Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells
Figure 5
Loss of the RasGRP1 binding region in SKAP-55 abrogates its ability to inhibit ERK activation and ELK transcriptional activity.
Panel A: RasGRP1 binding to SKAP-55 depends on the C-terminal domain of SKAP-55. Upper panel: deletion of the SKAP-55 SH3 domain prevents RasGRP1 binding. Lanes 1,2: myc-RasGRP1 expression in Jurkat cells transfected with SKAP-55 and SKAP-55ΔSH3. Lanes 3–5: association of RasGRP1 with SKAP-55 depends on the C-terminal domain of SKAP-55. Cell lysates: Lanes 1,2; anti-SKAP-55 precipitates: lanes 3–5. SKAP-55 transfection: Lanes 1, 4. SKAP-55ΔSH3 transfection: Lanes 2, 5. Lower panel: anti-SKAP-55 blot. Lane 1: transfection with myc-RasGRP1; lane 2: transfection with myc-RasGRP1 and SKAP-55; lane 3: transfection with myc-RasGRP1 and SKAP-55ΔSH3. Panel B: Loss of the RasGRP1 binding region in SKAP-55 abrogates its ability to inhibit ERK and ELK activation. Upper panel: anti-pERK blot of lysates from cells transfected with myc-RasGRP1 plus SKAP-55 or SKAP-55ΔSH3. Resting cells: lanes 1,3,5,7; anti-CD3: lanes 2,4,6,8. Middle panel: anti-ERK blot; Lower panel: anti-pELK blot. Panel C: Loss of the RasGRP1 binding region in SKAP-55 abrogates its ability to inhibit ELK transcriptional activity. Left panel: histogram showing inhibition of ELK transcription activity by SKAP-55 and lack of inhibition by SKAP-55ΔSH3. Right panel: anti-SKAP-55 (upper) and anti-myc (myc-RasGRP1) blot of cell lysates.