Structure of Cdc42 in a complex with the GTPase-binding domain of the cell polarity protein,Par6

Cdc42 is a small GTPase that is required for cell polarity establishment in eukaryotes as diverse as budding yeast and mammals. Par6 is also implicated in metazoan cell polarity establishment and asymmetric cell divisions. Cdc42.GTP interacts with proteins that contain a conserved sequence called a CRIB motif. Uniquely, Par6 possesses a semi-CRIB motif that is not sufficient for binding to Cdc42. An adjacent PDZ domain is also necessary and is required for biological effects of Par6. Here we report the crystal structure of a complex between Cdc42 and the Par6 GTPase-binding domain. The semi-CRIB motif forms a beta-strand that inserts between the four strands of Cdc42 and the three strands of the PDZ domain to form a continuous eight-stranded sheet. Cdc42 induces a conformational change in Par6, detectable by fluorescence resonance energy transfer spectroscopy. Nuclear magnetic resonance studies indicate that the semi-CRIB motif of Par6 is at least partially structured by the PDZ domain. The structure highlights a novel role for a PDZ domain as a structural scaffold.     

A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation

BACKGROUND: Rac and Cdc42 are members of the Rho family of small GTPases. They modulate cell growth and polarity, and contribute to oncogenic transformation by Ras. The molecular mechanisms underlying these functions remain elusive, however. RESULTS: We have identified a novel effector of Rac and Cdc42, hPar-6, which is the human homolog of a cell-polarity determinant in Caenorhabditis elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKCzeta, and forms a stable ternary complex with Rac1 or Cdc42 and PKCzeta. This association results in stimulation of PKCzeta kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKCzeta-dependent pathway distinct from the pathway mediated by Raf. CONCLUSIONS: These findings indicate that Rac/Cdc42 can regulate cell growth through Par-6 and PKCzeta, and suggest that deregulation of cell-polarity signaling can lead to cell transformation.     

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.     

Cdc42 regulates the Par-6 PDZ domain through an allosteric CRIB-PDZ transition

Regulation of protein interaction domains is required for cellular signaling dynamics. Here, we show that the PDZ protein interaction domain from the cell polarity protein Par-6 is regulated by the Rho GTPase Cdc42. Cdc42 binds to a CRIB domain adjacent to the PDZ domain, increasing the affinity of the Par-6 PDZ for its carboxy-terminal ligand by approximately 13-fold. Par-6 PDZ regulation is required for function as mutational disruption of Cdc42-Par-6 PDZ coupling leads to inactivation of Par-6 in polarized MDCK epithelial cells. Structural analysis reveals that the free PDZ domain has several deviations from the canonical PDZ conformation that account for its low ligand affinity. Regulation results from a Cdc42-induced conformational transition in the CRIB-PDZ module that causes the PDZ to assume a canonical, high-affinity PDZ conformation. The coupled CRIB and PDZ architecture of Par-6 reveals how simple binding domains can be combined to yield complex regulation.     

Biochemical characterization of the Ras-related GTPases Rit and Rin.

We report the biochemical characterization of Rit and Rin, two members of the Ras superfamily identified by expression cloning. Recombinant Rit and Rin bind GTP and exhibit intrinsic GTPase activity. Conversion of Gln to Leu at position 79 (for Rit) or 78 (for Rin) (equivalent to position 61 in Ras) resulted in a complete loss of GTPase activity. Surprisingly, significant differences were found when the guanine nucleotide dissociation constants of Rit and Rin were compared with the majority of Ras-related GTPases. Both proteins display higher k(off) values for GTP than GDP in the presence of 10 mM Mg(2+). These GTP dissociation rates are 5- to 10-fold faster than most Ras-like GTPases. Despite these unique biochemical properties, our data support the notion that both Rit and Rin function as nucleotide-dependent molecular switches. To begin to address whether these proteins act as regulators of distinct signaling pathways, we examined their interaction with a series of known Ras-binding proteins by yeast two-hybrid analysis. Although Rit, Rin, and Ras have highly related effector domain sequences, Rit and Rin were found to interact with the known Ras binding proteins RalGDS, Rlf, and AF-6/Canoe but not with the Raf kinases, RIN1, or the p110 subunit of phosphatidylinositol 3-kinase. These interactions were GTP and effector domain dependent and suggest that RalGDS, Rlf, and AF-6 are Rit and Rin effectors. Their biochemical properties and interaction with a subset of known Ras effector proteins suggest that Rit and Rin may play important roles in the regulation of signaling pathways and cellular processes distinct from those controlled by Ras.     

