The Cool-2/α-Pix Protein Mediates a Cdc42-Rac Signaling Cascade

an SH3 domain, followed by a tandem arrangement of Background: Cloned-out of library-2 (Cool-2)/PAK-interDbl homology (DH) and Pleckstrin homology (PH) doactive exchange factor ( -Pix) was identified through mains [6]. This arrangement of DH and PH domains is its ability to bind the Cdc42/Rac target p21-activated characteristic of the Dbl family of guanine nucleotide kinase (PAK) and has been implicated in certain forms exchange factors (GEFs) for Rho-related GTP binding of X-linked mental retardation as well as in growth facproteins, and so it was assumed that the Cool/Pix protorand chemoattractant-coupled signaling pathways. teins would also function as GEFs. Our laboratory has We recently found that the dimeric form of Cool-2 is a characterized three members of the Cool/Pix family, specific guanine nucleotide exchange factor (GEF) for namely p50Cool-1, a longer splice variant called Rac, whereas monomeric Cool-2 is a GEF for Cdc42 as p85Cool-1 (identical to -Pix), and a distinct gene prodwell as Rac. However, unlike many GEFs, Cool-2 binds uct called Cool-2 (identical to -Pix). However, based to activated forms of Cdc42 and Rac. Thus, we have on these studies, it is clear that the Cool/Pix proteins investigated the functional consequences of these interdo not all simply function as GEFs but rather exhibit a actions. more complicated array of functional activities. In particResults: We show that the binding of activated Cdc42 ular, p50Cool-1 appears to block the stimulation of PAK to the Cool-2 dimer markedly enhances its ability to activity by Dbl and other GEFs [7], through an as yet associate with GDP bound Rac1, resulting in a signifiundetermined mechanism. The p85Cool-1 protein does cant activation of Rac-GEF activity. While the Rac-spenot interfere with PAK activity and is permissive for PAK cific GEF activity of Cool-2 is mediated through the Dbl activation, allowing activated Cdc42 or Rac molecules, homology (DH) domain from one monomer and the generated by different Dbl-related proteins, to stimulate Pleckstrin homology domain from the other, activated PAK [9]. Moreover, we recently made the rather unexCdc42 interacts with the DH domain, most likely oppopected finding that activated forms of Cdc42 bound to site the DH domain binding site for GDP bound Rac. p85Cool-1, and this interaction played an important role Activated Rac also binds to Cool-2; however, it strongly in the regulation of EGF receptor ubiquitination, as catainhibits the GEF activity of dimeric Cool-2. lyzed by the Cbl proteins [10]. Conclusions: We provide evidence for novel mechaUnlike the Cool-1 proteins, the expression of Cool-2 nisms of allosteric regulation of the Rac-GEF activity of in cells leads to PAK activation [9, 11, 12], apparently the Cool-2 dimer, involving stimulatory effects by Cdc42 arising from Cool-2’s ability to function as a GEF [13]. and feedback inhibition by Rac. These findings demonFull-length Cool-2 normally exists as a dimer and under strate that by serving as a target for GTP bound Cdc42 these conditions is a Rac-specific GEF [14]. Conditions and a GEF for Rac, Cool-2 mediates a GTPase cascade that lead to the dissociation of the dimer to the monowhere the activation of Cdc42 is translated into the actimer—for example, through the binding of a complex vation of Rac. containing PAK and G protein subunits to the SH3