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Biomedical subjects

James A Johnston

Publications and source records attributed to James A Johnston.

At least 19 recordsLinked to original sources
PubMed2006

SOCS3 targets Siglec 7 for proteasomal degradation and blocks Siglec 7-mediated responses.

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5-11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.

Animals↗
PubMed2006

CD33 responses are blocked by SOCS3 through accelerated proteasomal-mediated turnover.

CD33 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family of inhibitory receptors and a therapeutic target for acute myeloid leukemia (AML). CD33 contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), which can recruit SHP-1 and SHP-2. How CD33 expression is regulated is unclear. Suppressor of cytokine signaling 3 (SOCS3) is expressed in response to cytokines, LPS, and other PAMPs, and competes with SHP-1/2 binding to ITIMs of cytokine receptors, thereby inhibiting signaling. In this study, using peptide pull-down experiments, we found that SOCS3 can specifically bind to the phosphorylated ITIM of CD33. Additionally, following cross-linking SOCS3 can recruit the ECS E3 ligase resulting in accelerated proteasomal degradation of both CD33 and SOCS3. Our data suggest that the tyrosine motifs in CD33 are not important for internalization, while they are required for degradation. Moreover, SOCS3 inhibited the CD33-induced block on cytokine-induced proliferation. This is the first receptor shown to be degraded by SOCS3 and where SOCS3 and its target protein are degraded concomitantly. Our findings clearly suggest that during an inflammatory response, the inhibitory receptor CD33 is lost by this mechanism. Moreover, this has important clinical implications as tumors expressing SOCS3 may be refractory to alpha-CD33 therapy.

Amino Acid Motifs↗
PubMed2005

Mechanisms of SOCS3 phosphorylation upon interleukin-6 stimulation. Contributions of Src- and receptor-tyrosine kinases.

The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine signal transduction. SOCS3 is a key negative regulator of interleuking-6 (IL-6) signal transduction. Furthermore, SOCS3 was shown to be phosphorylated upon treatment of cells with IL-2, and this has been reported to regulate its function and half-life. We set out to investigate whether SOCS3 phosphorylation may play a role in IL-6 signaling. Tyrosine-phosphorylated SOCS3 was detected upon treatment of mouse embryonic fibroblasts with IL-6. Interestingly, the observed SOCS3 phosphorylation does not require SOCS3 recruitment to phosphotyrosine (Tyr(P)) 759 of gp130, and the kinetics of SOCS3 phosphorylation do not match the activation kinetics of the Janus kinases. This suggests that other kinases may be involved in SOCS3 phosphorylation. Using Src and Janus kinase inhibitors as well as Src kinase-deficient mouse embryonic fibroblasts, we provide evidence that Src kinases, which we found to be constitutively active in these cells, are involved in the phosphorylation of IL-6-induced SOCS3. In addition, we found that receptor-tyrosine kinases such as platelet-derived growth factor receptor or epidermal growth factor receptor can very potently phosphorylate IL-6-induced SOCS3. Taken together, these results suggest that SOCS3 phosphorylation is not a JAK-mediated phenomenon but is dependent on the activity of other kinases such as Src kinases or receptor-tyrosine kinases, which can either be constitutively active or activated by an additional stimulus.

Animals↗
PubMed2005

The DUB/USP17 deubiquitinating enzymes, a multigene family within a tandemly repeated sequence.

Here we report the identification of 10 human, 1 murine, and 2 rat ORFs, all of which represent additional members of the DUB/USP17 family of deubiquitinating enzymes. In addition, we demonstrate that this family constitutes part of a tandemly repeated sequence conserved throughout humans, mice, and rats. Furthermore, upon examination of the known family members we have found that the multiple genes observed, in contrast to other gene families, have arisen due to the independent expansion of an ancestral sequence within each species. This premise is further strengthened by the observation that the murine and rat genes span two exons while their human counterparts have one. These observations, in conjunction with previous work demonstrating that the DUB/USP17's are cytokine inducible and that they regulate both cell growth and survival, suggest that the DUB/USP17's are a large highly conserved family of genes that may play an important role in controlling cell fate.

Amino Acid Sequence↗
PubMed2005

SOCS2 can enhance interleukin-2 (IL-2) and IL-3 signaling by accelerating SOCS3 degradation.

