Fig. 4. Mapping of the TIAM1 caspase site in vitro and in vivo. A, HA-tagged forms of 945 and 993 (corresponding to the peptides generated by use of sites C and D, respectively, in Fig. 1
) were expressed in COS cells, and their electrophoretic mobilities were compared with that of the cleavage product produced in Jurkat cells by Fas treatment. Blots were probed with anti-TIAM antibody. B, TIAM1 C1199 or 993 was in vitro translated and subjected to protease reactions as described in the Fig. 3
legend. In vitro translated products were left untreated (-) or incubated with extracts prepared from Jurkat cells treated with anti-Fas for the indicated times. 35S-labeled peptides were detected by autoradiography. C, in vitro protease reactions were performed as described in the Fig. 3
legend using either C1199 or C1199(DN), in which the DETD993 motif was mutated, as substrate and analyzed by autoradiography. D, HMN1 cells were transfected with the indicated TIAM constructs and then treated with C2-ceramide for 2 h as indicated. Whole cell lysates were immunoblotted with anti-TIAM antibody. Arrowhead, caspase cleavage product produced in vivo or in vitro; closed arrow, HA-tagged 993; open arrow, degradation product of transfected TIAM mutants that was not observed in untransfected cells.