Figure 5. The Cdc25-EF linker inhibits RasGRP1.

(A) The RasGRP1 Cdc25 domain (left) is shown with the Cdc25-EF linker (red) and Ras modeled using the Ras-SOS complex (right). Tyr 64 of switch 2 of Ras makes crucial contacts at the base of the SOS helical hairpin. (B) Sequence alignment of the Cdc25-EF linker region of different RasGRP proteins reveals partial (light blue) and complete (yellow) conservation of amino acids important for autoinhibition. (C) The in vitro nucleotide exchange activities of different RasGRP1 proteins (10 μM) were compared with 500 nM Ras in solution. Error bars represent ± standard deviation. (D) FACS measurements were used to compare ERK phosphorylation in cells expressing full length RasGRP1 (wild type) or mutant proteins with two (Linker 2A), three (Linker 3D) or five mutations (Linker 5A) to the Cdc25-EF linker as a function of expression level. R271E is a catalytically dead mutant that is shown for reference. The average levels are shown with error bars that represent ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.00813.011

Figure 5—figure supplement 1. Cellular analysis of RasGRP1 nucleotide exchange activity.

(1) Jurkat cells expressing low endogenous levels of RasGRP1 are transfected with plasmid DNA encoding myc-tagged wild type RasGRP1 or RasGRP1 variants. (2) Cells rest for 5 hr after transfection. (3) Each cell displays a different level of myc-RasGRP1 protein, resulting in a population of cells with a range of expression levels. myc-RasGRP1 will activate Ras, which leads to an increased level of phosphorylated ERK (P-ERK). (4) Fluorochrome-conjugated antibodies are applied to detect myc-tagged RasGRP1 and P-ERK. (5) Fluorescence activated cell sorting (FACS) is used to measure expression levels of P-ERK and myc-tagged RasGRP1 in each cell. Each point in the dot plot represents the P-ERK level of a single cell as a function of the RasGRP1 expression level. (6) Cells are divided in four different groups based on the expression of myc-tagged RasGRP1: untransfected, low, medium (med), and high expression. (7) For each group of cells the geometric mean (mean fluorescence intensity) of the P-ERK level is determined and normalized to the P-ERK level in untransfected cells from the wild type RasGRP1 experiment. The normalized P-ERK levels for each bin are then plotted for each RasGRP1 protein.

DOI: http://dx.doi.org/10.7554/eLife.00813.012