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Expression cloning of RasGRP : a member of the CDC25 family of Ras exchangers Tognon, Cristina Esao

Abstract

The fact that Ras mutations are found in approximately 30% of all human malignancies points towards a role for Ras signalling pathways in the process of malignant transformation. An expression cloning strategy was used to identify genes that were equal to Ras in their ability to transform cells. Upon screening a T hybridoma cell cDNA library we identified a novel cDNA whose overexpression in NIH 3T3 cells caused transformation phenotypically similar to that of Ras overexpression. RasGRP, the protein encoded by this cDNA, contained several unique structural features, the foremost being regions of high sequence similarity to the CDC25 family of guanine nucleotide exchange factors (GNEFs). RasGRP not only contained the three structurally conserved regions found in these domains but also contained a Ras exchange motif (REM) box that is found in GNEFs that are capable of exchange on true Ras proteins. Overexpression of RasGRP caused hyperactivation of MAP kinases, specifically ERK 1 and 2. This data is compatible with the idea RasGRP is capable of causing Ras activation. Furthermore, the ability of RasGRP to transform cells and activate MAP kinase was eliminated by mutations affecting the exchange factor domain or by deletions of the REM box. RasGRP also contained a Cl domain which was very similar in sequence to diacylglycerol- and phorbol ester-binding Cl domains of protein kinase Cs (PKCs). RasGRP's Cl domain was found to be essential for fibroblast transformation and could be functionally replaced by a prenylation signal or by the Cl domain of PKCδ. RasGRP was found to localize to the ER as well as other membranes and this localization was dependent upon the Cl domain. The isolated Cl domain was also capable of re-localization in response to phorbol ester stimulation and to increases in diacylglycerol produced upon phosphatidylcholine-phospholipase C stimulation. These responses were identical to those of the PKC5-C1 domain. Taken altogether, these results provide the evidence on which to base the following model of Ras activation via RasGRP. It states that RasGRP's Cl domain facilitates the translocation of RasGRP to membranes enriched in diacylglycerol or phorbol ester. Once at the membrane, RasGRP is able to interact with Ras proteins tethered there by prenylation. GDP exchange then takes place via RasGRP's REM/GEF domains and the Ras pathway is activated. RasGRP also contains a pair of calcium binding EF hands, a proline rich region, and an a-helical domain. These domains were found to be non-essential for fibroblast transformation and it will be of interest to discover what roles they play in RasGRP function. RasGRP exhibits a unique pattern of expression when compared to other GNEFs capable of exchange on Ras. It is expressed extensively in brain, thymus, spleen, and bone marrow, which points to possible roles in neuronal and lymphoid signalling.

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