Cytokine responses can be regulated by a family of proteins termed suppressors of cytokine signaling (SOCS) which can inhibit the JAK/STAT pathway in a classical negative-feedback manner. While the SOCS are thought to target signaling intermediates for degradation, relatively little is known about how their turnover is regulated. Unlike other SOCS family members, we find that SOCS2 can enhance interleukin-2 (IL-2)- and IL-3-induced STAT phosphorylation following and potentiate proliferation in response to cytokine stimulation. As a clear mechanism for these effects, we demonstrate that expression of SOCS2 results in marked proteasome-dependent reduction of SOCS3 and SOCS1 protein expression. Furthermore, we provide evidence that this degradation is dependent on the presence of an intact SOCS box and that the loss of SOCS3 is enhanced by coexpression of elongin B/C. This suggests that SOCS2 can bind to SOCS3 and elongin B/C to form an E3 ligase complex resulting in the degradation of SOCS3. Therefore, SOCS2 can enhance cytokine responses by accelerating proteasome-dependent turnover of SOCS3, suggesting a mechanism for the gigantism observed in SOCS2 transgenic mice.

Animals↗
PubMed2004

The chemoattractants, IL-8 and formyl-methionyl-leucyl-phenylalanine, regulate granulocyte colony-stimulating factor signaling by inducing suppressor of cytokine signaling-1 expression.

Suppressors of cytokine signaling (SOCS) are encoded by immediate early genes known to inhibit cytokine responses in a classical feedback loop. SOCS gene expression has been shown to be induced by many cytokines, growth factors, and innate immune stimuli, such as LPS. In this paper, we report that the chemoattractants, IL-8 and fMLP, up-regulate SOCS1 mRNA in human myeloid cells, primary human neutrophils, PBMCs, and dendritic cells. fMLP rapidly up-regulates SOCS1, whereas the induction of SOCS1 upon IL-8 treatment is delayed. IL-8 and fMLP did not signal via Jak/STATs in primary human macrophages, thus implicating the induction of SOCS by other intracellular pathways. As chemoattractant-induced SOCS1 expression in neutrophils may play an important role in regulating the subsequent response to growth promoting cytokines like G-CSF, we investigated the effect of chemoattractant-induced SOCS1 on cytokine signal transduction. We show that pretreatment of primary human neutrophils with fMLP or IL-8 blocks G-CSF-mediated STAT3 activation. This study provides evidence for cross-talk between chemoattractant and cytokine signal transduction pathways involving SOCS proteins, suggesting that these chemotactic factors may desensitize neutrophils to G-CSF via rapid induction of SOCS1 expression.

Carrier Proteins↗
PubMed2004

The role of the inhibitors of interleukin-6 signal transduction SHP2 and SOCS3 for desensitization of interleukin-6 signalling.

The immediate early response of cells treated with IL-6 (interleukin-6) is the activation of the signal transducer and activator of transcription (STAT)3. The Src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP2 and the feedback inhibitor SOCS3 (suppressor of cytokine signalling) are potent inhibitors of IL-6 signal transduction. Impaired function of SOCS3 or SHP2 leads to enhanced and prolonged IL-6 signalling. The inhibitory function of both proteins depends on their recruitment to the tyrosine motif 759 within glycoprotein gp130. In contrast to inactivation, desensitization of signal transduction is regarded as impaired responsiveness due to prestimulation. Usually, after activation the sensing receptor becomes inactivated by modifications such as phosphorylation, internalization or degradation. We designed an experimental approach which allows discrimination between desensitization and inactivation of IL-6 signal transduction. We observed that pre-stimulation with IL-6 renders cells less sensitive to further stimulation with IL-6. After several hours, the cells become sensitive again. We show that not only signal transduction through previously activated receptors is affected by desensitization but signalling through receptors which were not targeted by the first stimulation was also attenuated ( trans -desensitization). Interestingly, in contrast to inhibition, desensitization does not depend on the presence of functional SHP2. Furthermore, cells lacking SOCS3 show constitutive STAT3 activation which is not affected by pre-stimulation with IL-6. All these observations suggest that desensitization and inhibition of signalling are mechanistically distinct.

Animals↗
PubMed2003

DUB-3, a cytokine-inducible deubiquitinating enzyme that blocks proliferation.

