Cdc42/Rac Interactive Binding Research Articles

Physcomitrium patens PpRIC, an ancestral CRIB-domain ROP effector, inhibits auxin-induced differentiation of apical initial cells.

RHO guanosine triphosphatases are important eukaryotic regulators of cell differentiation and behavior. Plant ROP (RHO of plant) family members activate specific, incompletely characterized downstream signaling. The structurally simple land plant Physcomitrium patens is missing homologs of key animal and flowering plant RHO effectors but contains a single CRIB (CDC42/RAC interactive binding)-domain-containing RIC (ROP-interacting CRIB-containing) protein (PpRIC). Protonemal P. patens filaments elongate based on regular division and PpROP-dependent tip growth of apical initial cells, which upon stimulation by the hormone auxin differentiate caulonemal characteristics. PpRIC interacts with active PpROP1, co-localizes with this protein at the plasma membrane at the tip of apical initial cells, and accumulates in the nucleus. Remarkably, PpRIC is not required for tip growth but is targeted to the nucleus to block caulonema differentiation downstream of auxin-controlled gene expression. These observations establish functions of PpRIC in mediating crosstalk between ROP and auxin signaling, which contributes to the maintenance of apical initial cell identity.

Mixed lineage kinase 3 requires a functional CRIB domain for regulation of blood pressure, cardiac hypertrophy, and left ventricular function.

Mixed lineage kinase 3 (MLK3) modulates blood pressure and left ventricular function, but the mechanisms governing these effects remain unclear. In the current study, we therefore investigated the role of the MLK3 Cdc42/Rac interactive binding (CRIB) domain in cardiovascular physiology. We examined baseline and left ventricular pressure overload responses in a MLK3 CRIB mutant (MLK3C/C) mouse, which harbors point mutations in the CRIB domain to disrupt MLK3 activation by Cdc42. Male and female MLK3C/C mice displayed increased invasively measured blood pressure compared with wild-type (MLK3+/+) littermate controls. MLK3C/C mice of both sexes also developed left and right ventricular hypertrophy but normal baseline LV function by echocardiography and invasive hemodynamics. In LV tissue from MLK3C/C mice, map3k11 mRNA, which encodes MLK3, and MLK3 protein were reduced by 74 ± 6% and 73 ± 7%, respectively. After 1-wk LV pressure overload with 25-gauge transaortic constriction (TAC), male MLK3C/C mice developed no differences in LV hypertrophy but displayed reduction in the LV systolic indices ejection fraction and dP/dt normalized to instantaneous pressure. JNK activation was also reduced in LV tissue of MLK3C/C TAC mice. TAC induced MLK3 translocation from cytosolic fraction to membrane fraction in LV tissue from MLK3+/+ but not MLK3C/C mice. These findings identify a role of the MLK3 CRIB domain in MLK3 regulation of basal blood pressure and cardiac morphology, and in promoting the compensatory LV response to pressure overload.NEW & NOTEWORTHY Here, we identified that the presence of two discrete point mutations within the Cdc42/Rac interaction and binding domain of the protein MLK3 recapitulates the effects of whole body MLK3 deletion on blood pressure, cardiac hypertrophy, and left ventricular compensation after pressure overload. These findings implicate the CRIB domain, and thus MLK3 activation by this domain, as critical for maintenance of cardiovascular homeostasis.

Cdc42/Rac Interactive Binding Containing Effector Proteins in Unicellular Protozoans With Reference to Human Host: Locks of the Rho Signaling.

