice failed to suckle, however, and died within 24 hours of birth, suggesting that NR6 is necessary for the recognition or processing of pheromonal signals or for the mechanics of suckling itself. In addition, NR6(-/-) mice had reduced numbers of haemopoietic progenitor cells, suggesting a potential role in the regulation of primitive haemopoiesis.

Amino Acid Sequence↗
PubMed1999

The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation.

The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.

Amino Acid Sequence↗
PubMed1999

Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction.

SOCS-1 (suppressor of cytokine signaling-1) is a representative of a family of negative regulators of cytokine signaling (SOCS-1 to SOCS-7 and CIS) characterized by a highly conserved C-terminal SOCS box preceded by an SH2 domain. This study comprehensively examined the ability of several SOCS family members to negatively regulate the gp130 signaling pathway. SOCS-1 and SOCS-3 inhibited both interleukin-6 (IL-6)- and leukemia inhibitory factor (LIF)-induced macrophage differentiation of murine monocytic leukemic M1 cells and LIF induction of a Stat3-responsive reporter construct in 293T fibroblasts. Deletion of amino acids 51-78 in the N-terminal region of SOCS-1 prevented inhibition of LIF signaling. The SOCS-1 and SOCS-3 N-terminal regions were functionally interchangeable, but this did not extend to other SOCS family members. Mutation of SH2 domains abrogated the ability of both SOCS-1 and SOCS-3 to inhibit LIF signal transduction. Unlike SOCS-1, SOCS-3 was unable to inhibit JAK kinase activity in vitro, suggesting that SOCS-1 and SOCS-3 act on the JAK-STAT pathway in different ways. Thus, although inhibition of signaling by SOCS-1 and SOCS-3 requires both the SH2 and N-terminal domains, their mechanisms of action appear to be biochemically different.

Animals↗
PubMed1999

Negative regulation of the JAK/STAT pathway.

Cytokines induce a variety of biological responses by binding to specific cell surface receptors and activating cytoplasmic signal transduction pathways, such as the JAK/STAT pathway. Although these responses are generally transient, few molecules have been characterised that switch the signal off. Several different steps of the signal transduction pathway appear to be targeted by negative regulators, including the receptor/ligand complex, JAK kinases, and STAT transcription factors. Negative regulation is achieved by dephosphorylation of signalling intermediates by protein tyrosine phosphatases such as SHP-1, and by proteolytic degradation. Recent studies have identified two new families of negative regulatory molecules, SOCS and PIAS, which function in novel ways to suppress signal transduction pathways. The duration and intensity of a cell's response to cytokine therefore appear to be determined by the net effect of several regulatory mechanisms.

Animals↗
PubMed1999

Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction.

SOCS-1 was originally identified as an inhibitor of interleukin-6 signal transduction and is a member of a family of proteins (SOCS-1 to SOCS-7 and CIS) that contain an SH2 domain and a conserved carboxyl-terminal SOCS box motif. Mutation studies have established that critical contributions from both the amino-terminal and SH2 domains are essential for SOCS-1 and SOCS-3 to inhibit cytokine signaling. Inhibition of cytokine-dependent activation of STAT3 occurred in cells expressing either SOCS-1 or SOCS-3, but unlike SOCS-1, SOCS-3 did not directly interact with or inhibit the activity of JAK kinases. Although the conserved SOCS box motif appeared to be dispensable for SOCS-1 and SOCS-3 action when overexpressed, this domain interacts with elongin proteins and may be important in regulating protein turnover. In gene knockout studies, SOCS-1(-/-) mice were born but failed to thrive and died within 3 weeks of age with fatty degeneration of the liver and hemopoietic infiltration of several organs. The thymus in SOCS-1(-/-) mice was small, the animals were lymphopenic, and deficiencies in B lymphocytes were evident within hemopoietic organs. We propose that the absence of SOCS-1 in these mice prevents lymphocytes and liver cells from appropriately controlling signals from cytokines with cytotoxic side effects.

Animals↗
PubMed1999

Negative regulators of cytokine signal transduction.

Enormous advances have been made over the last 10 years in unravelling cytokine signal transduction. This work has led to the recognition of the prime importance of Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). More recently, the importance of negative regulators of this pathway has begun to be realised. There is now evidence for at least three families of proteins that inhibit JAK/STAT signalling. The suppressors of cytokine signalling (SOCS), protein inhibitors of activated STATs (PIAS) and the SH2-containing phosphatase (SHP-1). This review describes some of the key features of SOCS proteins and contrasts their actions with other negative regulators, the PIAS proteins and SHP-1.

Animals↗
PubMed1999

Aberrant hematopoiesis in mice with inactivation of the gene encoding SOCS-1.