Previous studies have identified the DUB family of cytokine-regulated murine deubiquitinating enzymes, which play a role in the control of cell proliferation and survival. Through data base analyses and cloning, we have identified a human cDNA (DUB-3) that shows significant homology to the known murine DUB family members. Northern blotting has shown expression of this gene in a number of tissues including brain, liver, and muscle, with two transcripts being apparent (1.6 and 1.7 kb). In addition, expression was observed in cell lines including those derived from a number of hematopoietic tumors such as the Burkitt's lymphoma cell line RAJI. We have also demonstrated that DUB-3, which was shown to be an active deubiquitinating enzyme, is induced in response to interleukin-4 and interleukin-6 stimulation. Finally, we have demonstrated that constitutive expression of DUB-3 blocks proliferation and can initiate apoptosis in both IL-3-dependent Ba/F3 cells and NIH3T3 fibroblasts. These findings suggest that human DUB-3, like the murine DUB family members, is transiently induced in response to cytokines and can, when constitutively expressed, block growth factor-dependent proliferation.

Amino Acid Sequence↗
PubMed2003

Are SOCS suppressors, regulators, and degraders?

Suppressors of cytokine signaling have been identified as inhibitors of cytokine signaling and have been shown to act in a classical feedback loop. The prototype members of this family, cytokine-inducible Src homology 2-containing protein and suppressors of cytokine signaling SOC was cloned as cytokine-inducible immediate early gene that could inhibit the activation of signal transducer and activator of transcription factors and block biological responses to several cytokines. Although steady progress has been made in the identification of SOCS and their physiological importance, precisely how SOCS proteins function has not yet been discovered. Many recent findings indicate that the SOCS act as adaptors that regulate the turnover of certain substrates by interacting with and activating an E3 ubiquitin ligase. Here, I explore recent evidence (presented at the International Cytokine Society meeting in Dublin, Ireland, September 2003) that SOCS molecules may not act simply as regulators of cytokine responses but may also play an essential role in determining cell fate and controlling cell differentiation.

Animals↗
PubMed2003

SOCS-3 regulates onset and maintenance of T(H)2-mediated allergic responses.

Members of the suppressor of cytokine signaling (SOCS) family are involved in the pathogenesis of many inflammatory diseases. SOCS-3 is predominantly expressed in T-helper type 2 (T(H)2) cells, but its role in T(H)2-related allergic diseases remains to be investigated. In this study we provide a strong correlation between SOCS-3 expression and the pathology of asthma and atopic dermatitis, as well as serum IgE levels in allergic human patients. SOCS-3 transgenic mice showed increased T(H)2 responses and multiple pathological features characteristic of asthma in an airway hypersensitivity model system. In contrast, dominant-negative mutant SOCS-3 transgenic mice, as well as mice with a heterozygous deletion of Socs3, had decreased T(H)2 development. These data indicate that SOCS-3 has an important role in regulating the onset and maintenance of T(H)2-mediated allergic immune disease, and suggest that SOCS-3 may be a new therapeutic target for the development of antiallergic drugs.

Animals↗
PubMed2003

Tyrosine phosphorylation disrupts elongin interaction and accelerates SOCS3 degradation.

The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor-induced signal transduction. The C-terminal SOCS box region is thought to regulate SOCS protein stability most likely via an elongin C interaction. In the present study, we have found that phosphorylation of SOCS3 at two tyrosine residues in the conserved SOCS box, Tyr204 and Tyr221, can inhibit the SOCS3-elongin C interaction and activate proteasome-mediated SOCS3 degradation. Jak-mediated phosphorylation of SOCS3 decreased SOCS3 protein half-life, and phosphorylation of both Tyr204 and Tyr221 was required to fully destabilize SOCS3. In contrast, a phosphorylation-deficient mutant of SOCS3, Y204F,Y221F, remained stable in the presence of activated Jak2 and receptor tyrosine kinases. SOCS3 stability correlated with the relative amount that bound elongin C, because in vitro phosphorylation of a SOCS3-glutathione S-transferase fusion protein abolished its ability to interact with elongin C. In addition, a SOCS3/SOCS1 chimera that co-precipitates with markedly increased elongin C, was significantly more stable than wild-type SOCS3. The data suggest that interaction with elongin C stabilizes SOCS3 protein expression and that phosphorylation of SOCS box tyrosine residues disrupts the complex and enhances proteasome-mediated degradation of SOCS3.

Animals↗
PubMed2003

A role of suppressor of cytokine signaling 3 (SOCS3/CIS3/SSI3) in CD28-mediated interleukin 2 production.

