Interleukin-3 signals through multiple isoforms of Stat5.

The interleukin (IL)-3 family of cytokines mediates its numerous effects on myeloid growth and maturation by binding a family of related receptors. It has been shown recently that IL-3 induces the activation of two distinct cytoplasmic signal transducing factors (STFs) that are likely to mediate the induction of immediate early genes. In immature myeloid cells, IL-3 activates STF-IL-3a, which comprises two tyrosine-phosphorylated DNA binding proteins of 77 and 80 kDa. In mature myeloid cells, IL-3 and granulocyte-macrophage colony-stimulating factor activate STF-IL-3b, which consists of a 94 and 96 kDa tyrosine-phosphorylated DNA binding protein. Peptide sequence data obtained from the purified 77 and 80 kDa proteins (p77 and p80) indicate that they are closely related but are encoded by distinct genes. Both peptide and nucleotide sequence data demonstrate that these two proteins are the murine homologs of ovine mammary gland factor (MGF)/Stat5. The peptide data also indicate that p77 and p80 are phosphorylated on tyrosine 699, a position analogous to the tyrosine that is phosphorylated in Stat1 and Stat2 in response to interferon. Additionally, antiserum raised against bacterially expressed p77/p80 recognizes the 94 and 96 kDa protein components of STF-IL-3b, suggesting that these may be additional isoforms of Stat5. These studies indicate that the IL-3 family of ligands is able to activate multiple isoforms of the signal transducing protein Stat5.     

Direct interaction of STAT4 with the IL-12 receptor.

Signal transduction by interleukin-12 (IL-12) requires phosphorylation and activation of STAT4. Direct interaction of the SH2 domain of STAT4 with a phosphotyrosine residue in the IL-12 receptor has been proposed to be required for the subsequent STAT4 phosphorylation. The IL-12 receptor beta2 subunit contains three tyrosine residues in its cytoplasmic domain. To test the hypothesis that one of these tyrosines is involved in binding STAT4, phosphopeptides were synthesized according to the amino acid sequences surrounding each of these tyrosine residues. Only the phosphopeptide containing pTyr800 strongly bound to STAT4 in a cell-free binding assay. When this phosphopeptide was introduced into TALL-104 cells, it blocked IL-12-induced STAT4 phosphorylation by competing with the IL-12 receptor for binding to STAT4. A series of alanine replacements was performed in this phosphopeptide to elucidate which amino acids surrounding the pTyr800 residue are critical for STAT4 binding. To summarize, the site on the IL-12 receptor which binds STAT4 can be described as -T-X-X-G-pY(800)-L-, where the core G-pY(800)-L motif is critical for the binding; the threonine at the pY-4 position has only a minor contribution and X represents amino acids not critical for the binding. These results demonstrate that only a small region of the IL-12 receptor is critically involved in binding STAT4 and suggest the feasibility that small molecule inhibitors could be identified which interfere with IL-12 signal transduction for treatment of autoimmune diseases.     

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.     

Interleukin-4 mediates STAT6 activation in 3T3-L1 preadipocytes but not adipocytes

STAT6 is abundantly expressed in 3T3-L1 preadipocytes and adipocytes but activating ligands are not well defined. In this report, we provide evidence that interleukin 4 (IL-4) induced JAK2-mediated STAT6 tyrosine phosphorylation and DNA binding in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. Loss of IL-4-mediated STAT6 tyrosine phosphorylation occurred 2 days after preadipocytes were induced to differentiate into adipocytes but when cells remained phenotypically preadipocytes. 3T3-L1 adipocytes were still responsive to IL-4 through tyrosine phosphorylation of other cellular proteins. We conclude that IL-4 signals through STAT6 in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. This differentiation-dependent loss of STAT6 activation may be critical for distinct biological effects of IL-4 in 3T3-L1 preadipocytes and adipocytes.     

Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1

The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.     

