Historical Background and Taxonomy
Rac GTPases comprise one of the eight subfamilies of the Rho (Ras homology) GTPases family, itself a subgroup of the Ras superfamily of small G proteins (Burridge and Wennerberg 2004). They were first identified as a substrate for the bacterial C3-like transferases that block Rho by ADP-ribosylation (hence their name, Ras-related C3 botulinum toxin substrate 1–3), although the C3-like transferases act on Rac rather inefficiently. More effective are the large clostridial cytotoxins (with prototypes the Clostridium difficile toxin A and B) which glycosylate Rac at Thr35, inhibiting its functions by preventing effector coupling (Aktories et al. 2000). Rac GTPases are preferred targets for bacteria since they act as molecular switches in a multitude of signaling processes, regulating many fundamental cellular functions, including actin cytoskeleton, cell adhesion, motility and migration, vesicular transport pathways and cytokinesis, reactive oxygen species (ROS) production via NADPH oxidase, as well as cell proliferation and survival (Hall 1998).
The Rac GTPase subfamily consists of four members: Rac1, Rac2, Rac3, and RhoG. They all have high homology, with the first three sharing over 90% amino acid sequence identity between them (Heasman and Ridley 2008). Despite this sequence similarity, studies using knockout mice indicate that many of their functions are nonredundant (Wang and Zheng 2007). Homologues of Rac GTPases have been found in all eukaryotic cells studied, but are totally absent in prokaryotes. In mammalian tissues, Rac1 is ubiquitously expressed while expression of Rac2 is mostly restricted to hematopoietic cells. Rac3 is abundant in the brain but has also been identified in a variety of other tissues including tissues of the male and female reproductive system, gastrointestinal tract, and skin. RhoG (Ras homology growth-related) is expressed in a variety of organs but reaches a particularly high level in hematopoietic cells and lymph nodes. Deletion of the Rac1 gene in mouse germ line produces an early embryonic lethal phenotype and thus studies of Rac1 function have utilized tissue-specific conditional knockout. In contrast, Rac2, Rac3, and RhoG knockout mice are viable, fertile, and do not exhibit obvious developmental defects. Nevertheless, they do exhibit cell-type-specific functional defects. For example, the Rac3-null mice demonstrate neurological defects (Wang and Zheng 2007; Heasman and Ridley 2008).
Activation of Rac GTPases and Downstream Signaling
Rac GTPases bind to and activate the p21-activating kinases (PAK1, PAK2, PAK3), serine/threonine kinases, which drive cytoskeletal remodeling (lamellipodia and membrane ruffling), cell adhesion and proliferation, and gene transcription. While PAK can activate c-Jun NH2-terminal kinase (JNK), Rac1 activates JNK mostly independently of PAK (Westwick et al. 1997) and mainly through mixed lineage kinases (MLKs). Pathways downstream of PAK and MLK stimulate AP1-dependent gene expression. AP1 can upregulate the expression of genes that control cell cycle progression, such as cyclin D1 and c-myc, proteins which when overexpressed are associated with cell transformation and cancer (Bosco et al. 2009). A pathway connecting sequentially Rac1-PAK1-Raf-MEK1-ERK, as well as activation of the pro-survival Akt Ser/Thr kinase via PAK2 has also been reported (Wang et al. 2010). Through PAK, Rac GTPases phosphorylate and activate LIMK, which in turn phosphorylates and inhibits cofilin, an actin-filament-severing protein, hence inducing actin polymerization into lamellipodia and membrane ruffling. Additionally, Rac stimulates the Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE) complex, which in turn activates actin-related proteins 2/3 complex (Arp2/3) that nucleates unbranched actin filaments (Heasman and Ridley 2008). Rac regulation of cell contractility includes PAK-mediated phosphorylation of myosin light-chain kinase (MLCK) and hence its inactivation causes decreased phosphorylation of the myosin regulatory light chain (MRLC) and reduced