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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 1. Overexpression of an oncogenic mutant form of Src(E378G) increases the shedding of ADAM17 substrates in mouse embryonic fibroblasts.

Figure 1

(A, B) Immortalized wild type mouse embryonic fibroblasts (mEFs) on six-well plates were transiently transfected with an alkaline phosphatase (AP)-tagged TGFα and the inactive Src(K295A) or the constitutively active Src(E378G) (Levy et al., 1986; Ma et al., 2000; Sun et al., 2007). Six hours after transfection the cells were incubated with the Src inhibitor PP2 (10μM, Calbiochem, San Diego, CA) (A) or the metalloproteinase inhibitor Marimastat (MM, 5μM, kindly provided by Dr. O. Ouerfelli, MSKCC, New York) (B) for 8 hours. Cells were lysed on ice in TBS Triton X-100 (1%), 1 mmol/L EDTA and glycoproteins were precipitated with the lectin Con-A overnight and eluted the next day with 0.5 mM Methyl α-D Manno-Pyranoside. All reagents were from Sigma-Aldrich, St. Louis, MO, unless otherwise indicated. For the evaluation of the AP activity in the cell lysates, equal amounts of total protein were separated by 8% SDS-PAGE and then incubated with NBT/BCIP (Roche, San Francisco, CA) substrate solution for 10 min (Sahin et al., 2006). Cells expressing Src(E378G) showed strongly decreased levels of TGFα in comparison to cells transfected with Src(K295A). The expression of TGFα in cells co-transfected with Src(E378G) could be restored by incubation with PP2 (A) or Marimastat (B). (C, D) Shedding of TGFα (C) or betacellulin (BTC) (D) into the supernatant of wild type mEFs was analyzed by colorimetric assay for alkaline phosphatase activity. For photometric quantitation of the AP-activity in the culture supernatant, 100 μl of conditioned medium was mixed with 100 μl of a 2 mg/ml solution of the alkaline phosphatase substrate 4-nitrophenyl phosphate in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 20 mM MgCl2. After incubation at 37°C for 30 minutes, the amount of product was quantitated by determining the A405. The shedding of TGFα into the supernatant of mEFs was enhanced by co-transfection of Src(E378G) and v-Src compared to co-transfection with the inactive Src(K295A) or MAD2, used as a control. (D) The shedding of BTC, used here as a readout for the activity of ADAM10 (Sahin et al., 2004), was not affected by the co-expression of Src(E378G) or v-Src compared to Src(K295A) or MAD2. (E) When we compared shedding of the ADAM17 substrates ICAM-1 (Weskamp et al., 2010), amphiregulin (AMP) (Sahin et al., 2004) or TNFα (Black et al., 1997; Horiuchi et al., 2007a; Moss et al., 1997) in the presence of Src(K295A) or Src(E378G), we found that the transforming form of Src increased the shedding of all three ADAM17 substrates. Results were obtained from three independent experiments and expressed as means ± SEM. Pairwise comparison was performed by Mann-Whitney rank sum test, multiple comparisons versus a control group by Dunnett’s method using SigmaStat 3.1 software (SYSTAT, Erkrath, Germany). P values of <0.05 were considered statistically significant, and samples with significant changes compared to controls are marked by an asterisk.