Small GTPases are the key to actin cytoskeleton signaling, which opens the lock of effector proteins to forward the signal downstream in several cellular pathways. Actin cytoskeleton assembly is associated with cell polarity, adhesion, movement and other functions in eukaryotic cells. Rho proteins, specifically Cdc42 and Rac, are the primary regulators of actin cytoskeleton dynamics in higher and lower eukaryotes. Effector proteins, present in an inactive state gets activated after binding to the GTP bound Cdc42/Rac to relay a signal downstream. Cdc42/Rac interactive binding (CRIB) motif is an essential conserved sequence found in effector proteins to interact with Cdc42 or Rac. A diverse range of Cdc42/Rac and their effector proteins have evolved from lower to higher eukaryotes. The present study has identified and further classified CRIB containing effector proteins in lower eukaryotes, focusing on parasitic protozoans causing neglected tropical diseases and taking human proteins as a reference point to the highest evolved organism in the evolutionary trait. Lower eukaryotes’ CRIB containing proteins fall into conventional effector molecules, PAKs (p21 activated kinase), Wiskoit-Aldrich Syndrome proteins family, and some have unique domain combinations unlike any known proteins. We also highlight the correlation between the effector protein isoforms and their selective specificity for Cdc42 or Rac proteins during evolution. Here, we report CRIB containing effector proteins; ten in Dictyostelium and Entamoeba, fourteen in Acanthamoeba, one in Trypanosoma and Giardia. CRIB containing effector proteins that have been studied so far in humans are potential candidates for drug targets in cancer, neurological disorders, and others. Conventional CRIB containing proteins from protozoan parasites remain largely elusive and our data provides their identification and classification for further in-depth functional validations. The tropical diseases caused by protozoan parasites lack combinatorial drug targets as effective paradigms. Targeting signaling mechanisms operative in these pathogens can provide greater molecules in combatting their infections.

Bond swapping from a charge cloud allows flexible coordination of upstream signals through WASP: Multiple regulatory roles for the WASP basic region

Wiskott–Aldrich syndrome protein (WASP) activates the actin-related protein 2/3 homolog (Arp2/3) complex and regulates actin polymerization in a physiological setting. Cell division cycle 42 (Cdc42) is a key activator of WASP, which binds Cdc42 through a Cdc42/Rac-interactive binding (CRIB)-containing region that defines a subset of Cdc42 effectors. Here, using site-directed mutagenesis and binding affinity determination and kinetic assays, we report the results of an investigation into the energetic contributions of individual WASP residues to both the Cdc42–WASP binding interface and the kinetics of complex formation. Our results support the previously proposed dock-and-coalesce binding mechanism, initiated by electrostatic steering driven by WASP's basic region and followed by a coalescence phase likely driven by the conserved CRIB motif. The WASP basic region, however, appears also to play a role in the final complex, as its mutation affected both on- and off-rates, suggesting a more comprehensive physiological role for this region centered on the C-terminal triad of positive residues. These results highlight the expanding roles of the basic region in WASP and other CRIB-containing effector proteins in regulating complex cellular processes and coordinating multiple input signals. The data presented improve our understanding of the Cdc42–WASP interface and also add to the body of information available for Cdc42–effector complex formation, therapeutic targeting of which has promise for Ras-driven cancers. Our findings suggest that combining high-affinity peptide-binding sequences with short electrostatic steering sequences could increase the efficacy of peptidomimetic candidates designed to interfere with Cdc42 signaling in cancer.

CRIB effector disorder: exquisite function from chaos.

The CRIB (Cdc42/Rac interactive binding) family of small G-protein effectors contain significant regions with intrinsic disorder. The G-protein-binding regions are contained within these intrinsically disordered regions. Most CRIB proteins also contain stretches of basic residues associated with their G-protein-binding regions. The basic region (BR) and G-protein-binding region together allow the CRIB effectors to bind to their cognate G-protein via a dock- and coalesce-binding mechanism. The BRs of these proteins take on multiple roles: steering G-protein binding, interacting with elements of the membrane and regulating intramolecular regulatory interactions. The ability of these regions of the CRIBs to undergo multivalent interactions and mediate charge neutralizations equips them with all the properties required to drive liquid-liquid phase separation and therefore to initiate and drive signalosome formation. It is only recently that the structural plasticity in these proteins is being appreciated as the driving force for these vital cellular processes.