actomyosin assembly and contraction (Bishop and Hall 2000). PAK can also inhibit the microtubule destabilizing activity of OP18/stathmin by phosphorylation. Other functions of Rac GTPases mediated by PAK are interactions with the myosin heavy chain, also leading to decreased actomyosin filaments, with filamin A to promote membrane ruffling, as well as with components of the paxillin-GIT/PKL-P1X complex to regulate cell adhesion and motility (Schwartz 2004). Independently of PAK, Rac binds to the actin-binding protein IQGAP (named GAP because of some homology with Ras GAP, but actually a Rac effector) which oligomerizes and cross-links F-actin in vitro and has been shown to arrange actin filaments into the cytokinetic contractile ring in yeast (Bishop and Hall 2000). Rac GTPases bind and activate phosphatidylinositol-4-phosphate 5-kinase (PIP5K) leading to production of phosphatidylinositol (4,5)-bisphosphate (PIP2) and activation of the ERM (ezrin, radixin, moesin) complex of proteins. ERM proteins have actin-binding domains as well as domains that bind to cytosolic domains of plasma membrane integral proteins and mediate the association of F-actin to plasma membrane (Schwartz 2004). Another mode of action of Rac1 on the cytoskeleton is through CLASPs (cytoplasmic linker-associated proteins). CLASPs are a family of microtubule-associated proteins that bind to growing microtubule plus ends (+TIPs) in the cell body. In the leading edge and lamellipodia, however, CLASPs dynamically attach to the entire microtubule lattice, a function regulated downstream of Rac1 through a pathway involving GSK3β inactivation as shown in migrating PtK1 epithelial cells (Wittmann and Waterman-Storer 2005).
Phosphatidylinositol 3-kinase (PI3K) is also a Rac effector with multiple actions. It stimulates WASP and Arp2/3, thus inducing actin polymerization and produces 3′-phosphorylated lipids that bind to and stimulate Rac GEFs, creating a positive feedback loop that maintains cell migration. In addition, it also activates Akt to support cell survival (Schwartz 2004; Bosco et al. 2009). Rac GTP also binds to the p67phox component of NADPH oxidase, activating the enzyme to produce superoxide. Superoxide and other reactive oxygen species (ROS) have multiple roles and effects on cells and tissues, including signaling and stimulation of NFκB-dependent gene expression (Schwartz 2004; Hordijk 2006).
RhoG participates in the regulation of the actin cytoskeleton by activating Rac1 through the recruitment of the ELMO/Dock180 GEF complex (Katoh and Negishi 2003). Through this, RhoG participates in functions such as phagocytosis of apoptotic cells, the trans-endothelial migration of leukocytes and even the axonal and dendritic differentiation of neurons (Schumacher and Franke 2013).
Rac GTPases in Hematopoiesis
In neutrophils, Rac1 regulates cell spreading and adhesion, while Rac2 regulates directed migration and superoxide production (Gu et al. 2003). Rac2-deficient mice exhibit a phagocyte immunodeficiency syndrome. Interestingly, after the description of this phenotype, the case of a patient with leukocytosis and neutrophilia but multiple, recurrent, life-threatening infections in infancy was described. A dramatic decrease of neutrophil infiltration (absence of pus) in areas of infections was noted. The neutrophils of the patient exhibited decreased chemotaxis and impaired superoxide generation in response to fMLP (N-formyl-methionyl-leucyl-phenylalanine), as well as reduced rolling on the L-selectin ligand GlyCAM-1, the latter a defect that had been observed in Rac2−/− mouse neutrophils. After LAD (leucocyte adhesion disorder) was ruled out with normal presence of CD11b, CD11c, and CD18, the patient was found to have a p.Asp57Asn (D57N) mutation of Rac2 (Williams et al. 2000). This is a highly conserved position in Rac GTPases as well as in the Ras superfamily as a whole, since it is located in the GTP-binding pocket of the GTPase. The mutation creates a dominant negative protein that is not only dysfunctional but also antagonizes Rac1 and Rac3 for GTP.
