Rac-GEF Activity Research Articles

Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1.

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

A Docker Instead of a Dbl

Overexpression of Dock180 can stimulate nucleotide exchange on the guanosine triphosphatase (GTPase) Rac, promoting such cellular responses as membrane ruffling and phagocytosis, and this effect is enhanced if two other proteins are also expressed, ELMO1 and the adaptor CrkII. These data suggested that Dock180 is a guanine exchange factor (GEF) for Rac; however, Dock180 lacks the Dbl homology (DH) domain responsible for GEF activity in all other known GEFs. Brugnera et al. investigated in vitro and in transfected cells Dock180's GEF activity and identified a new domain, which they call Docker, that bound to Rac and promoted GTP loading. The docker domain is conserved in other members of the mammalian Dock180 family and in a protein encoded by a yeast gene implicated in the control of filamentous growth. These domains from Dock2 and the protein encoded in the yeast gene also interacted with Rac in vitro. Mutations in the Docker domain or the ELMO binding domain prevented the stimulation of membrane ruffles or particle engulfment in cells transfected with the mutated Dock180 and ELMO1. Coprecipitation of Dock180 and Rac in transfected cells was enhanced if ELMO was also transfected into the cells, suggesting that ELMO1 may promote the Dock180-Rac interaction. However, the mechanism by which these proteins work together to regulate Rac activity remains unknown. E. Brugnera, L. Haney, C. Grimsley, M. Lu, S. F. Walk, A.-C. Tosello-Trampont, I. G. Macara, H. Madhani, G. R. Fink, K. S. Ravichandran, Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex. Nat. Cell Biol. 4 , 574-582 (2002). [Online Journal]

Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex.

Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. We can also detect a trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac.

P-Rex1, a PtdIns(3,4,5)P 3- and Gβγ-Regulated Guanine-Nucleotide Exchange Factor for Rac

Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a PtdIns(3,4,5)P 3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by PtdIns(3,4,5)P 3 and Gβγs both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in PtdIns(3,4,5)P 3 and Gβγ signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.

Mechanisms through which Sos-1 coordinates the activation of Ras and Rac.

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1–Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1–Grb2 (S/G) or a Sos-1–E3b1–Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1–Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.

Induction of Rac-Guanine Nucleotide Exchange Activity of Ras-GRF1/CDC25Mm following Phosphorylation by the Nonreceptor Tyrosine Kinase Src

Ras-GRF1/CDC25(Mm) has been implicated as a Ras-guanine nucleotide exchange factor (GEF) expressed in brain. Ras-GEF activity of Ras-GRF1 is augmented in response to Ca(2+) influx and G protein betagamma subunit (Gbetagamma) stimulation. Ras-GRF1 also acts as a GEF toward Rac, but not Rho and Cdc42, when activated by Gbetagamma-mediated signals. Tyrosine phosphorylation of Ras-GRF1 is critical for the induction of Rac-GEF activity as evidenced by inhibition by tyrosine kinase inhibitors. Herein, we show that the nonreceptor tyrosine kinase Src phosphorylates Ras-GRF1, thereby inducing Rac-GEF activity. Ras-GRF1 transiently expressed with v-Src was tyrosine-phosphorylated and showed significant GEF activity toward Rac, but not Rho and Cdc42, which was comparable with that induced by Gbetagamma. In contrast, Ras-GEF activity remained unchanged. The recombinant c-Src protein phosphorylated affinity-purified glutathione S-transferase-tagged Ras-GRF1 in vitro and thereby elicited Rac-GEF activity. Taken together, tyrosine phosphorylation by Src is sufficient for the induction of Rac-GEF activity of Ras-GRF1, which may imply the involvement of Src downstream of Gbetagamma to regulate Ras-GRF1.

From Ras to Rac

Cytoskeletal rearrangements can occur when cells respond to certain extracellular stimuli. One of the signaling cascades implicated in this process involves communication between the small GTP-binding proteins Ras and Rac. Scita et al. now show that fibroblast cell lines derived from knockout mice that lack the protein Eps8 do not remodel actin when treated with growth factors. Rac activity was regulated by a complex containing the proteins Eps8, E3b1, and Sos-1. The authors suggest that the formation of this complex stimulates the Rac-GEF activity of Sos-1. In addition, Eps8 activity was localized downstream of phosphatidylinositol 3-phosphate kinase, an immediate downstream effector of Ras. Hence, this complex may facilitate the transfer of signals between Ras and Rac. Scita, G., Nordstrom, J., Carbone, R., Tenca, P., Giardina, G., Gutkind, S., Bjarnegard, M., Betsholtz, C., and Di Fiore, P.P. (1999) EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401 : 290-293. Online Journal