Calmodulin-dependent protein kinases phosphorylate gp130 at the serine-based dileucine internalization motif

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the low affinity LIF receptor (LIFR) and gp130. We previously demonstrated that LIF stimulation caused phosphorylation of gp130 at Ser782, adjacent to a dileucine internalization motif, and that transient expression of a mutant receptor lacking Ser782 resulted in increased cell surface expression and increased LIF-stimulated gene expression compared to wild-type receptor. Phosphorylation of Ser782 on gp130 fusion protein by LIF-stimulated 3T3-L1 cell extracts was inhibited 61% by autocamtide-2-related inhibitory peptide (AIP), a highly specific and highly effective inhibitor of calmodulin-dependent protein kinase type II (CaMKII). Purified rat forebrain CaMKII was also able to phosphorylate gp130 fusion protein at Ser782 in vitro. Furthermore, antibodies targeting CaMKII and CaMKIV were able to immunoprecipitate gp130 phosphorylating activity from LIF-stimulated 3T3-L1 lysates. While pretreatment of cells with the MAPKK inhibitors PD98059 and U0126 blocked phosphorylation of Ser782 prior to LIF stimulation, these inhibitors did not block Ser782 phosphorylation by LIF-stimulated 3T3-L1 cell extracts in vitro. These results show that CaMKII and possibly CaMKIV phosphorylate Ser782 in the serine-based dileucine internalization motif of gp130 via a MAPK-dependent pathway.     

Calmodulin-dependent protein kinases phosphorylate gp130 at the serine-based dileucine internalization motif

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the low affinity LIF receptor (LIFR) and gp130. We previously demonstrated that LIF stimulation caused phosphorylation of gp130 at Ser782, adjacent to a dileucine internalization motif, and that transient expression of a mutant receptor lacking Ser782 resulted in increased cell surface expression and increased LIF-stimulated gene expression compared to wild-type receptor. Phosphorylation of Ser782 on gp130 fusion protein by LIF-stimulated 3T3-L1 cell extracts was inhibited 61% by autocamtide-2-related inhibitory peptide (AIP), a highly specific and highly effective inhibitor of calmodulin-dependent protein kinase type II (CaMKII). Purified rat forebrain CaMKII was also able to phosphorylate gp130 fusion protein at Ser782 in vitro. Furthermore, antibodies targeting CaMKII and CaMKIV were able to immunoprecipitate gp130 phosphorylating activity from LIF-stimulated 3T3-L1 lysates. While pretreatment of cells with the MAPKK inhibitors PD98059 and U0126 blocked phosphorylation of Ser782 prior to LIF stimulation, these inhibitors did not block Ser782 phosphorylation by LIF-stimulated 3T3-L1 cell extracts in vitro. These results show that CaMKII and possibly CaMKIV phosphorylate Ser782 in the serine-based dileucine internalization motif of gp130 via a MAPK-dependent pathway.     

Protein phosphatase type 2A, PP2A, is involved in degradation of gp130

The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp 130 at Ser-782 and degradation of gp 130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp 130. Taken together, present results strongly suggest that degradation of gp 130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp 130 is a potential therapeutic target in cancers.     

Protein phosphatase type 2A,PP2A,is involved in degradation of gp130

The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp130 at Ser-782 and degradation of gp130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp130. Taken together, present results strongly suggest that degradation of gp130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp130 is a potential therapeutic target in cancers. (Mol Cell Biochem 269: 183–187, 2005)     

Regulation of neurokine receptor signaling and trafficking

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are neurally active cytokines, or neurokines. LIF signals through a receptor consisting of gp130 and the low affinity LIF receptor (LIFR), while the CNTF receptor consists of gp130, LIFR, and the low affinity CNTF receptor (CNTFR). Ser1044 of the LIFR is phosphorylated by Erk1/2 MAP kinase. Stimulation of neural cells with growth factors which strongly activate Erk1/2 decreases LIF-mediated signal transduction due to increased degradation of the LIFR as a consequence of Erk1/2-dependent phosphorylation of the receptor at Ser1044.     

