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 beta-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.

FIG. 1

Schematic overview of gp130 truncation and deletion mutants. Several truncation (A) and deletion (B) mutants of gp130 were cloned as chimeric receptor molecules as described in Materials and Methods; the numbers of amino acids refer to wild-type gp130. The locations of tyrosine residues and the STAT3 binding region at tyrosine 814 are indicated. The homology boxes are shaded gray as boxes 1 to 3. All truncation (t) and deletion (d) mutants, as well as the wild-type gp130 (Eg) bearing C-terminal His and myc tags for expression in Cos-7 cells (or His and V5 tags for Baf-B03 cell experiments), are indicated.

FIG. 2

Deletion of 41 aa inhibits Hck binding. Different gp130 mutants were expressed together with Hck cDNA in Cos7 cells. The expression of recombinant proteins was controlled by Western blotting with specific antibodies against the His tag (C and G) or Hck (D and H), respectively. The association of Hck to gp130 was tested by IP with Hck antibody and subsequent blotting with His-tag antibody for the detection of coprecipitated EPOR-gp130 fusion protein (A and E) Aliquots of the IP reaction mixtures were loaded on an additional gel and blotted with Hck antibody to demonstrate equal precipitation of Hck (B and F). In lanes 1 of panels A, B, E, and F, Hck interaction was blocked by preincubation with a specific blocking peptide. The molecular mass markers and the immunoglobulin G (Ig G) bands are indicated. α-Hck, anti-Hck antibody.

FIG. 3

Stimulation of gp130 but not d771-811 induces activation of Hck in the growth factor-dependent cell line Baf-B03. Baf-B03 cells were transfected with expression vectors for Hck (pDpuro Hck) and for Eg and d771-811, respectively (pcDNA6/V5-His). These cells were then either stimulated with 40 U of EPO/ml (+) or with medium only (−). The activity of Hck was tested by performing in vitro kinase assays in the presence of radiolabeled ATP and the external Src kinase substrate Sam 68 as described in the text. After separation with SDS–10% PAGE the EPO-induced phosphorylation of Sam 68 was assessed by autoradiography (upper panel). Aliquots of the Hck immunoprecipitate were blotted with anti-Hck antibody (α-Hck) to control for comparable precipitation. The results were quantified by using phosphorimaging analysis software. Normalized induction levels of kinase activity were as indicated.

FIG. 4

Deletion of the acidic domain of gp130 does not reduce the activation of STAT3. Baf-B03 cells were transfected with cDNAs for Hck and Eg, Y814F, or d771-811. These cells were either not treated (−) or stimulated with 40 U of EPO/ml (+). The lysates were tested for endogenous STAT3 expression by blotting with anti-STAT3 antibody (A), for the detection of the receptor constructs with V5 antibody (B), and for the detection of Hck with anti-Hck antibody (α-Hck) (C).Thereafter, lysates were used for precipitation experiments with anti-STAT3 antibody (right panel). (D) For control of STAT3 precipitation, aliquots of the IP reaction mixtures were incubated with anti-STAT3 antibody, and the specificity of STAT3 antiserum was tested by preincubation of anti-STAT3 with a specific blocking peptide (lane 7). (E and F) To check the STAT3 phosphorylation, separated lysates were blotted with the antiphosphotyrosine antibody PY99 (E) and then stripped and blotted with an antibody directed against serine 727-phosphorylated STAT3 (F).

FIG. 5

The tyrosine kinase inhibitor PP2 reduces gp130-mediated proliferation. (A) The IL-6-dependent murine plasmocytoma cell line 7TD-1 was kept in the absence (□) or presence (▪) of the tyrosine kinase inhibitor PP2 (10 μmol). After 3 days of IL-6 stimulation with the indicated amounts the cell proliferation was assessed by microscopic counting. (B) Baf-B03 cells expressing the indicated proteins were stimulated with 8 U of EPO/ml (solid bars, upper panel), 4 U of IL-3/ml (solid bars, lower panel), or medium alone (open bars, both upper and lower panels) for 72 h. Cells were kept in the absence (left) or presence (right) of 10 μmol of PP2. Proliferation was assessed by counting the cells under the microscope after trypan blue staining. Triplicate results from two different clones are shown, with the standard deviations indicated by the error bars.

FIG. 6

Deletion of the Hck binding domain impairs gp130-mediated cell proliferation. Clonally derived Baf-B03 cells expressing the indicated proteins were stimulated with the indicated amounts (in units per milliliter) of EPO (upper panels) or IL-3 (lower panels) for 72 h. Proliferation was assessed by microscopic counting after trypan blue staining. Triplicate results from two different clones are shown, with the standard deviations indicated by the error bars.

FIG. 7

Pyk2 and Erk regulation is impaired by the disruption of the Hck binding site. (A) Baf-B03 cells were cotransfected with the expression vectors for Eg (lanes 3 and 4) or d771-811 (lanes 5 and 6) and Hck. After stimulation with medium or EPO, cells were lysed as described in the text. Lysates were separated on 10% SDS gels. After blotting, the membranes were incubated with antibodies against phosphorylated Pyk2 (upper panels) or with several control antibodies as indicated (lower panels). (B) Baf-B03 cells expressing the indicated proteins were stimulated for the indicated times with 40 U of EPO/ml. Thereafter, membranes were subjected to Western blots by using antibodies specific for activated Erk1/2 (upper panels). Equal loading was confirmed by blotting with normal Erk antibody (lower panels).

FIG. 8

Model of Hck involvement in gp130 signaling. Binding of IL-6 to the IL-6R results in the activation of Hck via gp130. Hck can then act on downstream signaling events by creating docking sites for signaling molecules such as Grb2 or Shp-2 and/or by activating adapter molecules such as Shc. The recruitment of Shp-2 to the Hck/gp130 complex might then lead to the dephosphorylation of Pyk2, resulting a block in apoptotic signaling in MM cells. Grb2 forms stable complexes with Sos, a GDP-GTP exchange factor for Ras. Thus, the binding of Grb2 to Shc, Pyk2, and/or Shp-2 via its SH2 domain can lead to the activation of Ras and Erk.