The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation

CTLA-4 is a negative regulator of T cell responses. Sequence analysis of this molecule reveals the presence of two cytoplasmic tyrosine residues at positions 165 and 182 that are potential Src homology (SH)-2 domain binding sites. The role of phosphorylation of these residues in CTLA-4-mediated signaling is unknown. Here, we show that sole TCR ligation induces zeta-associated protein (ZAP)-70-dependent tyrosine phosphorylation of CTLA-4 that is important for cell surface retention of this molecule. However, CTLA-4 tyrosine phosphorylation is not required for down-regulation of T cell activation following CD3-CTLA-4 coengagement. Specifically, inhibition of extracellular signal-regulated kinase (ERK) activation and of IL-2 production by CTLA-4-mediated signaling occurs in T cells expressing mutant CTLA-4 molecules lacking the cytoplasmic tyrosine residues, and in lck-deficient or ZAP-70-deficient T cells. Therefore, CTLA-4 function involves interplay between two different levels of regulation: phosphotyrosine-dependent cell surface retention and phosphotyrosine-independent association with signaling molecules.     

Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.     

IL-3 and IL-4 activate cyclic nucleotide phosphodiesterases 3 (PDE3) and 4 (PDE4) by different mechanisms in FDCP2 myeloid cells

In FDCP2 myeloid cells, IL-4 activated cyclic nucleotide phosphodiesterases PDE3 and PDE4, whereas IL-3, granulocyte-macrophage CSF (GM-CSF), and phorbol ester (PMA) selectively activated PDE4. IL-4 (not IL-3 or GM-CSF) induced tyrosine phosphorylation of insulin-receptor substrate-2 (IRS-2) and its association with phosphatidylinositol 3-kinase (PI3-K). TNF-alpha, AG-490 (Janus kinase inhibitor), and wortmannin (PI3-K inhibitor) inhibited activation of PDE3 and PDE4 by IL-4. TNF-alpha also blocked IL-4-induced tyrosine phosphorylation of IRS-2, but not of STAT6. AG-490 and wortmannin, not TNF-alpha, inhibited activation of PDE4 by IL-3. These results suggested that IL-4-induced activation of PDE3 and PDE4 was downstream of IRS-2/PI3-K, not STAT6, and that inhibition of tyrosine phosphorylation of IRS molecules might be one mechnism whereby TNF-alpha could selectively regulate activities of cytokines that utilized IRS proteins as signal transducers. RO31-7549 (protein kinase C (PKC) inhibitor) inhibited activation of PDE4 by PMA. IL-4, IL-3, and GM-CSF activated mitogen-activated protein (MAP) kinase and protein kinase B via PI3-K signals; PMA activated only MAP kinase via PKC signals. The MAP kinase kinase (MEK-1) inhibitor PD98059 inhibited IL-4-, IL-3-, and PMA-induced activation of MAP kinase and PDE4, but not IL-4-induced activation of PDE3. In FDCP2 cells transfected with constitutively activated MEK, MAP kinase and PDE4, not PDE3, were activated. Thus, in FDCP2 cells, PDE4 can be activated by overlapping MAP kinase-dependent pathways involving PI3-K (IL-4, IL-3, GM-CSF) or PKC (PMA), but selective activation of PDE3 by IL-4 is MAP kinase independent (but perhaps IRS-2/PI3-K dependent).     

Crosstalk between PKCzeta and the IL4/Stat6 pathway during T-cell-mediated hepatitis

PKCzeta is required for nuclear factor kappa-B (NF-kappaB) activation in several cell systems. NF-kappaB is a suppressor of liver apoptosis during development and in concanavalin A (ConA)-induced T-cell-mediated hepatitis. Here we show that PKCzeta-/- mice display inhibited ConA-induced NF-kappaB activation and reduced damage in liver. As the IL-4/Stat6 pathway is necessary for ConA-induced hepatitis, we addressed here the potential role of PKCzeta in this cascade. Interestingly, the loss of PKCzeta severely attenuated serum IL-5 and liver eotaxin-1 levels, two critical mediators of liver damage. Stat6 tyrosine phosphorylation and Jak1 activation were ablated in the liver of ConA-injected PKCzeta-/- mice and in IL-4-stimulated PKCzeta-/- fibroblasts. PKCzeta interacts with and phosphorylates Jak1 and PKCzeta activity is required for Jak1 function. In contrast, Par-4-/- mice have increased sensitivity to ConA-induced liver damage and IL-4 signaling. This unveils a novel and critical involvement of PKCzeta in the IL-4/Stat6 signaling pathway in vitro and in vivo.     

