PMC

Phosphorylation of RasGRP3 Thr-133. (A) Wild-type RasGRP3 or its T133A mutant was coexpressed with PKC-β in 293T cells, and immunoprecipitated RasGRP3 was blotted with anti-pT133 Ab (Left). RasGRP3 or its mutant was immunoprecipitated from RasGRP3-deficient DT40 B cells expressing exogenous wild-type RasGRP3 or its T133A mutant () and blotted with anti-pT133 Ab (Right). (B) Wild-type or PLC-γ²-deficient DT40 B cells were stimulated, immunoprecipitated, and blotted with anti-pT133 Ab (Left). Wild-type DT40 cells were treated with Go6976 for 10 min or left untreated, then stimulated by means of BCR (Right).

Effect of RasGRP3 T133A mutation on its activation. (A) DT40 cells expressing wild-type RasGRP3-EGFP or its T133A mutant were stimulated by means of BCR, and subcellular localization of fusion proteins was visualized by using a confocal microscope. Among 50 cells counted for membrane localization, the numbers that showed clear membrane localization were 37 for DT40 cells expressing wild-type RasGRP3-EGFP and 32 for those expressing T133A mutant (Left). The amount of fusion proteins expressed in each transfectants was evaluated by flow cytometric analysis (Right). (B) Wild-type RasGRP3 or its mutants were coexpressed with PKC-β in 293T cells, and the Ras activation status was measured by using RBD-bound beads.

Model structure of RasGRP3. The RasGRP3 structure was built by homology modeling based on a crystal structure of Sos. (A) Ribbon models of Ras exchange motif (cyan) and cdc25 (pink) domains of chicken RasGRP3. Ras (orange) is put on an equivalent position by superimposition of the model on Sos structures. Sphere (green) indicates the phosphorylation site at Thr-133. (B) Molecular surface of RasGRP3 is shown. Red and blue indicate negatively charged residues, Glu and Asp, and positively charged residues, Lys and Arg, respectively. The orientation is same as in A.

Phosphorylation of RasGRP3 by BCR stimulation and its correlation with Ras activation. DT40 cells were incubated at 37°C with or without 20 μM Go6976 for 10 min, followed by stimulation with 5 μg/ml M4 for 3 min. Cell lysates were prepared and incubated with anti-chicken RasGRP3 Ab-coupled beads for 1 h. Beads were either left untreated or treated with 20 units of calf intestine alkaline phosphatase for 30 min at 37°C. Proteins bound to beads were eluted and subjected to Western blot analysis using anti-chicken RasGRP3 Ab. A total of 1 × 10⁷ cells per lane was used (Top). For measuring active GTP-bound Ras, lysates (1 × 10⁷ cells per lane) were incubated with RBD-bound beads for 30 min. Protein eluted from beads was subjected to Western blot analysis using anti-Ras mAb (Middle). As demonstrated in ref. , the upper and lower bands of Ras are likely to represent K-Ras and H-Ras, respectively. The lysates (1 × 10⁶ cells per lane) also were subjected directly to Western blot analysis for measuring total amount of Ras protein (Bottom).

Thr-133 in RasGRP3 is crucial for BCR-mediated Ras activation. (A) Schematic diagram of RasGRP3 mutants. By homology modeling based on structures of Sos, an N-terminal ≈400-residue catalytic module of chicken RasGRP3 is thought to be sufficient for the Ras-specific nucleotide exchange activity. This segment of RasGRP3 includes a core region known as the cdc25 domain (residues 130–399) and an additional domain, known as the Ras exchange motif domain, located N-terminal to the cdc25 domain (residues 1–129). RasGRP3 cDNAs encoding these mutations were transfected into RasGRP3-deficient DT40 cells. A single clone expressing each RasGRP3 mutant was extensively analyzed and represented, although some critical experiments were carried out, at least, using two different clones. EFh, EF hand; C1, protein kinase C conserved region 1 domain. (B) Expression of surface BCR on various mutant DT40 cells. Filled and opened histograms represent unstained cells and cells stained with FITC-anti-chicken IgM, respectively. (C) Expression of RasGRP3 in various mutant DT40 cells. Whole-cell lysates (2 × 10⁶ cells per lane) were subjected to Western blot analysis using anti-chicken RasGRP3 Ab (Upper) or anti-extracellular signal-regulated kinase Ab (Lower). (D) BCR-mediated Ras activation in various mutant DT40 cells. DT40 B cells (1 × 10⁷ cells per lane) were stimulated with M4 (5 μg/ml) for the indicated period. Lysates were incubated with RBD-bound beads for measuring GTP-bound Ras. Proteins eluted from beads were resolved in SDS/PAGE followed by Western blot analysis using anti-Ras mAb (Upper). For measuring total Ras, parts of the lysates (1 × 10⁶ cells per lane) were subjected to Western blot analysis without incubation with beads (Lower). (E) Comparison of protein sequences of the RasGRP family surrounding Thr-133 (denoted by an asterisk).