Signaling through a Novel Domain of gp130 Mediates Cell Proliferation and Activation of Hck and Erk Kinases

Abstract

Interleukin-6 (IL-6) induces the activation of the Src family kinase Hck, which is associated with the IL-6 receptor ␤-chain, gp130. Here we describe the identification of an "acidic" domain comprising amino acids 771 to 811 of gp130 as a binding region for Hck, which mediates proliferative signaling. The deletion of this region of gp130 (i.e., in deletion mutant d771-811) resulted in a significant reduction of Hck kinase activity and cell proliferation upon stimulation of gp130 compared to wild-type gp130. In addition, d771-811 disrupted the growth factor-stimulated activation of Erk and the dephosphorylation of Pyk2. Based on these findings, we propose a novel, acidic domain of gp130, which is responsible for the activation of Hck, Erk, and Pyk2 and signals cell proliferation upon growth factor stimulation.To exert its biological effects, interleukin-6 (IL-6) must bind to the IL-6 receptor (IL-6R), composed of two ␣-chains (IL-6R␣, 80 kDa) and two ␤-chains (IL-6R␤ or gp130, 130 kDa). Two moieties of IL-6 and two pairs of these receptor chains form a functional hexameric IL-6R complex (42,43,55). The subsequent intracellular signaling events are activated via gp130, which is the common ␤-chain of the receptors for cardiotrophin 1, ciliary neurotrophic factor, oncostatin M, leukemia inhibitory factor, . Activation of the IL-6R stimulates at least two major signaling pathways, the Src homology 2 (SH2) domain containing protein tyrosine phosphatase 2 (Shp-2)/mitogen-activated protein kinase (MAPK) signaling cascade (8,26,31,32,41,46) and the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathway (6,18,25,45). It was shown in recent in vivo studies that gp130-mediated signals were regulated by a balance between these two pathways (33). However, the signaling cascades mediating IL-6-induced cell growth are not fully defined. It was shown that Jak and STAT proteins are activated by IL-6 in multiple myeloma (MM) cells independently of the proliferative response. In contrast, MAPK was activated only in cells showing a proliferative response to IL-6 (32). Moreover, the physical separation of gp130 and Shp-2 reduced cell proliferation (26).We have shown previously that at least three members of the Src family of tyrosine kinases, i.e., Fyn, Hck, and Lyn, coprecipitate with gp130 in lysates of MM cells (20). Stimulation of cells with IL-6 increased the activity of these kinases. The association of Hck kinase with gp130 appeared to be stronger than either of the other two kinases. Therefore, we decided to focus on the Hck kinase to elucidate the mechanism(s) and biological significance of the IL-6-mediated Src kinase activation. To identify the gp130 binding domain for Hck, several mutants of gp130 were constructed. These mutants were based on a chimeric receptor consisting of the extracellular part of the erythropoietin receptor (EPOR) and the intracellular part of human gp130 (23). These EPOR/gp130 receptor chimeras (Eg) allowed study of the activation of gp130 by erythropoietin (EPO) after tr...

Inhibition of IL-6-dependent growth of myeloma cells by an acidic peptide repressing the gp130-mediated activation of Src family kinases

“…In a similar manner, the SFK Hck was shown to be associated with the AD of gp130 (Schaeffer et al, 2001), the central portion of which shows some similarity to consensus substrate sequences of protein tyrosine kinases, although it does not contain a tyrosine. In MM cell lines IL-6 stimulation increases the activity of Fyn, Hck and Lyn , as was confirmed here for INA-6 cells, where the IL-6 induced activation was inhibited by peptide 18AD (Figure 7a).…”

“…In contrast, the growth of OKT1 cells was not affected by any of the tested peptides. As the concept of an Hck-binding region had been derived from observations of cloned chimeric EpoRgp130 (Schaeffer et al, 2001), the impact of peptides 18AD and 18sc on Ba/F3 cells overexpressing Hck and EpoR-gp130 was investigated. Ba/F3 cells grow IL-3 dependently and growth induction by Epo was observed in the genetically engineered derivative cell line, BaF-EH, but not in the parent cell line.…”

“…For transient expression of chimeric EpoR-gp130 and Hck, constructs employing the vectors pCDNA6 and pApuro, respectively, were used (Schaeffer et al, 2001). (Schaeffer et al, 2001), BaF-EH, was grown under selection by 8 mg of blasticidin/ml and 5 mg of puromycin/ml.…”

“…Recently, we were able to map the Hck-binding region of gp130 to positions 771-811 (Schaeffer et al, 2001). This region was termed acidic domain (AD) owing to its high content of acidic residues, in analogy with the acidic region of the IL-2Rb subunit for interaction with the SFK p56 lck (Hatakeyama et al, 1991).…”

“…For analysis of gp130-mediated signalling pathways, we determined the influence of heterologously expressed mutants of human Hck on the IL-6-dependence of 7TD1-derived recombinant cell lines and monitored IL-6-induced STAT3-phosphorylation. To exclude that the observed effects of peptide 18AD on cell growth and survival were restricted to 7TD1 cells, the mouse pro-B cell line Ba/F3 expressing chimeric erythropoietin receptor (EpoR)-gp130 and human Hck (Schaeffer et al, 2001) and the human myeloma cell lines INA-6 (Burger et al, 2001) and MM1.S (Greenstein et al, 2003) were employed as additional model systems. In IL-6-dependent INA-6 cells, we investigated the effects of peptide 18AD on IL-6-induced activation of the SFKs Hck, Lyn, Fyn and Src.…”