Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity.

Abstract

Growth factor-triggered activation of Ras proteins is believed to be mediated by guanine nucleotide exchange factors (CDC25/GRF and SOS1/2) that promote formation of the active Ras GTP-bound state. Although the mechaism(s) of guanine nucleotide exchange factor regulation is unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. To evaluate this model, we generated constructs that encode the catalytic domains of human CDC25 or mouse SOS1, either alone (designated cCDC25 and cSOS1, respectively) or terminating in the carboxyl-terminal CAAX membrane-targeting sequence from K-Ras4B (designated cCDC25-CAAX and cSOS1-CAAX, respectively; in CAAX, C is Cys, A is an aliphatic amino acid, and X is Ser or Met). We then compared the transforming potential of cCDC25 and cSOS1 with their membrane-targeted counterparts. We observed that addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their focus-forming activity (10-to 50-fold) in NIH 3T3 transfection assays. Similarly, we observed that the membrane-targeted versions showed a 5-to 10-fold enhanced ability to induce transcriptional activation from the Ets/AP-1 Rasresponsive element. Furthermore, whereas cells that stably expressed cCDC25 or cSOSl exhibited the same morphologies as untransformed NIH 3T3 cells, cells expressing cCDC25-CAAX or cSOSl-CAAX displayed transformed morphologies that were indis hable from the elongated and refractile morphology of oncogenic Ras-transformed cells. Thus, these results suggest that membrane translocation alone is sufficient to potentiate guanine nucleotide exchange factor activation of Ras.Ras proteins are GDP/GTP-binding proteins that function as molecular switches to mediate downstream signaling from a diverse variety of extracellular stimuli that influence cell growth and differentiation (1). Ras activity is controlled by a GDP/GTP cycle that is negatively regulated by GTPase-activating proteins (GAPs; p120 and NF1 GAP) that stimulate the intrinsic GTPase activity of Ras to promote formation of the inactive GDP-bound state of Ras and positively regulated by guanine nucleotide exchange factors (GEFs; CDC25/GRF and SOS1/2) that promote exchange of GDP for GTP to attain the active conformation (2). While mitogen-stimulated elevations in Ras-GTP are believed to occur primarily as a consequence of the activation of Ras GEFs, the precise mechanism for this activation remains unresolved (9). Recent studies identifying the role of SOS1 in mediating epidermal growth factor (EGF)-triggered activation of Ras have suggested a mechanism whereby recruitment to the plasma membrane may be sufficient to cause Ras activation (10-17). In quiescent Rat-i fibroblasts, SOS1 was found almost exclusively in the cytosolic fraction. However, upon EGF stimulation, Buday and Downward (12) observed that the majority of SOS1 was translocated to the particulate fraction and was found to be associated with...

Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-sevenless 1 (mSos1)

“…These characteristics of the mSos1 CDC25H domain are consistent with those reported for the CDC25H domains of CDC25 Mm and Cdc25p (Cen et al, 1993;Garreau et al, 1996). It was shown in the present study that the arti®cial connection of a CAAX sequence to mSos1 fragments increases their Ras signaling activities only at low expression levels, which is consistent with previous observations (Aronheim et al, 1994;Quilliam et al, 1994).…”

EGF induced SOS phosphorylation in PC12 cells involves P90 RSK-2

“…The mechanism by which SOS activates Ras is still unclear since the intrinsic guanine nucleotide exchange activity of SOS is not enhanced by growth factor stimulation (Buday and Downward, 1993a). The evidence to date suggests that translocation of the Grb2-SOS complex to the plasma membrane may be su cient to lead to Ras activation since either constitutively membrane targeted mutants (Quilliam et al, 1994;Aronheim et al, 1994) or conditional membrane targeted alleles of SOS (Holsinger et al, 1995) can directly lead to Ras activation. In addition, studies done in Drosophila and in mammalian cells indicate that sequences contained within the amino-terminal region of SOS, including the PH and DBL domains, may be involved in stimulating the exchange activity Karlovich et al, 1995;Byrne et al, 1996).…”

Constitutive membrane association potentiates activation of Bruton tyrosine kinase

“…Other cytosolic proteins interact with membrane associated signaling molecules such as Ras which are downstream of receptors in signal transduction pathways (Vojtek et al, 1993). In many cases, cytosolic signal transducers can be arti®cially activated by the introduction of membrane targeting motifs such as lipid addition signals, retroviral Gag sequences, or the extracellular and transmembrane domains of cell surface proteins (Stokoe et al, 1994;Leevers et al, 1994;Aronheim et al, 1994;Quilliam et al, 1994;Klippel et al, 1996;Burgering et al, 1995;Kolanus et al, 1993;Sakai et al, 1995).…”