Rac Activation: P-Rex1 — A Convergence Point for PIP3 and Gβγ?

Small GTPases of the Rho family regulate a vast spectrum of functions in eukaryotic cells, from cyto-skeletal rearrangements to vesicle transport to transcriptional regulation. Rho GTPases act as molecular switches, cycling between an ‘inactive’ GDP-bound form and an ‘active’ GTP-bound form. Activation is accomplished by guanine-nucleotide exchange factors (GEFs) which catalyze GDP dissociation, thereby facilitating GTP loading. Members of the Rac subfamily of Rho GTPases play an important role in transcriptional activation, the production of reactive oxygen species, and actin polymerization during lamellipodial formation [1]. A recent study [2] has identified a novel Rac activator, P-Rex1, which may act as a coincidence detector for signals transduced by phosphoinositide lipids and trimeric G proteins. The road to the discovery of PRex1 began with a biochemical assay for the production of reactive oxygen species in neutrophil lysates. Reactive oxygen species are produced downstream of G-protein-coupled receptors in neutrophils and other innate immune cells, and represents an essential component of the pathogen killing process [3]. Both phosphatidylinositol 3-kinase (PI 3-kinase) and Rac have been implicated in reactive oxygen species production, and activated Rac is sufficient to drive production of reactive oxygen species. Welch et al. [2] found that the PI 3-kinase lipid product PIP3 can also stimulate reactive oxygen species formation in neutrophil lysates, and this stimulation is blocked by proteins that sequester GEFs for Rac. These data suggested that PIP3 activates exchange factors for Rac in neutrophil lysates. Using a direct assay for PIP3 activation of Rac, Welch et al. [2] purified a novel exchange factor for Rac, which they named PRex1, for PIP3-dependent Rac exchanger [2]. P-Rex1 was the major PIP3-dependent Rac GEF in neutrophil lysates, representing about 65% of the total Rac GEF activity. As Gβγ also suffices to stimulate reactive oxygen species production in neutrophil lysates, Welch et al. [2] tested whether Gβγ can activate PRex1. Surprisingly, they found that Gβγ directly activates P-Rex1, and synergizes with PIP3 for P-Rex1 activation in vitro and in vivo. Antisense-mediated knock-down of P-Rex1 levels in a neutrophil-like cell line decreased agonist-induced formation of reactive oxygen species by 40–45%, suggesting that P-Rex1 is necessary for full reactive oxygen species production in vivo [2]. The identification of P-Rex1 is particularly significant in several respects. This is the first biochemical purification of an exchange factor for a Rho GTPase based on its activity. Most other mammalian Rho GEFs have been identified either as oncogene products or by homology to known GEFs identified by genetic screens in a model species. The best known Rho GEF activators have been protein kinases. PI 3-kinase has been reported to regulate several of these GEFs, but it primarily appears to do so indirectly, by affecting GEF phosphorylation by protein kinases. Where PIP3 has been reported to directly regulate GEFs in vitro, the observed degree of GEF activation was significantly lower than for P-Rex1: for example, PIP3 activated Vav1 [4] and Pix [5] less than 2.5-fold, compared to 20-fold activation of P-Rex1 by PIP3 [2]. Finally, P-Rex1 is the first example of an exchange factor for Rho GTPases that is directly stimulated by Gβγ. Other Rho GTPase exchange factor are known to act downstream of Gβγ, but the yeast GEF Cdc24 requires adaptor proteins to bind to and be activated by Gβγ [6], and the mammalian GEF RasGRF is regulated indirectly by Gβγ through tyrosine phosphorylation [7]. How does P-Rex1 fit into the known picture of Rac activation in neutrophils? In neutrophils and other haematopoietic cells, Gβγ release and PIP3 production are linked by the lipid kinase PI 3-kinase-γ and its adapter protein p101 (Figure 1A) [8]. Therefore, both coactivators of P-Rex1 are naturally produced upon activation of a G-protein-coupled receptor. In contrast, activation of some tyrosine kinase receptors which fail to generate Gβγ can lead to significant production of PIP3 but no detectable Rac activation. In some [9,10] but not all [11] experiments, Rac activation in neutrophils is dependent on PI 3-kinase, possibly reflecting the relative importance of Gβγ versus PIP3 at different time points for Rac activation. The fact that Gβγ and PIP3 synergize in P-Rex1 activation suggests that they bind separate domains of P-Rex1, though neither domain has yet been identified. The pleckstrin homology (PH) domain would seem the most obvious candidate for interaction with PIP3, although P-Rex1’s PH domain is not very similar to those of known PIP3-binding proteins, such as Akt, Grp1 and Btk. For interaction with Gβγ, the DEP domain is a good possibility as it is found in ‘regulator of G protein signalling’ (RGS) proteins, which interact with the Gα subunit of heterotrimeric G proteins and other signaling proteins that act at the plasma membrane (Figure 1B). Identifying the protein domains used by PRex1 to interact with Gβγ and PIP3 will be instrumental for identifying additional GEFs and other proteins Dispatch Current Biology, Vol. 12, R429–R431, June 25, 2002, ©2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)00917-X