白血病抑制因子与胚胎干细胞

白血病抑制因子对细胞的生长和分化有多种作用,通过与其受体结合传导信号,gp130与LIF受体β链的结合激活JAK激酶(JAK1和JAK2),JAK激酶磷酸化STAT信号转录子,STAT3的磷酸化对于阻止体外培养的干细胞的分化具有十分重要的作用。     

Involving of the cytoplasmic region of leukemia inhibitory factor receptor α subunit,IL-6 related signal transducer-gp130 or Fas death domain for MAPK p42/44 activation in HL-60 cell with LIF or anti-Fas IgG

The chimeric receptors were prepared by exchanging the cytoplasmic region between leukemia inhibitory factor (LIF) receptor subunit (gp190) and the other subunit-gp130 (190/130,130/190) and separately transduced into leukemia line HL-60 (to have the wild type subunit). The purpose is to investigate which subunit for activating MAPK p42/44 in leukemia cell while the cytoplasmic region homodimerization (190cyt-190cyt, 130cyt-130cyt) was induced by LIF. The results showed that MAPK p42/44 expression level after LIF stimulation 5 h was lower in the transformants with pED 130/190 (190cyt-190cyt) (p < 0.01) and higher in the transformants with pED 190/130 (130cyt-130cyt) (p < 0.05) than those in the parent cells. Meanwhile, MAPK p42/44 phosphorylation (Thr202/Tyr204) was ascended and the highest at 10 min in the 190/130 and descended in the 130/190. It suggests that gp130 activate MAPK p42/44 and gp190 indirectly regulate its expression and function. In order to analyses the relation of the subunit oligomerization and MAPK p42/44 we also prepared the recombination of the extracellular and transmembrane region of Fas and the cytoplasmic region of each LIFR subunit (Fas/190, Fas/130). After transduction into HL-60 with lipofection and induction by anti-Fas IgG, we found that MAPK p42/44 expression levels were lower in the Fas/190 than in the Fas/130 and parent cells (p < 0.01) and no difference between the Fas/130 and the wild type receptor. However, phospho-MAPK p42/44 were increased in the Fas/130 than the parent cells. It suggests that the oligomerization of the cytoplasmic regions of gp130 be potential to normally initiate MAPK p42/44 for the signal of HL-60 proliferation. We also determine that the separated oligomerization FasDD (no dimerization) can initiate the corresponding signal molecules, then regulate MAPK p42/44 expression and phosphorylation in leukemia cells.     

Differential response of neuronal cells to a fusion protein of ciliary neurotrophic factor/soluble CNTF-receptor and leukemia inhibitory factor.

Ciliary neurotrophic factor (CNTF) displays neurotrophic activities on motor neurons and neural cell populations both in vivo and in vitro. On target cells lacking intrinsic expression of specific receptor alpha subunits cytokines of the IL-6 family only act in the presence of their specific agonistic soluble receptors. Here, we report the construction and expression of a CNTF/soluble CNTF-receptor (sCNTF-R) fusion protein (Hyper-CNTF) with enhanced biological activity on cells expressing gp130 and leukemia inhibitory factor receptor (LIF-R), but not membrane-bound CNTF-R. At the cDNA level, the C-terminus of the extracellular domain of human CNTF-R (amino acids 1-346) was linked via a single glycine residue to the N-terminus of human CNTF (amino acids 1-186). Recombinant Hyper-CNTF protein was expressed in COS-7 cells. Hyper-CNTF efficiently induced dose-dependent STAT3 phosphorylation and proliferation of BAF-3 cells stably transfected with gp130 and LIF-R cDNAs. While on BAF3/gp130/LIF-R cells, Hyper-CNTF and LIF exhibited similar biological responses, the activity of Hyper-CNTF on pheochromocytoma cells (PC12 cells) was quite distinct from that of LIF. In contrast to LIF, Hyper-CNTF stimulated neurite outgrowth of PC12 cells in a time- and dose-dependent manner correlating with the ability to phosphorylate MAP kinases. These data indicate that although LIF and Hyper-CNTF use the same heterodimeric receptor complex of gp130 and LIFR, only Hyper-CNTF induces neuronal differentiation. The therapeutic potential of Hyper-CNTF as a superagonistic neurotrophin is discussed.     

