http://genomebiology.com/2002/3/2/reviews/0002.1 Review The evolutionary history of effectors downstream of Cdc42 and Rac comment Sophie Cotteret and Jonathan Chernoff Address: ox Chase Cancer Center, 7701 Burholme Ave, Philadelphia, PA 19111, USA. Correspondence: Jonathan Chernoff. E-mail: [email protected] reviews Published: 14 January 2002 Genome Biology 2002, 3(2):reviews0002.1–0002.8 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2002/3/2/reviews/0002 © BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914) Abstract reports Cdc42 and Rac - ancient, highly conserved, small GTPases - mediate extracellular signals, triggering changes in transcription and in the actin cytoskeleton. Although dozens of proteins act downstream of these GTPases, a comparison of effector proteins from evolutionarily diverse organisms suggests that six groups of proteins serve as the core machinery for signaling from Cdc42 and Rac. deposited research Reorganization of the actin cytoskeleton plays crucial roles in signaling mechanisms of two of these three subgroups, many cellular functions, including morphology, motility, and Cdc42 and Rac, as they are often linked in their physiological adhesion. Regulation of actin polymerization and depolymer- functions and have several effectors in common. ization is orchestrated by small GTPases of the Rho family; these proteins are also important in regulating gene transcrip- Cdc42 and/or Rac homologs are found in fruit flies, round- tion. The basic signaling properties of two major subgroups of worms and budding yeast. Interestingly, small GTPases Rho GTPases - the Cdc42 and Rac subfamilies - are highly con- related to Rac are found even in organisms that lack Ras, refereed research served amongst all eukaryotes, but the means by which they such as plants. This broad distribution across widely diver- act are not well understood. In an effort to understand the fun- gent eukaryotic species suggests that Cdc42 and Rac GTPases damental signaling elements, or core machinery, required for have an ancient origin, perhaps even predating that of their the function of these GTPases, we describe here the conserva- cousin Ras. The analysis of Cdc42 and Rac function in evolu- tion and functional similarities of Cdc42 and Rac effectors in tionarily distant organisms is useful as a tool to uncover the five different species: plant, yeast, fruit fly, roundworm and basic activities of these proteins. In all systems, these human. We focus on six core effectors that operate in almost GTPases are best known for their effects on the polymeriza- all these species - members of the Pak, WASP/WAVE, tion and distribution of actin in the cell cortex [1]. This activ- formin, lipid-kinase, IQGAP and NADPH oxidase families. ity is strongly conserved and probably represents a interactions Additional modules that have been added during the evolution primordial function of these proteins. By regulating filamen- of particular lineages are not considered in detail here. tous actin, Cdc42 and Rac exert a profound effect on cell shape, polarity, migration, cell:cell and cell:matrix adhesion, protein traffic, and cytokinesis. In addition, they play impor- Core functions of Rho GTPases tant roles in gene transcription (via activation of mitogen Rho GTPases are small (20-30 kDa) GTP-binding proteins of activated protein (MAP) kinase pathways and, in higher the Ras superfamily. The Rho subfamily is divided in three eukaryotes, the transcription factor NkB), generation of information main subgroups - Cdc42, Rac, and Rho - examples of which are reactive oxygen species, apoptosis, and cell-cycle progression. represented in all eukaryotes from plants to man. The mam- malian Rho family consists of at least sixteen distinct members: the Cdc42 subgroup (Cdc42, TC10, Chp, and Wrch-1); the Rac Core GTPase effectors subgroup (Rac1-Rac3); and the Rho subgroup (RhoA-RhoE, In mammalian cells, as many as twenty types of protein have RhoG, RhoH, Rnd1, and Rnd2). In this article, we focus on the been reported to bind to activated Cdc42 [2]. A roughly 2 Genome Biology Vol 3 No 2 Cotteret and Chernoff similar number of proteins are recruited to activated Rac [3]. activities of the GTPase. If we apply such an evolutionary Among this large group of effectors are protein kinases; lipid filter to Cdc42 and Rac effectors, including only those effec- kinases, hydrolases, and phosphatases; actin-bundling pro- tors that are shared in mammals and yeast or plants, we find teins; coatomers (vesicle coat proteins); adaptor proteins; that only a small subset of proteins emerge as potential com- and proteins that interact with members of the Arp2/3 ponents of a core apparatus. These are p21-activated kinases complex and hence the actin cytoskeleton (Table 1). Many, (Paks), Wiskott-Aldrich syndrome proteins (WASPs) and but not all, of these effectors contain a conserved 18 amino- their relatives the WAVEs, formins, lipid kinases, IQGAPs, acid