Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Abstract

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H-and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester-and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol-and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerolenriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.

Early endosome localization and activity of RasGEF1b, a toll-like receptor-inducible Ras guanine-nucleotide exchange factor

“…These signals include the phosphorylation of proteins and lipids, calcium fluxes, and the generation of cyclic nucleotides or diacylglycerol. [20][21][22][23] In the structure of RasGEF1b exists two main domains: one for interaction with Ras (RasGEFN) and one cdc25-like domain spanning the region of amino acid at position 204 through 448, 5 which may be essential for full GEF catalytic activity. Among its functions, it is likely that RasGEF1b gene is a signalling component expressed during the immune response, as expression of RasGEF1b mRNA is enhanced in vitro in macrophages activated with TLR agonist or in activated T cells, as well as in vivo in animals that received microbial challenge.…”

Mast Cells in Airway Hyporesponsive C3H/HeJ Mice Express a Unique Isoform of the Signaling Protein Ras Guanine Nucleotide Releasing Protein 4 That Is Unresponsive to Diacylglycerol and Phorbol Esters

“…All normal variants of mouse, rat, and human RasGRPs have a 50-mer domain in their C-terminal portions that is weakly homologous to the C1, DAG-binding domain in protein kinase C. Recombinant hRasGRP3 can bind 12-[ 3 H]phorbol-13-dibutyrate efficiently in the presence of phospholipids (15). Thus, it has been speculated that the interaction with DAG or PMA is needed for the efficient movement of the varied RasGRPs from their cytosolic compartments to the plasma membrane (14,15,36,37). Although the location of the DAG-binding site has not been deduced experimentally in any RasGRP, it has been assumed that the site is the C1-like sequence that resides in the C-terminal domain of these GEFs.…”

Transgenic Expression of RasGRP1 Induces the Maturation of Double-Negative Thymocytes and Enhances the Production of CD8 Single-Positive Thymocytes

“…RasGRP1 is activated by recruitment to diacylglycerol or phorbol ester-enriched membranes (27,28,30,31) and thus can provide a route to Ras activation downstream of any phospholipase C-coupled receptor, including TCR or pre-TCR. In the Jurkat T cell line, a high proportion of Ras activation in response to TCR cross-linking is mediated by RasGRP1, and overexpression of RasGRP1 in these cells prolongs the ERK activation response to TCR cross-linking (31).…”

“…RasGRP1 (also known as RasGRP or CalDAG-GEFII) is an exchange factor that is expressed in the thymus and can activate H-Ras, K-Ras, and N-Ras and the Ras-related GTPases R-Ras and TC21 (27)(28)(29)(30). RasGRP1 is activated by recruitment to diacylglycerol or phorbol ester-enriched membranes (27,28,30,31) and thus can provide a route to Ras activation downstream of any phospholipase C-coupled receptor, including TCR or pre-TCR.…”

“…Total RNA was isolated from thymi of 4-wk-old PBS-or anti-CD3⑀-injected RAG-2 Ϫ/Ϫ mice or tissues of adult C57BL/6 mice, separated on a 1% agarose formaldehyde gel, and analyzed by Northern blot analysis as previously described (33). Blots were probed using a full-length murine RasGRP1 cDNA probe (30) or elongation factor-1␣ as a loading control. For RT-PCR analysis, thymocytes from 6-to 8-wk-old C57BL/6 mice were sorted using a FACStar Plus flow cytometer (BD Biosciences) into DP, CD4 SP, and CD8 SP fractions as previously described (34).…”