PAR3beta,a novel homologue of the cell polarity protein PAR3, localizes to tight junctions

The cell polarity protein PAR3, conserved from the nematode to the vertebrate, forms a complex with PAR6 and atypical protein kinase C (aPKC), and the protein complex occurs at the tight junctions in mammalian epithelial cells. Here we have cloned human cDNA for a novel PAR3 homologue, designated PAR3beta, whose messages are present in a variety of tissues and most abundantly expressed in the adult and fetal kidneys. The encoded protein of 1,205 amino acids contains a region homologous to the aPKC-binding domain of PAR3alpha, another human homologue previously identified, and three PDZ domains; the first PDZ domain of PAR3alpha is considered to interact with PAR6. Unexpectedly, in contrast to other PAR3s found in various species, PAR3beta is incapable of binding to any isotypes of PAR6 or aPKC. Nevertheless PAR3beta, expressed intrinsically or extrinsically, localizes to the tight junctions, indicating that the localization does not require the ternary complex formation.     

Internal recognition through PDZ domain plasticity in the Par-6-Pals1 complex

PDZ protein interaction domains are typically selective for C-terminal ligands, but non-C-terminal, 'internal' ligands have also been identified. The PDZ domain from the cell polarity protein Par-6 binds C-terminal ligands and an internal sequence from the protein Pals1/Stardust. The structure of the Pals1-Par-6 PDZ complex reveals that the PDZ ligand-binding site is deformed to allow for internal binding. Whereas binding of the Rho GTPase Cdc42 to a CRIB domain adjacent to the Par-6 PDZ regulates binding of C-terminal ligands, the conformational change that occurs upon binding of Pals1 renders its binding independent of Cdc42. These results suggest a mechanism by which the requirement for a C terminus can be readily bypassed by PDZ ligands and reveal a complex set of cooperative and competitive interactions in Par-6 that are likely to be important for cell polarity regulation.     

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 distorts?a conserved element of PDZ ligand recognition. The polarity regulator Par-6 assembles a conserved multiprotein complex and is directly modulated by the?Rho 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.     

Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal-binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin-CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.     

A novel function for the PAR complex in subcellular morphogenesis of tracheal terminal cells in Drosophila melanogaster

The processes that generate cellular morphology are not well understood. To investigate this problem, we use Drosophila melanogaster tracheal terminal cells, which undergo two distinct morphogenetic processes: subcellular branching morphogenesis and subcellular apical lumen formation. Here we show these processes are regulated by components of the PAR-polarity complex. This complex, composed of the proteins Par-6, Bazooka (Par-3), aPKC, and Cdc42, is best known for roles in asymmetric cell division and apical/basal polarity. We find Par-6, Bazooka, and aPKC, as well as known interactions between them, are required for subcellular branch initiation, but not for branch outgrowth. By analysis of single and double mutants, and isolation of two novel alleles of Par-6, one of which specifically truncates the Par-6 PDZ domain, we conclude that dynamic interactions between apical PAR-complex members control the branching pattern of terminal cells. These data suggest that canonical apical PAR-complex activity is required for subcellular branching morphogenesis. In addition, we find the PAR proteins are downstream of the FGF pathway that controls terminal cell branching. In contrast, we find that while Par-6 and aPKC are both required for subcellular lumen formation, neither Bazooka nor a direct interaction between Par-6 and aPKC is needed for this process. Thus a novel, noncanonical role for the polarity proteins Par-6 and aPKC is used in formation of this subcellular apical compartment. Our results demonstrate that proteins from the PAR complex can be deployed independently within a single cell to control two different morphogenetic processes.     