CDC42 binds PAK4 via an extended GTPase-effector interface

The p21-activated kinase (PAK) group of serine/threonine kinases are downstream effectors of RHO GTPases and play important roles in regulation of the actin cytoskeleton, cell growth, survival, polarity, and development. Here we probe the interaction of the type II PAK, PAK4, with RHO GTPases. Using solution scattering we find that the full-length PAK4 heterodimer with CDC42 adopts primarily a compact organization. X-ray crystallography reveals the molecular nature of the interaction between PAK4 and CDC42 and shows that in addition to the canonical PAK4 CDC42/RAC interactive binding (CRIB) domain binding to CDC42 there are unexpected contacts involving the PAK4 kinase C-lobe, CDC42, and the PAK4 polybasic region. These additional interactions modulate kinase activity and increase the binding affinity of CDC42 for full-length PAK4 compared with the CRIB domain alone. We therefore show that the interaction of CDC42 with PAK4 can influence kinase activity in a previously unappreciated manner.

A dock and coalesce mechanism driven by hydrophobic interactions governs Cdc42 binding with its effector protein ACK

Cdc42 is a Rho-family small G protein that has been widely studied for its role in controlling the actin cytoskeleton and plays a part in several potentially oncogenic signaling networks. Similar to most other small G proteins, Cdc42 binds to many downstream effector proteins to elicit its cellular effects. These effector proteins all engage the same face of Cdc42, the conformation of which is governed by the activation state of the G protein. Previously, the importance of individual residues in conferring binding affinity has been explored for residues within Cdc42 for three of its Cdc42/Rac interactive binding (CRIB) effectors, activated Cdc42 kinase (ACK), p21-activated kinase (PAK), and Wiskott–Aldrich syndrome protein (WASP). Here, in a complementary study, we have used our structure of Cdc42 bound to ACK via an intrinsically disordered ACK region to guide an analysis of the Cdc42 interface on ACK, creating a panel of mutant proteins with which we can now describe the complete energetic landscape of the Cdc42-binding site on ACK. Our data suggest that the binding affinity of ACK relies on several conserved residues that are critical for stabilizing the quaternary structure. These residues are centered on the CRIB region, with the complete binding region anchored at each end by hydrophobic interactions. These findings suggest that ACK adopts a dock and coalesce binding mechanism with Cdc42. In contrast to other CRIB-family effectors and indeed other intrinsically disordered proteins, hydrophobic residues likely drive Cdc42–ACK binding.

PAK6 targets to cell-cell adhesions through its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape.

The six serine/threonine kinases in the p21-activated kinase (PAK) family are important regulators of cell adhesion, motility and survival. PAK6, which is overexpressed in prostate cancer, was recently reported to localize to cell-cell adhesions and to drive epithelial cell colony escape. Here we report that PAK6 targeting to cell-cell adhesions occurs through its N-terminus, requiring both its Cdc42/Rac interactive binding (CRIB) domain and an adjacent polybasic region for maximal targeting efficiency. We find PAK6 localization to cell-cell adhesions is Cdc42-dependent, as Cdc42 knockdown inhibits PAK6 targeting to cell-cell adhesions. We further find the ability of PAK6 to drive epithelial cell colony escape requires kinase activity and is disrupted by mutations that perturb PAK6 cell-cell adhesion targeting. Finally, we demonstrate that all type II PAKs (PAK4, PAK5 and PAK6) target to cell-cell adhesions, albeit to differing extents, but PAK1 (a type I PAK) does not. Notably, the ability of a PAK isoform to drive epithelial colony escape correlates with its targeting to cell-cell adhesions. We conclude that PAKs have a broader role in the regulation of cell-cell adhesions than previously appreciated.

CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa.

Cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. Asexual spores (conidia) of the mold Neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (CATs), which exhibit negative and positive chemotropism, respectively. We provide the first evidence that shared and separate functions of the Rho-type GTPases CDC-42 and RAC-1 regulate these opposite chemotropisms. We demonstrate that RAC-1 is essential for CAT formation and cell fusion, whereas CDC-42 is necessary and sufficient for normal germ tube development. Cdc42-Rac-interactive-binding (CRIB) reporters were constructed to exclusively label locally activated GTP-bound GTPases. Time course analyses showed that repositioning of these activated GTPase clusters within germ tube and CAT tip apices controls directional growth in the absence of a tip-localized vesicle supply center (Spitzenkörper). We propose a model in which the local assembly of a plasma-membrane-associated GTPase-PAK-MAPK signaling platform regulates chemoattractant perception and secretion in order to synchronize oscillatory cell-cell communication and directional CAT tip growth.

