Skip to main content
. 2002 Jun;13(6):2031–2044. doi: 10.1091/mbc.01-11-0561

Figure 4.

Figure 4

Phosphorylation of TACE by Erk. (A) Primary sequence of a region of the mouse TACE intracellular domain indicating (arrow) a potential MAPK phosphorylation site. Bottom, an autoradiogram of an in vitro phosphorylation of TACE by Erk. Assays were performed by using bacterially produced GST-Erk or anti-Erk immunoprecipitates (IP anti-Erk) as the enzyme and 10 μg of GST, GST-Elk or GST-TACE as the substrate. To obtain activated Erk from HeLa cell lysates, cells were treated with PMA for 30 min before lysis and immunoprecipitated with the anti-Erk antibody. (B) Results from an in vitro kinase assay using bacterially produced GST-TACE or GST-TACE-T735A. Lysates from control or PMA-treated HeLa cells were immunoprecipitated with the anti-Erk antibody, and the immunoprecipitates were tested for their ability to phosphorylate in vitro 10 μg of GST-TACE or GST-TACE-T735A. The amounts of the recombinant proteins used in the kinase assays were verified by Coomassie staining of gels run in parallel (Díaz-Rodríguez, Montero, Esparís-Ogando, Yuste, and Pandiella, unpublished data). (C) Phosphoamino acid analysis of an experiment identical to that shown in B. The phosphorylated bands were excised, proteins were eluted and hydrolyzed, and phosphoamino acids were identified by two-dimensional electrophoresis. (D) In vivo phosphorylation of TACE by PMA. Top, 293 cells labeled with 32P were treated with PMA for 30 min where indicated. TACE was immunoprecipitated with the anti-TACE antibody, and the immunoprecipitates were analyzed by SDS-PAGE, followed by autoradiography. An additional PMA-treated sample was incubated with an excess (10 μg) of the peptide used to raise the anti-TACE antiserum. The asterisks indicate the position of two bands of unknown identity. Bottom, effect of the Erk pathway inhibitor PD98059 on TACE phosphorylation. Where indicated, cells labeled with 32P were incubated with PD98059 (50 μM) for 30 min before PMA treatment. Lysates were immunoprecipitated with the anti-TACE antibody, followed by SDS-PAGE and autoradiography.