Assembly of epithelial tight junctions is negatively regulated by Par6

Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts-claudins, occludin, and junctional adhesion molecule (JAM)-plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Here, we show that Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca(2+) depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs, but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells.     

Direct interaction of two polarity complexes implicated in epithelial tight junction assembly

Tight junctions help establish polarity in mammalian epithelia by forming a physical barrier that separates apical and basolateral membranes. Two evolutionarily conserved multi-protein complexes, Crumbs (Crb)-PALS1 (Stardust)-PATJ (DiscsLost) and Cdc42-Par6-Par3-atypical protein kinase C (aPKC), have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. Here we identify a biochemical and functional link between these two complexes that is mediated by Par6 and PALS1 (proteins associated with Lin7). The interaction between Par6 and PALS1 is direct, requires the amino terminus of PALS1 and the PDZ domain of Par6, and is regulated by Cdc42-GTP. The transmembrane protein Crb can recruit wild-type Par6, but not Par6 with a mutated PDZ domain, to the cell surface. Expression of dominant-negative PALS1-associated tight junction protein (PATJ) in MDCK cells results in mis-localization of PALS1, members of the Par3-Par6-aPKC complex and the tight junction marker, ZO-1. Similarly, overexpression of Par6 in MDCK cells inhibits localization of PALS1 to the tight junction. Our data highlight a previously unrecognized link between protein complexes that are essential for epithelial polarity and formation of tight junctions.     

The signaling adaptor gab1 regulates cell polarity by acting as a par protein scaffold

Cell polarity plays a key role in development and is disrupted in tumors, yet the molecules and mechanisms that regulate polarity remain poorly defined. We found that the scaffolding adaptor GAB1 interacts with two polarity proteins, PAR1 and PAR3. GAB1 binds PAR1 and enhances its kinase activity. GAB1 brings PAR1 and PAR3 into a transient complex, stimulating PAR3 phosphorylation by PAR1. GAB1 and PAR6 bind the PAR3 PDZ1 domain and thereby compete for PAR3 binding. Consequently, GAB1 depletion causes PAR3 hypophosphorylation and increases PAR3/PAR6 complex formation, resulting in accelerated and enhanced tight junction formation, increased transepithelial resistance, and lateral domain shortening. Conversely, GAB1 overexpression, in a PAR1/PAR3-dependent manner, disrupts epithelial apical-basal polarity, promotes multilumen cyst formation, and enhances growth factor-induced epithelial cell scattering. Our results identify GAB1 as a negative regulator of epithelial?cell polarity that functions as a scaffold for modulating PAR protein complexes on the lateral membrane.     

Investigation of the Interaction between Cdc42 and Its Effector TOCA1: HANDOVER OF Cdc42 TO THE ACTIN REGULATOR N-WASP IS FACILITATED BY DIFFERENTIAL BINDING AFFINITIES*

Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery.     

Compartmentalized Ras proteins transform NIH 3T3 cells with different efficiencies

Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively.     

Nerve growth factor-dependent activation of the small GTPase Rin

The Rit and Rin proteins comprise a distinct and evolutionarily conserved subfamily of Ras-related small GTPases. Although we have defined a role for Rit-mediated signal transduction in the regulation of cell proliferation and transformation, the function of Rin remains largely unknown. Because we demonstrate that Rin is developmentally regulated and expressed in adult neurons, we examined its role in neuronal signaling. In this study, we show that stimulation of PC6 cells with either epidermal growth factor or nerve growth factor (NGF) results in rapid activation of Rin. This activation correlates with the onset of Ras activation, and dominant-negative Ras completely inhibits Rin activation induced by NGF. Further examination of Ras-mediated Rin activation suggests that this process is dependent upon neuronally expressed regulatory factors. Expression of mutationally activated H-Ras fails to activate Rin in non-neuronal cells, but results in potent stimulation of Rin-GTP levels in a variety of neuronal cell lines. Furthermore, although constitutively activated Rin does not induce neurite outgrowth on its own, both NGF-induced and oncogenic Ras-induced neurite outgrowth were inhibited by the expression of dominant-negative Rin. Together, these studies indicate that Rin activation is a direct downstream effect of growth factor-dependent signaling in neuronal cells and suggest that Rin may function to transduce signals within the mature nervous system.     