STUDY ON THE BIOLOGICAL FUNCTIONS OF P21-ACTIVATED KINASE 5

ObjectivesPAK5 is a member of P21-activated kinase (PAK) family, which characterised by a highly conserved amino-terminal Cdc42/Rac interactive binding (CRIB) domain and a carboxyl terminal kinase domain. However, the role...

A Conformational Switch in the CRIB-PDZ Module of Par-6

Here, we report a novel mechanism of PDZ (PSD-95/Dlg/ZO-1) domain regulation that distortsa conserved element of PDZ ligand recognition. The polarity regulator Par-6 assembles a conserved multiprotein complex and is directly modulated by theRho GTPase Cdc42. Cdc42 binds the adjacent Cdc42/Rac interactive binding (CRIB) and PDZ domains of Par-6, increasing C-terminal ligand binding affinity by 10-fold. By solving structures of the isolated PDZ domain and a disulfide-stabilized CRIB-PDZ, we detected a conformational switch that controls affinity by altering the configuration of the conserved "GLGF" loop. As a result, lysine 165 is displaced from the PDZ core by an adjacent hydrophobic residue, disrupting coordination of the PDZ ligand-binding cleft. Stabilization of the CRIB:PDZ interface restores K165 to its canonical location in the binding pocket. We conclude that a unique "dipeptide switch" in the Par-6 PDZ transmits a signal for allosteric activation to the ligand-binding pocket.

Dimeric Plant RhoGAPs Are Regulated by Its CRIB Effector Motif to Stimulate a Sequential GTP Hydrolysis

RopGAPs are GTPase-activating proteins (GAPs) for plant Rho proteins (ROPs). The largest RopGAP family is characterized by the plant-specific combination of a classical RhoGAP domain and a Cdc42/Rac interactive binding (CRIB) motif, which, in animal and fungi, has never been found in GAPs but in effectors for Cdc42 and Rac1. Very little is known about the molecular mechanism of the RopGAP activity including the regulatory role of the CRIB motif. Previously, we have shown that they are dimeric and form a 2:2 complex with ROPs. Here, we analyze the kinetics of the GAP-mediated GTP hydrolysis of ROPs using wild-type and mutant RopGAP2 from Arabidopsis thaliana. For an efficient GAP activity, RopGAP2 requires both the catalytic Arg159 in its GAP domain indicating a similar catalytic machinery as in animal RhoGAPs and the CRIB motif, which mediates high affinity and specificity in binding. The dimeric RopGAP2 is unique in that it stimulates ROP·GTP hydrolysis in a sequential manner with a 10-fold difference between the hydrolysis rates of the two active sites. Using particular CRIB point and deletion mutants lead us to conclude that the sequential mechanism is likely due to steric hindrance induced by the Arg fingers and/or the CRIB motifs after binding of two ROP molecules.

Rho GTPases and their role in organizing the actin cytoskeleton

Cells receive extracellular stimuli in various ways: in the form of soluble molecules (growth factors, cytokines and hormones) that interact with cell-surface receptors; from adhesive interactions with the extracellular matrix; and from cell–cell adhesions. These stimuli act to generate changes in

The unique plant RhoGAPs are dimeric and contain a CRIB motif required for affinity and specificity towards cognate small G proteins

Plant Rho proteins (ROPs) are inactivated by specific GTPase activating proteins, called RopGAPs. Many of these comprise the exclusive combination of a classic, catalytic Arg-containing RhoGAP domain, and a Cdc42/ Rac interactive binding (CRIB) motif which in animal and fungi has been identified in effectors for Cdc42 and Rac1, but never in any GAP protein. Both elements are required for an efficient RopGAP activity. Here, we analyzed the effect of the CRIB motif on the complex formation and the binding reaction with plant and human Rho proteins by using kinetic and equilibrium methods. We show that RopGAP2 from Arabidopsis thaliana dimerizes via its GAP domain and forms a 2:2 complex with ROP. The CRIB effector motif mediates high affinity and specificity in binding. The catalytic Arg in the context of the CRIB motif is inhibitory for binding. The unusually slow association and dissociation reactions suggest a major conformational change whereby the CRIB motif functions as a lid for binding and/or release of ROP. We propose a two-site interaction model where ROP binds to the CRIB motif as described for the human CRIB effectors and to the catalytic GAP domain as described for animal RhoGAPs.

