The Ras-GRF1 Exchange Factor Coordinates Activation of H-Ras and Rac1 to Control Neuronal Morphology

Abstract

The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRF⌬N, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRF⌬C, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRF⌬C binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size. INTRODUCTIONThe Ras superfamily of GTPases are regulated switches that control many intracellular pathways. The Ras family, which includes H-, K-, and N-Ras and other closely related isoforms, has been particularly associated with the control of proliferation in cells such as fibroblasts and epithelia (Lowy and Willumsen, 1986). This action is thought to be of particular relevance to the common involvement of activated Ras in human cancer (Barbacid, 1987), which can occur by mutational activation (Taparowsky et al., 1982), by inappropriate activation of other elements in the Ras activation pathway, such as the overexpression or aberrant stimulation of growth factor receptors (Malaney and Daly, 2001), or by loss of a deactivating GTPase-activating protein (GAP), such as in type 1 neurofibromatosis (DeClue et al., 1991). Ras proteins are also, however, highly involved in the function of terminally differentiated cells such as neurons of the CNS (Weeber et al., 2002). The Rho family small GTPases, which include Rac1 and many other members, have multiple cellular functions during both cellular differentiation (Beqaj et al., 2002;Sordella et al., 2003) and in the mature phenotype, including regulation of the cytoskeleton and cellular morphology, and coupling to transcription factor pathways (Aznar and Lacal, 2001). There is increasing evidence that the functions of Ras and Rho family small GTPases can be coordinated to produce regulation of cellular phenotypes, with most models suggesting that Ras activation occurs before the activation of Rho proteins (Sarner et al., 2000;Men...

Internal tandem duplication of FLT3 (FLT3/ITD) induces increased ROS production, DNA damage, and misrepair: implications for poor prognosis in AML

“…These pathways may also contribute to ROS production. Of interest, RAC1 may be downstream of RAS, 57 and we have previously shown that RAS activation leads to ROS production via RAC1. 44 Thus, other signaling pathways may also contribute to ROS production and genomic instability in FLT3/ITD-positive AML.…”

ATDC (Ataxia Telangiectasia Group D Complementing) Promotes Radioresistance through an Interaction with the RNF8 Ubiquitin Ligase

“…The bacteria were harvested and lysed by sonication on ice in 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.2% sodium deoxycholate, and 0.1% Triton X-100. The lysate was cleared by centrifugation, and RNF8 prebound glutathione-Sepharose beads were prepared according to protocol as described previously (21). FLAG-ATDC was in vitro translated directly from the PCR products that contain the T7 promoter by TNT quick coupled transcription/translation system according to manufacturer's protocol (Promega).…”

Differential Roles of NMDA Receptor Subtypes NR2A and NR2B in Dendritic Branch Development and Requirement of RasGRF1

“…These results prompted us to examine whether a particular downstream signaling pathway that is coupled to NR1NR2B-containing receptors is required to induce dendritic branch development. Given that RasGRF1 is a key signaling component in the regulation of several forms of neuronal plasticity, specifically associates with the NR2B subunit (Krapivinsky et al 2003;Li et al 2006), and controls neurite extension in PC12 cells (Yang and Mattingly 2006), we hypothesized that the binding of RasGRF1 to NMDARs is necessary for inducing branch formation. To impede the interaction between the NR2B subunit and RasGRF1, VSCNs were transfected with DsRed2 and NR2B (Fig.…”