Studies in gene-targeted mice demonstrated that Rac1 and Rac2 play an overlapping but essential role in organizing the erythrocyte cytoskeleton. Mice with combined deficiency of Rac1 and Rac2 GTPases in their hematopoietic cells develop hemolytic anemia, as evidenced by concurrent reticulocytosis. Rac1−/−;Rac2−/− red blood cells exhibit a disorganized membrane cytoskeleton with increased actin-to-spectrin ratio, F-actin aggregates and meshwork gaps, irregular clamping of band 3, decreased content of the proteins adducin and dematin, and decreased cellular deformability (Kalfa et al. 2006). These mice develop successful stress erythropoiesis in the spleen, while homeostatic erythropoiesis in the bone marrow is significantly compromised, implying different signaling pathways for homeostatic and stress erythropoiesis (Kalfa et al. 2010). Rac GTPases were also shown to play a role in enucleation by using constitutively active and dominant negative mutants of Rac1 and Rac2; both inhibited enucleation in cultured mouse fetal liver erythroblasts indicating that either inhibition or excessive activation of Rac GTPases inhibits enucleation via disruption of the contractile actin ring in enucleating erythroblasts (Ji et al. 2008). More recent studies, utilizing the Rac1−/−;Rac2−/− mouse model, showed that upregulation of Rac3 can compensate for the loss of Rac1 and Rac2 in enucleation; however inhibition of all three Rac GTPases inhibits enucleation as it results in the disruption of the contractile actomyosin ring that is necessary for enucleation (Konstantinidis et al. 2012).
Combined Rac1 and Rac2 deficiency has also been shown to impair T and B cell development, proliferation, survival, adhesion, and migration, while Rac1 deficiency compromises platelet aggregation, lamellipodia formation, granule secretion, and clot retraction (Mulloy et al. 2010).
In platelets, Rac1 and RhoG are the main Rac GTPases expressed (Aslan and McCarty 2013). Genetic and pharmacological inhibition of Rac1 shows that Rac1 plays a role in platelet aggregation, lamellipodia formation, granule secretion, and PAK activation downstream of thrombin. RhoG−/− platelets have reduced granule secretion, and RhoG−/− mice have impaired thrombus formation, with reduced integrin-mediated platelet activation and aggregation, but a normal response to thrombin (Goggs et al. 2013).
Rac GTPases in Disease
Rac GTPases have been implicated in cellular transformation, oncogenesis, cancer invasiveness, and metastasis. Rac1 can be induced by oncogenes like Ras and collaborates with p53 loss of function to promote transformation in primary fibroblasts. Overexpression or increased activity of Rac1 has been found in breast, lung, and colon cancer (Bosco et al. 2009), while through the use of high-throughput exome sequencing techniques, mutations affecting proline 29 (P29) of Rac1, and at a lower frequency Rac2, were found in 5–10% of melanoma cases. Rac1 P29 mutation was also associated with head and neck cancers, as well as breast cancers (Alan and Lundquist 2013). Activated Rac3 was detected in the malignant precursor B-lymphoblasts in p190-BCR/ABL transgenic mice (Cho et al. 2005), while Rac1 and Rac2 gene targeting was found to significantly delay or abrogate disease development in a p210-BCR/ABL mouse model of chronic myelogenous leukemia (CML) (Thomas et al. 2007). These data suggest that targeting modulation of Rac GTPases activity may provide clinical benefit for patients with CML, Ph-positive ALL, or other cancers.
Since Rac1 is important in platelet activation, and thus initiation of thrombosis, it could also be considered as a therapeutic target against thrombotic events (Carrizzo et al. 2014). By being a component of NADPH oxidases, increased Rac1 activity may also contribute in diseases aggravated by oxidative damage by NADPH-mediated production of reactive oxygen species (ROS) as in endothelial dysfunction, myocardial disease, diabetes mellitus, and sickle cell disease (George et al. 2013; Carrizzo et al. 2014).
The Rac subfamily of Rho GTPases consists of four members: Rac1, Rac2, Rac3, and RhoG. Although they exhibit high sequence similarity, a great number of their functions are nonredundant. Via proteins that activate them (guanosine exchange factors, GEFs) or deactivate them (GTPase-activating proteins, GAPs), they receive signals from the cell surface, after soluble ligand-receptor binding, interaction with the extracellular matrix or mechanical stress on cell surface receptors and propagate them through the appropriate downstream signaling pathways. They regulate many fundamental cellular functions, including actin cytoskeleton organization, cell adhesion, motility and migration, vesicular transport pathways and cytokinesis, ROS production via NADPH oxidase, gene transcription, and cell proliferation and survival. They have been implicated in many physiological and pathological processes, including hematopoiesis and cancer, and their role continues to be investigated using gene-targeted mouse models.
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