SHP-1 regulates Rac.

We used a heterologous COS-7 cell system and p21 activated kinase (PAK) binding domain pull-down assay to evaluate ITAM receptor–induced activation of Rac34 in the presence or absence of SHP-1 cotransfection. The transfection condition for each group is shown above the lanes. Lanes 1, 4, and 7 show no stimulation (NS); lanes 2, 5, and 8, sRBC stimulation for 1 minute at 37°C; lanes 3, 6, and 9, stimulation of transfected COS7 cells for 5 minutes with sRBC. Lane 10 shows a positive control for GTP-Rac, a COS7 cell lysate incubated with GTPγS. (A) SHP-1 blocks FcγRIIA-induced conversion of GDP-Rac to its GTP-bound state. Western blot was performed with anti-Rac1 antiserum on glutathione-S transferase PAK binding domain (GST-PBD) fusion protein pull-down to detect levels of GTP-Rac1 in COS7 cell lysates following sRBC stimulation. (B) Anti–SHP-1 Western blot analysis of cell lysates shown in panel A. Lane 1, no transfection; lane 2, transfection with FcγRIIA and EGFP-SYK; lane 3, transfected with empty vector plasmids; lane 4, transfected with FcγRIIA, EGFP-SYK, and SHP-1.