Suppressor of cytokine signaling (SOCS)3 has been characterized as a negative feedback regulator in cytokine-mediated Janus kinase signal transducer and activator of transcription signaling. However, this study shows that T cells from transgenic mice expressing SOCS3 exhibit a significant reduction in interleukin (IL)-2 production induced by T cell receptor cross-linking when T cells are costimulated with CD28. Decreased protein expression in SOCS3(+/-) mice enhanced CD28-mediated IL-2 production, clearly indicating the correlation between expression level of SOCS3 and IL-2 production ability. The SOCS3 protein interacted with phosphorylated CD28 through its SH2 domain but not the kinase inhibitory region. In addition, a point mutation in the SOCS3 SH2 domain attenuated the inhibition of CD28 function in IL-2 promoter activation. Committed T helper (Th)2 cells exclusively expressed SOCS3 and production of Th2 cytokines, such as IL-4 and IL-5, was much less dependent on CD28 costimulation compared with interferon gamma and IL-2 production in Th1 cells. Consistent with this notion, the expression level of SOCS3 in early T cell activation influenced the ability of IL-2 production induced by CD28 costimulation. Therefore, the SOCS3 may play an alternative role in prohibiting excessive progression of CD28-mediated IL-2 production.

Animals↗
PubMed2003

New interleukins: are there any more?

PURPOSE OF REVIEW: It is now well established that cytokines play a critical role in the regulation of the immune system. Processes such as lymphoid development, differentiation, homeostasis, tolerance and memory are all regulated by cytokines that bind the type I family of cytokine receptors. Like the interferons, which bind receptors designated as the type II cytokine receptor family, type I cytokines also have essential functions in host defence. RECENT FINDINGS: Recently, a number of new interleukins and their receptors have been discovered and their role in mounting an appropriate immune response is currently being studied. In this review we will describe the new interleukin-12 family of cytokines, which now includes two other members: interleukins 23 and 27. We will also review the newly discovered interleukins 28 and 29, also known as interferon-lambdas, which bind to the type II family of cytokine receptors, their structure and the structure of their receptors, their biological activities and modes of signalling. SUMMARY: These new molecules will certainly be the focus of active research in the immediate future. Their discovery opens the door to new therapeutic approaches to the treatment of various diseases ranging from infections from intracellular pathogens to viral infections.

Humans↗
PubMed2003

Deacetylase activity is required for recruitment of the basal transcription machinery and transactivation by STAT5.

The signal transducer and activator of transcription STAT5 plays a major role in the cellular response to cytokines, but the mechanism by which it activates transcription remains poorly understood. We show here that deacetylase inhibitors (trichostatin A, suberoylanilide hydroxamic acid, and sodium butyrate) prevent induction of endogenous STAT5 target genes, implying that a deacetylase activity is required for that process. Microarray analyses revealed that this requirement is common to all STAT5 target genes. Using chromatin immunoprecipitation, we show that, following STAT5 DNA binding, deacetylase inhibitors block transcription initiation by preventing recruitment of the basal transcription machinery. This inhibition is not due to effects on histone H3 and H4 acetylation or chromatin remodeling within the promoter region. This novel mechanism of transactivation by STAT5 provides a rationale for the use of deacetylase inhibitors for therapeutic intervention in STAT5-associated cancers.

Amidohydrolases↗
PubMed2002

Suppressor of cytokine signaling-3 is recruited to the activated granulocyte-colony stimulating factor receptor and modulates its signal transduction.

G-CSF is a polypeptide growth factor used in treatment following chemotherapy. G-CSF regulates granulopoiesis and acts on its target cells by inducing homodimerization of the G-CSFR, thereby activating intracellular signaling cascades. The G-CSFR encompasses four tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins. Suppressor of cytokine signaling 3 (SOCS-3), also referred to as cytokine-inducible Src homolgy 2-containing protein 3, is a member of a recently discovered family of feedback inhibitors that have been shown to inhibit the Janus kinase/STAT pathway. In this study, we demonstrate that human SOCS-3 is rapidly induced by G-CSF in polymorphonuclear neutrophils as well as in the myeloid precursor cell line U937 and that SOCS-3 negatively regulates G-CSFR-mediated STAT activation. Most importantly, we show that SOCS-3 is recruited to the G-CSFR in a phosphorylation-dependent manner and we identify phosphotyrosine (pY)729 as the major recruitment site for SOCS-3. Furthermore, we demonstrate that SOCS-3 directly binds to this pY motif. Surface plasmon resonance analysis reveals a dissociation constant (K(D)) for this interaction of around 2.8 microM. These findings strongly suggest that the recruitment of SOCS-3 to pY729 is important for the modulation of G-CSFR-mediated signal transduction by SOCS-3.

Carrier Proteins↗