Coexpression of oncostatin M and its receptors and evidence for STAT3 activation in human ovarian carcinomas

The expression of oncostatin M and leukemia inhibitory factor (LIF), JAK-STAT activators and members of the interleukin-6 family of cytokines, were examined in a series of primary ovarian carcinomas using immunohistochemistry. The malignant epithelial cells of all 29 ovarian carcinomas examined expressed oncostatin M; none expressed LIF. Oncostatin M can activate two related receptors, one consisting of a low-affinity LIF receptor subunit, LIFR beta, which forms a heterocomplex with the gp130 signal transducing protein and can recognize both oncostatin M and LIF, and a second heterocomplex consisting of a subunit that specifically recognizes oncostatin M, OSMR beta, and the gp130 protein. By immunohistochemistry, 25 of 25 ovarian carcinomas examined expressed the LIFR beta subunit in the malignant epithelial cells (all samples express gp130), and two-thirds the ovarian carcinomas studied expressed OSMR beta mRNA as determined by RT-PCR. Thus oncostatin M and its receptors are commonly coexpressed in malignant ovarian epithelial cells, and represent a potential autocrine loop in this tumor type. STAT3, of one the signaling proteins downstream of the oncostatin M/LIF receptors, was found in its phosphorylated, activated form (phosphotyrosine 705 STAT3) in the malignant epithelial cells of 17 of 23 ovarian carcinomas examined (74%) as determined by immunohistochemistry; this suggests that this protein is constitutively activated in most ovarian carcinomas, as it is in many other human malignancies. Recombinant human Oncostatin M (rhOSM) can induce the transient tyrosine 705 phosphorylation of STAT3 in serum-starved LIFR beta/OSMR beta expressing ovarian carcinoma cell lines, but does not alter cell growth and effects only a modest increase in the apoptotic rate in these cultured cells. Oncostatin M and its receptors may be part of a network of cytokine systems within ovarian carcinomas that may act to maintain STAT3 in its activated form, a phenomenon associated with the malignant phenotype.     

Novel inhibitors for murine and human leukemia inhibitory factor based on fused soluble receptors

Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases.     

Binding Interactions of Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor with the Different Subunits of Their High Affinity Receptors

Abstract: Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) share common components in their multimeric receptors. Both cytokine receptors contain gp130/interleukin-6-receptor transducer as well as gp190/low-affinity LIF receptor. For CNTF, addition of a third subunit, or α subunit, defines the high-affinity CNTF receptor. In the present study, we analyzed the binding interactions of LIF and CNTF in human cell lines and showed a mutual displacement for LIF and CNTF toward the trimeric high-affinity CNTF receptor. Similar results were obtained in the JEG cell line, which only expressed the gp130/gp190 high-affinity LIF receptor, by adding a soluble form of the αCNTF receptor to the system to reconstitute the high-affinity-type CNTF receptor. The different receptor subunits were then expressed separately in transfected cells and their binding capacities analyzed. The results showed that the heterocomplex CNTF/αCNTF receptor bound to gp130 with an affinity of 3–5 × 10⁻¹⁰ M , whereas LIF interacted mainly with gp190. In summary, the observed competition between LIF and CNTF does not result from the binding to a common site or receptor subunit, but rather to the interaction of the three receptor components to create a conformational site common to both LIF and CNTF.     

Interleukin-6 signal transducer gp130 mediates oncostatin M signaling.

Oncostatin M (OM) is a multifunctional cytokine that is structurally and functionally related to interleukin 6 (IL-6) and leukemia inhibitory factor (LIF). The specific receptor for OM has been demonstrated (by chemical cross-linking) to be a 150-kDa protein in a number of cell lines. The IL-6 signal transducer, gp130, is also an affinity converter for the LIF receptor. It does not bind to either IL-6 or LIF, but associates with the alpha subunits of the receptors and transduces the signals. We examined the possible involvement of gp130 in OM binding and signaling. We demonstrate that: (a) anti-gp130 monoclonal antibodies (mAbs) block the inhibitory effect of OM on A375 cell growth, (b) the binding and cross-linking of 125I-OM to H2981 cells are completely abolished by anti-gp130 mAbs, (c) the cross-linked OM-receptor complex is immunoprecipitated by anti-gp130 mAbs, and (d) COS-7 cells transfected with the full-length cDNA encoding gp130 exhibit increased OM binding and cross-linking, which are also blocked by anti-gp130 mAbs. Therefore, we conclude that the 150-kDa OM binding protein previously characterized in a variety of cell lines is gp130. OM is the natural ligand for gp130 and gp130 mediates the biological responses of OM.     