Desensitization of CXC chemokine receptor 4, mediated by IL-16/CD4, is independent of p56lck enzymatic activity

CCR5 and CXC chemokine receptor 4 (CXCR4) are coreceptors for CD4 as defined by HIV-1 glycoprotein (gp) 120 binding. Pretreatment of T cells with gp120 results in modulation of both CCR5 and CXCR4 responsiveness, which is dependent upon p56(lck) enzymatic activity. The recent findings that pretreatment of T cells with a natural CD4 ligand, IL-16, could alter cellular responsiveness to macrophage-inflammatory protein-1ss (MIP-1ss) stimulation, prompted us to investigate whether IL-16 could also alter CXCR4 signaling. These studies demonstrate that IL-16/CD4 signaling in T lymphocytes also results in loss of stromal derived factor-1alpha (SDF-1alpha)/CXCR4-induced chemotaxis; however, unlike MIP-1ss/CCR5, the effects were not reciprocal. There was no effect on eotaxin/CCR3-induced chemotaxis. Desensitization of CXCR4 by IL-16 required at least 10-15 min pretreatment; no modulation of CXCR4 expression was observed, nor was SDF-1alpha binding altered. Using murine T cell hybridomas transfected to express native or mutated forms of CD4, it was determined that IL-16/CD4 induces a p56(lck)-dependent inhibitory signal for CXCR4, which is independent of its tyrosine catalytic activity. By contrast, IL-16/CD4 desensitization of MIP-1ss/CCR5 responses requires p56(lck) enzymatic activity. IL-16/CD4 inhibition of SDF-1alpha/CXCR4 signals requires the presence of the Src homology 3 domain of p56(lck) and most likely involves activation of phosphatidylinositol-3 kinase. These studies indicate the mechanism of CXCR4 receptor desensitization induced by a natural ligand for CD4, IL-16, is distinct from the inhibitory effects induced by either gp120 or IL-16 on CCR5.     

Interleukin-4 inhibits platelet-derived growth factor-induced preadipocyte proliferation

Interleukin-4 (IL-4) activates STAT6 in 3T3-L1 preadipocytes but its functional role is not known. In this report, we first assessed interleukin-4 receptor alpha (IL-4Ralpha) expression during adipogenesis. IL-4Ralpha was highly expressed in proliferating 3T3-L1 preadipocytes. Receptor expression was down-regulated in post-confluent growth arrested preadipocytes. Induction of differentiation led to a transient 36-h increase in expression, but then levels decreased to undetectable amounts 3-8 days after induction of differentiation. Depending on the cell type, IL-4 either increases or decreases cell proliferation. In growth arrested preconfluent 3T3-L1 preadipocytes, IL-4 alone had no effect on preadipocyte proliferation. In contrast, IL-4 inhibited platelet-derived growth factor (PDGF-BB) induced preadipocyte proliferation. PDGF-BB, but not IL-4, induced STAT3 tyrosine and AKT serine phosphorylation. Both PDGF-BB and IL-4 induced STAT6 tyrosine phosphorylation, but the bands showed distinct electrophoretic migration patterns. IL-4 alone and IL-4 added to the differentiation cocktail had no effect on adipocyte formation or PPARgamma expression. Collectively, these studies demonstrate that IL-4 inhibits PDGF-BB-induced preadipocyte proliferation, possibly through STAT6 activation. The pattern of IL-4 receptor expression suggests that the effects of IL-4 are targeted primarily towards preadipocytes.     

Interleukin 2-induced activation of Ras requires two domains of interleukin 2 receptor beta subunit, the essential region for growth stimulation and Lck-binding domain.

Interleukin 2 (IL-2) can stimulate the proliferation of various kinds of T-cell lines. The receptor for IL-2 is composed of at least two subunits (alpha and beta), of which beta subunit plays the major role in transducing growth signals into the cells. A nonreceptor-type tyrosine kinase, Lck, is associated with IL-2 receptor beta subunit, and the binding of IL-2 to its receptor induces the activation of Lck. On the other hand, it has been shown that stimulation of T-cells with IL-2 causes rapid activation of Ras protein. In this paper, we describe that both of the two regions in IL-2 receptor beta subunit, the indispensable region for the induction of cell growth (serine-rich region) and the binding region of Lck protein (acidic region), are required for the activation of Ras. These two regions are also required for tyrosine phosphorylation of an 85-kDa cellular protein (p85) and the accumulation of fos and jun mRNAs. This observation suggests also that the activation of a receptor-associated tyrosine kinase in response to IL-2-stimulation is primarily responsible for subsequent activation of the pathway through Ras to Fos and Jun.