FIG. 6.
The isolated catalytic domain of SOS requires the allosteric pocket and RasGRP1 to efficiently trigger a Ras-ERK-CD69 pathway. (A) Ten micrograms of vector, SOS1-cat, SOS1-cat-F929A, or SOS1-cat-W729E expression constructs was transiently transfected into Jurkat or JPRM441 cells together with 10 μg of GFP plasmid. Sixteen hours later, cells were analyzed by FACS and Western blotting as described before. Numbers inside the plots indicate percentages of cell populations in each quadrant. (B) Wild-type Jurkat T cells were transiently cotransfected with 4 μg vector and 10 μg GFP plasmids or with 5 μg mouse Rasgrp1 or human RasGRP1 siRNA together with 4 μg SOS1-cat and 10 μg GFP plasmids. Transfected cells were analyzed for CD69 and GFP expression by FACS and for protein expression by Western blot analysis 40 h after transfection. The percentage of CD69-positive cells over all transfected cells was determined in three independent experiments and is plotted as mean values with error bars. Small amounts of SOS1-cat plasmid were introduced to minimize SOS1-cat expression before RasGRP1 knockdown occurs. (SOS1-cat starts to be expressed 4 h after transfection, but RasGRP1 knockdown is maximal only after 40 h.) (C) JPRM441 cells were transiently transfected with 4 μg vector and 10 μg GFP plasmids or with 5 μg mouse Rasgrp1 or human RasGRP1 siRNA together with 4 μg SOS1-cat and 10 μg GFP plasmids and analyzed as in panel B. As controls, wild-type Jurkat cells were transfected with 4 μg vector or SOS1-cat together with 5 μg vector and 10 μg GFP plasmids. Panel C is a representative example of two independent experiments. (D) Jurkat-CD25ζζ, JPRM441-CD25ζζ, or stably reconstituted JPRM441-CD25ζζ-wtRasGRP1 cells were transiently transfected with 30 μg of vector (vec) or SOS1-cat plasmids together with 15 μg of GFP expression construct and analyzed as in panel A at 4, 6, and 8 h posttransfection. The percentage of CD69-positive cells over all transfected cells was plotted for the various time points as a bar graph. Protein expression was determined as before using NP-40 lysates prepared at the indicated time points.