Stimulation of leukemia inhibitory factor receptor degradation by extracellular signal-regulated kinase

Leukemia inhibitory factor (LIF) signals via the heterodimeric receptor complex comprising the LIF receptor alpha subunit (LIFRalpha) and the common signal transducing subunit for interleukin-6 cytokine receptors, gp130. This study demonstrates that in different cell types, the level of LIFRalpha decreases during treatment with LIF or the closely related cytokine oncostatin M (OSM). Moreover, insulin and epidermal growth factor induce a similar LIFRalpha down-regulation. The regulated loss of LIFRalpha is specific since neither gp130 nor OSM receptor beta shows a comparable change in turnover. LIFRalpha down-regulation correlates with reduced cell responsiveness to LIF. Using protein kinase inhibitors and point mutations in LIFRalpha, we demonstrate that LIFRalpha down-regulation depends on activation of extracellular signal-regulated kinase 1/2 and phosphorylation of the cytoplasmic domain of LIFRalpha at serine 185. This modification appears to promote the endosomal/lysosomal pathway of the LIFRalpha. These results suggest that extracellular signal-regulated kinase-activating factors like OSM and growth factors have the potential to lower specifically LIF responsiveness in vivo by regulating LIFRalpha half-life.     

Protein Tyrosine Phosphatase 2 (SHP-2) Moderates Signaling by gp130 but Is Not Required for the Induction of Acute-Phase Plasma Protein Genes in Hepatic Cells

Signals propagated via the gp130 subunit of the interleukin-6 (IL-6)-type cytokine receptors mediate, among various cellular responses, proliferation of hematopoietic cells and induction of acute-phase plasma protein (APP) genes in hepatic cells. Hematopoietic growth control by gp130 is critically dependent on activation of both STAT3 and protein tyrosine phosphatase 2 (SHP-2). To investigate whether induction of APP genes has a similar requirement for SHP-2, we constructed two chimeric receptors, G-gp130 and G-gp130(Y2F), consisting of the transmembrane and cytoplasmic domains of gp130 harboring either a wild-type or a mutated SHP-2 binding site, respectively, fused to the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor. Rat hepatoma H-35 cells stably expressing the chimeric receptors were generated by retroviral transduction. Both chimeric receptors transmitted a G-CSF-induced signal characteristic of that triggered by IL-6 through the endogenous gp130 receptor; i.e., both activated the appropriate JAK, induced DNA binding activity by STAT1 and STAT3, and up-regulated expression of the target APP genes, those for α-fibrinogen and haptoglobin. Notwithstanding these similarities in the patterns of signaling responses elicited, mutation of the SHP-2 interaction site in G-gp130(Y2F) abrogated ligand-activated receptor recruitment of SHP-2 as expected. Moreover, the tyrosine phosphorylation state of the chimeric receptor, the associated JAK activity, and the induced DNA binding activity of STAT1 and STAT3 were maintained at elevated levels and for an extended period of time in G-gp130(Y2F)-expressing cells following G-CSF treatment compared to that in cells displaying the G-gp130 receptor. H-35 cells ectopically expressing G-gp130(Y2F) were also found to display an enhanced sensitivity to G-CSF and a higher level of induction of APP genes. Overexpression of the enzymatically inactive SHP-2 enhanced the signaling by the wild-type but not by the Y2F mutant G-gp130 receptor. These results indicate that gp130 signaling for APP gene induction in hepatic cells differs qualitatively from that controlling the proliferative response in hematopoietic cells in not being strictly dependent on SHP-2. The data further suggest that SHP-2 functions normally to attenuate gp130-mediated signaling in hepatic (and, perhaps, other) cells by moderating JAK action.     