Mice with homozygous inactivation of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) protein die within 21 days of birth with low body weight, fatty degeneration and necrosis of the liver, infiltration of the lung, pancreas, heart and skin by macrophages and granulocytes and a profound depletion of T- and B-lymphocytes. In the present study, SOCS-1 -/- mice were found to have a moderate neutrophilia, and reduced platelet and hematocrit levels. Replacement of the SOCS-1 gene by a lac-Z reporter gene allowed documentation by FACS sorting that at least a proportion of granulocyte-macrophage progenitor cells transcribe SOCS-1. Most hematopoietic progenitor cell frequencies were normal in -/- marrow as were the size and cellular content of colonies formed by -/- progenitor cells in response to various stimulating factors. However, there was an increased frequency of macrophage progenitor cells in -/- mice and, abnormally, one quarter of all progenitor cells were located in the liver. Progenitor cells from -/- mice were hyper-responsive to stimulation by GM-CSF but not by M-CSF or Multi-CSF (IL-3). Progenitor cells from -/- mice were also hypersensitive to inhibition by interferon-gamma (IFN-gamma), the degree of inhibition varying markedly with the stimulating factor used. The suppressive effects of IFN-gamma therefore appear to involve interactions with particular growth factor-initiated signals in -/- cells--interactions that are strongly modulated by the action of the SOCS-1 protein.

Animals↗
PubMed1999

Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins.

In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS proteins it was found that the ability of SOCS proteins to inhibit GH-mediated transcription was located in the amino-terminal 40-80 amino acids. In SOCS-3, 46 amino acids C-terminal to the SH2 domain were required for the inhibitory activity, while a truncated SOCS-1 having only 2 amino acids C-terminal to the SH2 domain was able to inhibit GH-mediated transcription. Both SOCS-1 and SOCS-3 were able to inhibit GH-induced STAT5 (signal transducer and activator of transcription) activation. SOCS-1 inhibited the tyrosine kinase activity of Janus kinase 2 (JAK2) directly, while SOCS-3 only inhibited JAK2 when stimulated by the GH receptor. All four SOCS proteins were able to bind to a tyrosine-phosphorylated glutathione-S-transferase-GH receptor fusion protein, and SOCS-3 required the same 46 C-terminal amino acids for GH receptor binding as it did for inhibition of GH-mediated transcription and STAT5 activation. These data suggest that SOCS-1 and -3 can suppress GH-induced transcriptional activity, presumably by inhibiting the kinase activity of JAK2 either directly in the case of SOCS-1 or via binding to the tyrosine-phosphorylated GH receptor in the case of SOCS-3.

Amino Acid Sequence↗
PubMed1998

The box-1 region of the leukemia inhibitory factor receptor alpha-chain cytoplasmic domain is sufficient for hemopoietic cell proliferation and differentiation.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that acts on a variety of cell types and regulates cell proliferation and differentiation. The functional receptor for LIF is composed of LIFR alpha-chain (LIFRalpha) and gp130 both of which are shared in the functional receptors for oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. By using stable transfection of wild-type or cytoplasmic deletion mutants of LIFRalpha together with full-length gp130 into Ba/F3 cells, we found that cells expressing gp130 and an extensively deleted mutant LIFRalpha containing only the box-1 region were capable of proliferating in response to LIF, although LIF-dependent long term growth of these cells was seriously impaired. Using a similar strategy to generate WEHI-3BD+ cells expressing gp130 and wild-type or truncation mutants of LIFRalpha, studies revealed that the box-1 region of the LIFRalpha was also sufficient for LIF-dependent induction of different aspects of differentiation, including up-regulation of macrophage surface marker expression, morphological change, and cell migration in agar culture. However, the C-terminal region of the LIFRalpha, although not essential for intracellular signaling, was important for efficient receptor-mediated ligand internalization. In summary, the membrane-proximal box-1 region plays a dominant role in LIF-induced signal transduction of both proliferation and differentiation.

Cell Differentiation↗
PubMed1998

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1.

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1(-/-) mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1(-/-) mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

Abnormalities, Multiple↗
PubMed1998

The immunoglobulin-like module of gp130 is required for signaling by interleukin-6, but not by leukemia inhibitory factor.

The transmembrane protein gp130 is a shared component of the receptor complexes for the interleukin-6 (IL-6)-type cytokines, which include IL-6, leukemia inhibitory factor (LIF) and oncostatin M (OSM). In addition to its role in the generation of high affinity receptors, gp130 is required for signal transduction by these cytokines. In the present study we have examined the role of the N-terminal located, extracellular immunoglobulin (Ig)-like module of gp130 in signal transduction by IL-6 and LIF. We have expressed wild-type human gp130 or three mutants in murine myeloid M1-UR21 cells that lack functional endogenous gp130 but express the IL-6 receptor (IL-6R) and the LIF receptor (LIFR). By measuring cellular responses, such as morphological changes upon differentiation, soft agar colony formation, and induction of tyrosine phosphorylation of the signal transducer and activator of transcription, STAT3, we show that signaling by IL-6, but not LIF, is significantly reduced by mutations in the Ig-like module of gp130. However, the binding of 125I-labeled IL-6 or LIF is not affected by these mutations. We also present evidence that the Ig-like module forms part of the epitope of an anti-gp130 monoclonal antibody that neutralizes the bioactivity of IL-6, but not of LIF or OSM. The data suggest that gp130-activation by IL-6 and LIF requires different regions of gp130, that the Ig-like module of gp130 may be required for IL-6-induced gp130 dimerization, and that the stoichiometry of the high affinity IL-6 receptor-complex differs from those of the receptor-complexes for LIF and OSM.

Animals↗