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. 2005 Feb 15;102(7):2328-33.
doi: 10.1073/pnas.0409675102. Epub 2005 Feb 3.

Suppressor of cytokine signaling (SOCS)-5 is a potential negative regulator of epidermal growth factor signaling

Sandra E Nicholson  1 , Donald Metcalf, Naomi S Sprigg, Ruth Columbus, Francesca Walker, Anabel Silva, Dale Cary, Tracy A Willson, Jian-Guo Zhang, Douglas J Hilton, Warren S Alexander, Nicos A Nicola
Affiliations

Suppressor of cytokine signaling (SOCS)-5 is a potential negative regulator of epidermal growth factor signaling

Sandra E Nicholson et al. Proc Natl Acad Sci U S A. .

Abstract

The suppressors of cytokine signaling (SOCS) proteins are a family of SH2 domain-containing intracellular inhibitors of cytokine signal transduction that act by several different mechanisms. Recent evidence suggests that the action of the SOCS proteins may extend beyond the cytokine receptors to signaling initiated by members of the tyrosine kinase receptor family. In this study, the ability of SOCS-5 to negatively regulate signaling cascades downstream of the epidermal growth factor receptor (EGF-R) has been examined by using an EGF-responsive cell line engineered to constitutively express the EGF-R and SOCS-5 or SOCS-5 mutants. SOCS-5 associated with the EGF-R complex in an EGF-independent manner, and the mitogenic response to EGF of all SOCS-5-expressing cell lines was dramatically inhibited when compared with control cell lines. Furthermore, this effect was abrogated after deletion of the SOCS-5 SOCS box. This result suggests that the inhibition of signaling occurs through enhanced proteasomal degradation of the EGF-R through SOCS box recruitment of E3 ubiquitin ligase activity.

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Figures

Fig. 1.
Fig. 1.
Generation of SOCS-5-expressing lines. (A) FACS analysis of BaF/ERX lines showing EGF-R expression levels. Parental Ba/F3 cells, which had not been transfected with EGF-R cDNA, are shown to the left in histogram A. EGF-R levels are shown to the right of histogram A and in subsequent panels. (A Upper) Puromycin-resistant control lines. (A Lower) SOCS-5-expressing lines. (B) SOCS-5 expression levels. Equal cell numbers of BaF/ERX puromycin-resistant lines and BaF/ERX cells expressing SOCS-5 were lysed and analyzed by immunoprecipitation and Western blot by using anti-Flag antibody. Lanes: A–E, independent puromycin-resistant lines; F–J, independent SOCS-5 clones.
Fig. 2.
Fig. 2.
SOCS-5 inhibits EGF proliferative responses. (A) A representative experiment showing the number of cells obtained per well of the parental BaF/ERX line (▪), two puromycin-resistant lines (○), and three SOCS-5 clones (•) in response to a 3-day incubation with increasing concentrations of EGF. (B) A representative experiment showing the number of cells obtained per well in response to a 3-day incubation with increasing concentrations of IL-3. (C) A representative experiment showing the number of cells obtained per well of the parental BaF/ERX line (▪), a SOCS-5 clone (•), and five clones expressing SOCS-5 lacking the N terminus (⋄) in response to a 3-day incubation with increasing concentrations of EGF.
Fig. 3.
Fig. 3.
Association of SOCS-5 with the EGF-R complex. BaF/ERX cells stably expressing Flag-tagged SOCS-5 (S5), SOCS-5 lacking the N terminus (ΔNT), and SOCS-5 with a mutated SH2 domain (mSH2) or with a deleted SOCS box (ΔSB) were incubated with (+) or without (-) 50 ng/ml EGF for 15 min on ice and lysed. EGF-R proteins were immunoprecipitated by using monoclonal antibody 528, and associated Flag-tagged SOCS proteins were detected by Western analysis with rat anti-Flag antibody (A). Total cell lysates were analyzed by Western blot with anti-phosphotyrosine antibodies (B). Western blots were reprobed by using anti-EGF-R monoclonal antibody 806 (C). Levels of SOCS-5 protein were determined by immunoprecipitation with anti-Flag antibodies conjugated to agarose (M2-beads) and Western blot with rat anti-Flag antibodies (D).
Fig. 4.
Fig. 4.
The SOCS-5 SOCS box is required for inhibition of EGF-R signaling. (A) Proliferation of BaF/ERX lines expressing SOCS-5 with a mutated (mSB) or deleted (ΔSB) SOCS box. The number of cells obtained per well is shown for the parental BaF/ERX line (▪), two SOCS-5 clones (•), two SOCS-5mSB clones (▴), and two SOCS-5ΔSB clones (⋄) in response to a 3-day incubation with increasing concentrations of EGF. (B) SOCS-5 expression levels. Equal cell numbers of two independent BaF/ERX lines expressing SOCS-5 or SOCS-5 with a mutated (mSB) or deleted SOCS box (ΔSB) were lysed and analyzed by immunoprecipitation and Western blot by using anti-Flag antibody. (C) SOCS-5-dependent down-regulation of EGF-R surface levels. BaF/ERX, puromycin-resistant BaF/ERX cells (Puro) and BaF/ERX cells expressing SOCS-5 or SOCS-5 with a mutated (mSB) or deleted SOCS box (ΔSB) were incubated with 50 ng/ml EGF for 4 h at 37°C and analyzed for EGF-R surface expression at 0 and 4 h (filled and unfilled histograms, respectively) by FACS with anti-EGF-R antibody 528. Data are shown for two independently derived clonal lines expressing SOCS-5, SOCS-5ΔSB, or SOCS-5mSB. Untransfected parental Ba/F3 cells are shown in gray in Upper Left. (D) Association of the SOCS-5 SOCS box with elongins B and C. 293T cells were transiently transfected with either vector alone (control) or plasmids expressing either Flag-tagged SOCS-5 or SOCS-5 with a deleted SOCS box (S5ΔSB). Flag-tagged proteins were immunoprecipitated and analyzed by SDS/PAGE and Western blot for the presence of elongins B and C (Left). Membranes were stripped and reprobed with anti-Flag antibodies (Right).
Fig. 5.
Fig. 5.
EGF-R degradation is enhanced in the presence of SOCS-5. Approach to steady state at 37°C for 125I-EGF interacting with BaF/ERX control cells (A) and BaF/ERX cells stably expressing SOCS-5 (B) is shown. 125I-EGF was added to prewarmed cells, and, at the indicated times, aliquots of cells were removed, centrifuged through chilled FCS to remove free 125I-EGF, and cell surface (• and ▪) or internalized (○ and □) 125I-EGF was determined. The lines drawn through the experimental points are the lines of best fit according to the computer program of Myers et al. (40). (C) Total cell-associated 125I-EGF (cell surface plus internalized cpm) is expressed as a percentage of maximal binding for control lines (▪, ▴, and ♦) and two independent SOCS-5-expressing lines (○ and □). Cell lines were assayed in duplicate.
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References

    1. Ihle, J. N., Thierfelder, W., Teglund, S., Stravapodis, D., Wang, D., Feng, J. & Parganas, E. (1998) Ann. N.Y. Acad. Sci. 865, 1-9. - PubMed
    1. Leonard, W. J. & O'Shea, J. J. (1998) Annu. Rev. Immunol. 16, 293-322. - PubMed
    1. Schlessinger, J. (2000) Cell 103, 211-225. - PubMed
    1. Yoshimura, A., Ohkubo, T., Kiguchi, T., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Hara, T. & Miyajima, A. (1995) EMBO J. 14, 2816-2826. - PMC - PubMed
    1. Starr, R., Willson, T. A., Viney, E. M., Murray, L. J., Rayner, J. R., Jenkins, B. J., Gonda, T. J., Alexander, W. S., Metcalf, D., Nicola, N. A. & Hilton, D. J. (1997) Nature 387, 917-921. - PubMed

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