The atypical SH3 domain-binding motif and the C-terminal SH3 domain of Grb2 reduce the ability of SLP-65 to activate AP1. (A) SLP-65–regulated activation of an AP1-driven luciferase reporter gene. SLP-65–deficient cells were reconstituted with either full-length SLP-65 (middle bars) or s-SLP-65 (right bars) or, as control, were transfected with the empty vector (left bars). Subsequently, the AP1 reporter construct was transiently introduced by electroporation together with a β-galactosidase expression plasmid (for determination of transfection efficiency). Enzymatic activities of luciferase and β-galactosidase were determined for resting cells (□) and cells stimulated without further treatment through their BCR for 6 hours (▪) or cells that were pretreated for 1 hour prior to BCR activation with 10 μM of either SB202190 to inhibit p38 (p38-i, ▨) or SP600125 to inhibit JNK (JNK-i, ▦). At least 3 independent clones were measured in 4 independent experiments and data are normalized according to the transfection efficiency. Error bars indicate SD. (B) Immunoblot analysis of BCR-mediated synthesis of c-Fos. DT40 cell lines described in panel A were left untreated or BCR-stimulated for 1 hour and lysed and c-Fos expression was determined by immunoblotting with anti–c-Fos antibodies (top panel). Equal loading of cellular lysates was controlled by immunoblotting with antiactin antibodies (bottom panel). The relative molecular masses of marker proteins are indicated on the left. (C) Impact of the C-terminal Grb2 SH3 domain on AP1 activation. DT40 cells expressing equal amounts of either wild-type or W193K mutant Grb2 (left and right bars, respectively; also Figure 1D) were transfected with the AP1 reporter and β-galactosidase constructs, and resulting enzymatic activities in resting and BCR-stimulated cells (□, ▪, respectively) were determined as described in panel A. Error bars indicate SD of 3 independent measurements.