binding motif that has been termed CRIB (Cdc42-Rac and NADPH oxidases (igure 1). interactive binding), PBD (p21-binding domain) or GBD (GTPase-binding domain) [4]. This motif is found in Paks Cdc42/Rac-associated proteins such as the protein kinases Paks are serine/threonine protein kinases that associate Pak, MRCK and Ack, the adaptor proteins Spec and WASP, with Cdc42, and usually also with Rac, via a conventional and, in degenerate form, in the kinases MLK, Mekk4, CRIB motif. The Pak family in mammals has six members, adaptor Par6, scaffold protein IRSp53, and the Borg pro- which can be further divided into two groups on the basis of teins. Interestingly, the CRIB motif also makes an appear- structure [7]. Plant genomes do not encode any Paks, but ance in plant proteins, but here it is attached to a both the budding and the fission yeast genomes encode mul- GTPase-activating protein, or GAP (reviewed in [5]). The tiple Pak homologs that are critical to cell polarity, mating, presence of such CRIB-containing proteins in plants sug- and cytokinesis. In the budding yeast Saccharomyces cere- gests that either the CRIB motif has a very ancient origin, visiae, the Pak homolog Ste20p phosphorylates the MEKK- and became associated with a variety of signaling proteins like protein kinase Ste11p, and thus activates the mating and over the course of evolution in various organisms, or else it filamentous growth MAP kinase pathways [11]. A similar arose independently in plants. The latter possibility seems arrangement is found in the fission yeast Schizosaccha- unlikely, however, as there are several Rac effectors that lack romyces pombe, in which Pak1 and Pak2 act upstream of a CRIB domain, indicating that this particular sequence of MAP kinase cascades [12,13]. Given that these two yeasts are amino acids is not a unique solution to the problem of how only distantly related (having diverged around 800 million to bind activated Rac. years ago), these data support the idea that Paks constitute a conserved and fundamental link from Rho GTPases to MAP Although the total number of Rac and Cdc42 effectors is not kinase cascades. This notion is supported by data from yet known for any organism, it is already sufficiently high mammals, in which Paks have also been shown to function that an analysis of the role of individual effectors is difficult. in MAP kinase activation, albeit in a more baroque manner or example, in mammalian cells, the identities and relative than in yeast. Here, the linkage is through the kinases Raf1 contributions of the Cdc42/Rac effectors that mediate acti- and Mek1, which Pak phosphorylates [14,15]. Such phospho- vation of the Jnk family of kinases are in dispute, with com- rylations are not in themselves sufficient for MAP kinase peting claims made for the Pak, MEKK and MLK kinases activation, but rather are permissive events: they promote and for the adaptor protein POSH [6,7]. Similarly, the rela- efficient coupling of Raf to Ras and of Mek to Raf, respec- tive contributions of various Cdc42/Rac-binding protein tively. There is also evidence that Paks may mediate Cdc42 kinases to actin reorganization are far from settled. Several and Rac signaling to stress-activated kinases such as Jnk and approaches have therefore been taken in an attempt to eluci- p38. The effects of Paks on these stress-kinase cascades are date the precise role of Cdc42/Rac effector proteins. or not robust, however, and there is as yet no evidence that Pak example, gene deletions have been used to analyze the role acts directly on any MEKKs. of Cdc42 effectors in budding yeast. In mammalian cells, loss-of-function studies of Cdc42 and Rac effectors are less In addition to their effects on MAP kinase cascades, Paks complete, and overexpression studies, using constitutively also play an important role in regulating cell morphology. active or dominant-negative mutants, have instead been the In budding yeast, the Pak Ste20p phosphorylates myosin I predominant methods of analysis. Several groups have also and is also associated with the scaffold proteins Ste5p and analyzed the signaling properties of Cdc42 and Rac mutants Bem1p, both of which interact with actin. One other Pak that selectively bind certain sets of effector proteins [8-10]. target of note in yeast is the guanine-nucleotide exchange In general, these kinds of studies have been more useful in factor (GE) Cdc24p, which is inactivated as a conse- excluding effectors from a given Cdc42/Rac function than in quence of phosphorylation. By phosphorylating Cdc24p, proving their involvement. the Pak homolog Cla4p provides a feedback inhibition to terminate Cdc42p action as the cell cycle progresses [16]. Another way to reduce this complexity to a more manage- Thus, Paks in budding yeast link a Rho family GTPase able level is to consider which small-GTPase effectors are (Cdc42p) to the cell polarity and morphology apparatus via shared among evolutionarily distant organisms.