Similar requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in diverse cell types

During animal development, a complex of Par3, Par6 and atypical protein kinase C (aPKC) plays a central role in cell polarisation. The small G protein Cdc42 also functions in cell polarity and has been shown in some cases to act by regulating the Par3 complex. However, it is not yet known whether Cdc42 and the Par3 complex widely function together in development or whether they have independent functions. For example, many studies have implicated Cdc42 in cell migrations, but the Par3 complex has only been little studied, with conflicting results. Here we examine the requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in a range of different developmental events. We found similar requirements in all tissues examined, including polarised growth of vulval precursors and seam cells, migrations of neuroblasts and axons, and the development of the somatic gonad. We also propose a novel role for primordial germ cells in mediating coalescence of the Caenorhabditis elegans gonad. These results indicate that CDC-42 and the PAR-3/PAR-6/aPKC complex function together in diverse cell types.     

SPECs, small binding proteins for Cdc42

The Rho GTPase, Cdc42, regulates a wide variety of cellular activities including actin polymerization, focal complex assembly, and kinase signaling. We have identified a new family of very small Cdc42-binding proteins, designated SPECs (for Small Protein Effector of Cdc42), that modulates these regulatory activities. The two human members, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acids, respectively. Both contain a conserved N-terminal region and a centrally located CRIB (Cdc42/Rac Interactive Binding) domain. Using a yeast two-hybrid system, we found that both SPECs interact strongly with Cdc42, weakly with Rac1, and not at all with RhoA. Transfection analysis revealed that SPEC1 inhibited Cdc42-induced c-Jun N-terminal kinase (JNK) activation in COS1 cells in a manner that required an intact CRIB domain. Immunofluorescence experiments in NIH-3T3 fibroblasts demonstrated that both SPEC1 and SPEC2 showed a cortical localization and induced the formation of cell surface membrane blebs, which was not dependent on Cdc42 activity. Cotransfection experiments demonstrated that SPEC1 altered Cdc42-induced cell shape changes both in COS1 cells and in NIH-3T3 fibroblasts and that this alteration required an intact CRIB domain. These results suggest that SPECs act as novel scaffold molecules to coordinate and/or mediate Cdc42 signaling activities.     

The Shank family of postsynaptic density proteins interacts with and promotes synaptic accumulation of the beta PIX guanine nucleotide exchange factor for Rac1 and Cdc42

The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and beta PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of beta PIX. Shank colocalizes with beta PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with beta PIX and beta PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of beta PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits beta PIX and PAK to spines for the regulation of postsynaptic structure.     

CDC-42 controls early cell polarity and spindle orientation in C. elegans.

BACKGROUND: Generation of asymmetry in the one-cell embryo of C. elegans establishes the anterior--posterior axis (A-P), and is necessary for the proper identity of early blastomeres. Conserved PAR proteins are asymmetrically distributed and are required for the generation of this early asymmetry. The small G protein Cdc42 is a key regulator of polarity in other systems, and recently it has been shown to interact with the mammalian homolog of PAR-6. The function of Cdc42 in C. elegans had not yet been investigated, however. RESULTS: Here, we show that C. elegans cdc-42 plays an essential role in the polarity of the one-cell embryo and the proper localization of PAR proteins. Inhibition of cdc-42 using RNA interference results in embryos with a phenotype that is nearly identical to par-3, par-6, and pkc-3 mutants, and asymmetric localization of these and other PAR proteins is lost. We further show that C. elegans CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs. CONCLUSIONS: Our results show that CDC-42 acts in concert with the PAR proteins to control the polarity of the C. elegans embryo, and provide evidence that the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit.