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. Author manuscript; available in PMC: 2012 May 2.
Published in final edited form as: Oncogene. 2010 Sep 27;30(5):611–618. doi: 10.1038/onc.2010.443

Figure 3. Activation of TGFα shedding by Src(E378G) is mediated by ADAM17 and does not require the cytoplasmic domain of ADAM17.

Figure 3

(A) Shedding of TGFα from Adam17−/− cells co-expressing wild type ADAM17 or the inactive ADAM17E>A mutant (Le Gall et al., 2009), and Src(K295A) or Src(E378G). The increased shedding of TGFα in the presence of Src(E378G) depended on ADAM17. (B) Adam17−/− cells transfected with TGFα and either wild type ADAM17 or the inactive ADAME>A were treated with 10 μM of PP3, PP2 or Desatinib or 5μM Marimastat. All inhibitors were pre-incubated for 10 minutes, and AP-activity in the supernatants (conditioned for 2 hours) and cell lysates was measured by colorimetry (Sahin et al., 2006). The effect of PP2, Desatinib and Marimastat depended on the presence of ADAM17. (C) Shedding of TGFα from Adam17−/− cells co-expressing inactive ADAM17E>A or the cytoplasmic deletion mutant of ADAM17 (ADAM17-Δcyto) (Le Gall et al., 2009), and Src(K295A) or Src(E378G). The activation of ADAM17 by Src(E378G) did not require the cytoplasmic domain of ADAM17. (D) Adam17−/− cells rescued with either wild type ADAM17 or ADAM17-Δcyto were treated with or without 10 μM of PP3 or PP2, Desatinib or 5 μM Marimastat. The effect of these inhibitors on TGFα shedding also did not depend on the presence of the cytoplasmic domain of ADAM17. Results were from three independent experiments and are expressed as means ± SEM. All pairwise multiple comparison procedures were performed by Tukey Test using SigmaStat 3.1 software. (A,C) + indicates a P-value of <0.05 with respect to the effect of ADAM17 or ADAM17-Δcyto on the shedding of TGFα compared to ADAM17E>A. * indicates a P-value <0.05 for the effect of Src(E378G) compared to Src(K295A) on the shedding of TGFα by overexpressed ADAM17. (B,D) * indicates a P-value of <0.05 for the effect of ADAM17 or ADAM17-Δcyto on the shedding of TGFα relative to ADAM17E>A, and + indicates a P-value of <0.05 for the effect of Src kinase inhibitors PP2 or Desatinib or the metalloprotease inhibitor Marimastat on the shedding of TGFα compared to untreated cells.