Rho GTPase, Rac1, regulates Skp2 levels, vascular smooth muscle cell proliferation, and intima formation in vitro and in vivo

Vascular smooth muscle cell (VSMC) proliferation contributes to intima formation after angioplasty or venous by-pass grafting, and during atherosclerosis. VSMC proliferation requires degradation of p27(Kip1) promoted by S-phase kinase-associated protein-2 (Skp2), an F-box protein component of the Skp-Cullin-F-box(Skp2) ubiquitin-ligase. We investigated the role of Rac(1) in the regulation of Skp2 in rat VSMC. Rat carotid balloon injury increased Rac(1) activity. Rho GTPase inhibition with Clostridium difficile Toxin B or specific Rac(1) inhibition with adenovirus-mediated expression of dominant-negative Rac(1) reduced Skp2 levels, and VSMC proliferation in vitro and intima formation in vivo following carotid balloon injury. Inhibition of Skp2 expression and proliferation by dominant-negative Rac(1) was reversed by exogenous Skp2. Elevation of endogenous adenosine 3',5'-cyclic monophosphate (cAMP) with forskolin-inhibited Rac(1) activity, reduced Skp2, increased p27(Kip1) and inhibited VSMC proliferation, effects that were reversed by constitutively active Rac(1). These effects were independent of Rac(1) Cdc42/Rac interactive binding (CRIB)-domain effector proteins but associated with Rac(1)-dependent actin polymerization. Rac(1) activity regulates VSMC proliferation by controlling Skp2 levels. Activation of Rac(1) induced by balloon injury in vivo increases Skp2 levels, which promotes VSMC proliferation and intima formation. Inhibition of this novel pathway underlies the negative effects of cAMP on VSMC proliferation.

Barley RIC171 interacts with RACBin plantaand supports entry of the powdery mildew fungus

RHO-like GTPases of plants (ROPs, also called RACs) are involved in plant development and interaction with the environment. The barley ROP protein RACB is involved in susceptibility to the fungal pathogen Blumeria graminis f.sp. hordei (Bgh). By screening barley sequence databases for potential protein interactors of plant RHO-like proteins, we identified a ROP-interactive CRIB (CDC42/RAC interactive binding) motif containing protein of 171 amino acids (RIC171). The protein interacted with constitutively activated RACB in a targeted yeast two-hybrid assay. By use of split yellow fluorescing protein fusions, we demonstrated that RIC171 interacts with constitutively activated (CA) RACB-G15V but not with dominant negative RACB-T20N in planta. Transient overexpression of RIC171, similar to overexpression of CA RACB-G15V, rendered epidermal cells more susceptible to penetration by Bgh. In contrast, expression of a 46-amino-acid RIC171-CRIB peptide, which was sufficient to interact with CA RACB-G15V, had a dominant negative effect and reduced susceptibility to Bgh. A red fluorescing DsRED-RIC171 fusion protein colocalized with green fluorescing GFP-RACB-G15V at the cell periphery. Coexpression with CA RACB-G15V but not with RACB-T20N increased peripheral localization of DsRED-RIC171. Additionally, DsRED-RIC171 accumulated at sites of fungal attack, suggesting enhanced ROP activity at sites of attempted fungal penetration.

Pom1 DYRK Regulates Localization of the Rga4 GAP to Ensure Bipolar Activation of Cdc42 in Fission Yeast