Differential regulation of leukemia inhibitory factor-stimulated neuronal gene expression by protein phosphatases SHP-1 and SHP-2 through mitogen-activated protein kinase-dependent and -independent pathways

The neurally active cytokine leukemia inhibitory factor (LIF) signals through a bipartite receptor complex composed of LIF receptor alpha (LIFR) and gp130. gp130 and LIFR contain consensus binding motifs for the protein tyrosine phosphatase SHP-2 surrounding tyrosines 118 and 115 (Y118 and Y115) of their cytoplasmic domains, respectively. These sites are necessary for maximal activation of mitogen-activated protein kinase (MAPK). Coexpression of catalytically inactive, but not wild-type, SHP-2 reduced LIFR- and gp130-mediated activation of MAPK up to 75%. Conversely, coexpression of the wild-type, but not catalytically inactive, SHP-1, a related phosphatase, reduced activity up to 80%, demonstrating that SHP-2 and SHP-1 have opposing effects on the MAPK pathway. Mutation of Y115 of the cytoplasmic domain of LIFR eliminates receptor-mediated tyrosine phosphorylation of SHP-2. In contrast, SHP-1 association with gp130 and LIFR is constitutive and independent of Y118 and Y115, respectively. SHP-1 has a positive regulatory role on LIF-stimulated vasoactive intestinal peptide (VIP) reporter gene expression in neuronal cells, whereas the effect of SHP-2 is negative. Furthermore, LIF-stimulated MAPK activation negatively regulates this VIP reporter gene induction. SHP-2 also negatively regulates LIF-dependent expression of choline acetyltransferase, but this regulation could be dissociated from its effects on MAPK activation. These data indicate that SHP-1 and SHP-2 are important regulators of LIF-dependent neuronal gene expression via both MAPK-dependent and -independent pathways.     

Desensitization of signaling by oncostatin M in human vascular cells involves cytoplasmic Tyr residue 759 in gp130 but is not mediated by either Src homology 2 domain-containing tyrosine phosphatase 2 or suppressor of cytokine signaling 3

Oncostatin M (OnM) signals through cell surface receptors, which utilize the gp130 subunit. In cultured human umbilical vein endothelial cells (HUVEC), OnM transiently elevates mRNA encoding for suppressor of cytokine signaling-3 (SOCS-3). By 1 h of OnM treatment, HUVEC become refractory to the restimulation by OnM, measured as failure to reinduce SOCS-3 mRNA. OnM-induced desensitization also prevents responses to other gp130-signaling cytokines (e.g. leukemia inhibitory factor and interleukin 11). OnM treatment does not affect gp130 expression levels and desensitizes signaling mediated by a transduced chimeric receptor containing extracellular domains of platelet-derived growth factor receptor-beta (PDGFRbeta) and the cytoplasmic region of gp130. Interestingly, a chimeric PDGFRbeta-gp130 mutant receptor, in which intracellular Tyr residue 759 of gp130 is replaced by a Phe residue, mediates prolonged signaling and is not cross-desensitized by OnM. Phospho-Tyr759 is the binding site for both SOCS-3 and for Src homology domain 2-containing tyrosine phosphatase 2 (SHP-2). In human aortic smooth muscle cells, neither prevention of SOCS-3 protein induction, using STAT3 or SOCS-3 antisense, nor prevention of SHP-2 expression, also with antisense, ablates desensitization. These data suggest that desensitization of vascular cells to OnM is mediated in trans and involves Tyr residue 759 in gp130 but is not mediated by either SOCS-3 or SHP-2, the only two proteins currently known to bind to gp130 at this site.     

白血病抑制因子受体细胞内区对HL-60细胞表达CD14和CD15的影响

观察白血病抑制因子 (LIF)受体gp190亚基完整的细胞内区和gp190胞内区C末端片段(190CT)对人白血病系HL 6 0表达CD14、CD15的影响 ,进一步了解LIF引发白血病细胞增殖抑制和分化的关系 .用基因重组技术将LIF另一亚基gp130的细胞内区换成gp190的细胞内区 ,用PCR技术扩增gp190细胞内区C末端的一个多肽的编码序列 ,构成嵌合体受体基因 130 /190及 190CT片段 ,并分别在HL 6 0细胞表达 .用免疫组化和流式细胞术检测分析在LIF的诱导下 ,HL 6 0细胞表达CD14、CD15的水平 .转染pcDNA130 /190的HL 6 0细胞 ,CD15表达量明显增高 ;转染pcDNA190CT的细胞 ,CD15的表达量降低 ;但 2组细胞的CD14表达量均较低且水平接近 .LIF可能诱导HL 6 0细胞向粒细胞而不向单核细胞分化 ,该效应是由gp190亚基细胞内区介导的 ,而gp190C末端片段可干扰LIFα受体介导的信号传导效应 .