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...

An acidic domain (AD) of gp130 was previously found to interact with the Src family kinase (SFK) Hck. Here, the influence of myristoylated peptides derived from this AD was assessed in the mouse myeloma cell line, 7TD1. The IL-6-dependent growth of 7TD1 cells was reduced by B75%, if 100 lM of myristoylated 18mer peptide (18AD) was included in the growth medium, but was unaffected by a control peptide with scrambled sequence (18sc). A similar differential inhibition by peptides 18AD and 18sc was observed for the erythropoietin-dependent growth of BaF-EH cells expressing chimeric erythropoietin receptor-gp130 and human Hck and for the human myeloma cell line INA-6. While the peptide 18AD concentration inhibiting 50% was B30 lM in 7TD1 and BaF-EH cells, peptide 18AD did not significantly inhibit growth of IL-6-independent MM1.S myeloma and OKT1 hybridoma cells or of BaF-EH cells supplied with IL-3. Treatment with 100 lM peptide 18AD caused the same degree or 60% of apoptosis induction as IL-6 deprivation in 7TD1 or INA-6 cells, respectively. Co-immunoprecipitation experiments revealed that peptide 18AD interfered with the association of Hck and gp130 in 7TD1 lysates in a concentrationdependent manner. IL-6-treatment of INA-6 cells induced the kinase activities of Fyn, Lyn and Hck, but not Src, and the IL-6-induced SFK activities were inhibited by peptide 18AD. Expression in 7TD1 cells of a kinaseinactive Hck mutant (K269R) elicited a dominantnegative effect on cell number increases providing further evidence that SFKs are required for gp130 signalling in myeloma cells.

Background: Src Family Kinases (SFKs) play pivotal roles in normal B-cell development and in B-cell neoplasias. Our aim is to elucidate the roles of SFKs in signaling cascades resulting in cell proliferation or anti-apoptosis in multiple myeloma (MM). The pleiotropic cytokine interleukin-6 (IL6) is one of the major growth factors for MM cells. We have previously shown that IL6 induces the activation of the SFKs Hck, Lyn and Fyn and that Hck is associated with the IL6R beta chain (gp130) via an acid domain (AD) in gp130. Aim: We observed that an 18mer peptide (18AD), which is derived from the AD, inhibited the IL-6-dependent growth of myeloma cells. In order to develop a lead structure for small molecule inhibitors we wish to elucidate the cellular and molecular effects of peptide 18AD. Results: On the cellular level, 50–100μM of a membrane-permeable myristoylated peptide 18AD inhibited factor-dependent proliferation in 7TD-1 (mouse) and INA-6 (human) cells by ~75%. Taking the percentage of annexinV-stained cells after IL6-withdrawal as the reference value for the level of maximal apoptosis, treatment of 7TD1 and INA-6 cells with peptide 18AD resulted in 100 and 60 % of the respective percentages of apoptotic cells. A control peptide (18sc) with an identical amino acid composition but an arbitrarily scrambled sequence had no effect on proliferation and apoptosis. Similar effects of peptides 18AD and 18sc were observed in Baf-EH cells, which overexpress a chimeric erythropoietin receptor-gp130 and human Hck and grew in an erythropoietin dependent manner. Peptide 18AD had no significant growth inhibiting effects on BaF-HE cells cultured in the presence of IL3 or on factor-independent OKT1 hybridoma cells. In 7TD1 cells the expression of a kinase-inactive Hck mutant lead to a reduced proliferative response to IL6. On the molecular level, immunoprecipitation experiments in 7TD1 cells showed that peptide 18AD decreased the complex formation between Hck and gp130 in a concentration dependent way. Immunocomplex kinase assays in INA-6 cells showed that the IL6 induced activation of Hck, Lyn and Fyn was blocked by peptide 18AD, but not by peptide 18sc. Conclusion: We characterized peptide 18AD, which inhibits the association of Hck and gp130 as well as IL6-induced SFK activity. Thus, the observed anti-proliferative and pro-apoptotic effects of peptide 18AD may be due to inhibition of SFK-mediated pathways.