In the fission yeast Schizosaccharomyces pombe, cell growth takes place exclusively at both ends of the cylindrical cell. During this highly polarized growth, microtubules are responsible for the placement of the cell-end marker proteins, the Tea1-Tea4/Wsh3 complex, which recruits the Pom1 DYRK-family protein kinase. Pom1 is required for proper positioning of growth sites, and the Deltapom1 mutation brings about monopolar cell growth. Pom1 kinase physically interacts with Rga4, which has a GAP (GTPase-activating protein) domain for Rho-family GTPase. Genetic and biochemical evidence indicates that Rga4 functions as GAP for the Cdc42 GTPase, an evolutionarily conserved regulator of F-actin. CRIB (Cdc42/Rac interactive binding)-GFP microscopy has revealed that GTP-bound, active Cdc42 is concentrated to growing cell ends accompanied by developed F-actin structures, where the Rga4 GAP is excluded. The monopolar Deltapom1 mutant fails to eliminate Rga4 from the nongrowing cell end, resulting in monopolar distribution of GTP-Cdc42 to the growing cell end. However, mutational inactivation of Rga4 allows Cdc42 to be active at both ends of Deltapom1 cells, suggesting that mislocalization of Rga4 in the Deltapom1 mutant contributes to its monopolar phenotype. Pom1 kinase recruited to cell ends by the Tea1-Tea4/Wsh3 complex is essential for proper localization of a GAP for Cdc42, Rga4, which ensures bipolar localization of GTP-bound, active Cdc42. Because of the established role of Cdc42 in F-actin formation, these observations provide a new insight into how the microtubule system achieves localized formation of F-actin to generate cell polarity.

Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP(2)-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase-dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein-protein and protein-membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex.

Cholesterol-sensitive Cdc42 activation regulates actin polymerization for endocytosis via the GEEC pathway.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present at the surface of living cells in cholesterol dependent nanoscale clusters. These clusters appear to act as sorting signals for the selective endocytosis of GPI-APs via a Cdc42-regulated, dynamin and clathrin-independent pinocytic pathway called the GPI-AP-enriched early endosomal compartments (GEECs) pathway. Here we show that endocytosis via the GEECs pathway is inhibited by mild depletion of cholesterol, perturbation of actin polymerization or overexpression of the Cdc42/Rac-interactive-binding (CRIB) motif of neural Wiskott-Aldrich syndrome protein (N-WASP). Consistent with the involvement of Cdc42-based actin nanomachinery, nascent endocytic vesicles containing cargo for the GEEC pathway co-localize with fluorescent protein-tagged isoforms of Cdc42, CRIB domain, N-WASP and actin; high-resolution electron microscopy on plasma membrane sheets reveals Cdc42-labelled regions rich in green fluorescent protein-GPI. Using total internal reflection fluorescence microscopy at the single-molecule scale, we find that mild cholesterol depletion alters the dynamics of actin polymerization at the cell surface by inhibiting Cdc42 activation and consequently its stabilization at the cell surface. These results suggest that endocytosis into GEECs occurs through a cholesterol-sensitive, Cdc42-based recruitment of the actin polymerization machinery.

Tobacco RhoGTPase ACTIVATING PROTEIN1 Spatially Restricts Signaling of RAC/Rop to the Apex of Pollen Tubes

Regulation by Rho-type small GTPases, such as RAC5, is important for the maintenance of polarity in tobacco (Nicotiana tabacum) pollen tubes. We previously showed that RhoGDI2 is necessary for RAC5 localization. Here, we describe the GTPase activating protein RhoGAP1 that controls the area of RAC5 activity. RhoGAP1 N-terminal and CRIB (for Cdc42/Rac-interactive binding) domains are both necessary for targeting yellow fluorescent protein-RhoGAP1 fusions to the plasma membrane close to, but not in, pollen tube apices. We propose that this localization restricts apical Rho-type GTPase activity from spreading toward the flanks, which ensures the maintenance of RAC signaling at the apex. The CRIB domain is not required but enhances in vitro RhoGAP1 activity toward the pollen tube-specific-RAC5. A mutation reducing GAP activity of RhoGAP1 leads to ballooning pollen tubes resembling those overexpressing RAC5. To ascertain the specific targeting mechanism of RhoGAP1, we isolated a 14-3-3 protein interacting with RhoGAP1. When overexpressed with RhoGAP1, it counteracts the growth-retarding effect of RhoGAP1 overexpression and attenuates RhoGAP1 membrane localization but, overexpressed alone, induces only small architectural changes. We propose that inactivation of RAC5 by the subapically localized RhoGAP1, together with dynamic relocalization of inactivated RAC5 from flanks to tip by RhoGDI2, leads to spatial restriction of RAC5 to pollen tube apices, thereby sustaining polar growth.