CRIB Domain Research Articles

PAR-6 is required for junction formation but not apicobasal polarization inC. elegansembryonic epithelial cells

Epithelial cells perform important roles in the formation and function of organs and the genesis of many solid tumors. A distinguishing feature of epithelial cells is their apicobasal polarity and the presence of apical junctions that link cells together. The interacting proteins Par-6 (a PDZ and CRIB domain protein) and aPKC (an atypical protein kinase C) localize apically in fly and mammalian epithelial cells and are important for apicobasal polarity and junction formation. Caenorhabditis elegans PAR-6 and PKC-3/aPKC also localize apically in epithelial cells, but a role for these proteins in polarizing epithelial cells or forming junctions has not been described. Here, we use a targeted protein degradation strategy to remove both maternal and zygotic PAR-6 from C. elegans embryos before epithelial cells are born. We find that PKC-3 does not localize asymmetrically in epithelial cells lacking PAR-6, apical junctions are fragmented, and epithelial cells lose adhesion with one another. Surprisingly, junction proteins still localize apically, indicating that PAR-6 and asymmetric PKC-3 are not needed for epithelial cells to polarize. Thus, whereas the role of PAR-6 in junction formation appears to be widely conserved, PAR-6-independent mechanisms can be used to polarize epithelial cells.

IQGAP1 Stimulates Actin Assembly through the N-Wasp-Arp2/3 Pathway

IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, beta-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.

A Novel Cdc42-interacting Domain of the Yeast Polarity Establishment Protein Bem1: IMPLICATIONS FOR MODULATION OF MATING PHEROMONE SIGNALING

In Saccharomyces cerevisiae, the Rho-type small GTPase Cdc42 is activated by its guanine-nucleotide exchange factor Cdc24 to polarize the cell for budding and mating. A multidomain protein Bem1 interacts not only with Cdc42 but also with Cdc24 and the effectors of Cdc42, including the p21-activated kinase Ste20, to function as a scaffold for cell polarity establishment. Although Bem1 interacts with Cdc24 and Ste20 via its PB1 and the second SH3 domains (SH3b), respectively, it is unclear how Bem1 binds Cdc42. Here we show that a region comprising the SH3b and its C-terminal flanking segment termed CI (SH3b-CI) directly interacts with Cdc42. A dual-bait reverse two-hybrid approach revealed that the CI is critical to the interaction: N253D substitution in the CI abolishes the binding of the SH3b-CI to Cdc42 but not to the proline-rich region of Ste20, whereas W192K substitution in the SH3b has the opposite effect. Nevertheless, the SH3b-CI interacts with Ste20 proline-rich region and Cdc42 in a mutually exclusive manner. The N253D substitution renders cellular growth temperature-sensitive and suppresses mating. The W192K-induced mating defect is exacerbated by the N253D substitution and suppressed by increasing the dosage of Ste20 provided that the CI is intact. Intriguingly, Cdc42 can mediate an indirect interaction of the SH3b-CI to the CRIB domain of Ste20. These results suggest that the SH3b and the CI collaborate in tethering of Ste20 to Bem1 to ensure efficient mating pheromone signaling.

Activation of the nonreceptor protein tyrosine kinase Ack by multiple extracellular stimuli

Ack/Ack1 is a nonreceptor protein tyrosine kinase that comprises a tyrosine kinase core, an SH3 domain, a Cdc42-binding region, a Ralt homology region, and a proline-rich region. Here we describe a detailed characterization of the Ack protein as well as the chromosomal localization of human Ack (chromosome 3q29) and the primary structure of murine Ack. We demonstrate that Ack is ubiquitously expressed, with highest expression seen in thymus, spleen, and brain. Activation of integrins by cell adhesion on fibronectin leads to strong tyrosine phosphorylation and activation of Ack. Upon cell stimulation with EGF or PDGF, Ack is tyrosine-phosphorylated and recruited to activated EGF or PDGF receptors, respectively. A pool of endogenous Ack molecules is constitutively tyrosine-phosphorylated, even in starved cells. Moreover, tyrosine-phosphorylated Ack forms a stable complex with the adapter protein Nck via its SH2 domain. Finally, we have characterized a membrane-targeting sterile alpha motif-like domain in the amino terminus of Ack. Using several Ack mutants, we show that the amino-terminal and CRIB domains are necessary for Ack autophosphorylation, whereas the SH3 domain appears to have an autoinhibitory role. These experiments suggest a functional role for Ack as an early transducer of multiple extracellular stimuli.

Rho GTPase signaling in Dictyostelium discoideum: Insights from the genome

Rho GTPases are ubiquitously expressed across the eukaryotes where they act as molecular switches participating in the regulation of many cellular processes. We present an inventory of proteins involved in Rho-regulated signaling pathways in Dictyostelium discoideum that have been identified in the completed genome sequence. In Dictyostelium the Rho family is encoded by 18 genes and one pseudogene. Some of the Rho GTPases (Rac1a/b/c, RacF1/F2 and RacB) are members of the Rac subfamily, and one, RacA, belongs to the RhoBTB subfamily. The Cdc42 and Rho subfamilies, characteristic of metazoa and fungi, are absent. The activities of these GTPases are regulated by two members of the RhoGDI family, by eight members of the Dock180/zizimin family and by a surprisingly large number of proteins carrying RhoGEF (42 genes) or RhoGAP (43 genes) domains or both (three genes). Most of these show domain compositions not found in other organisms, although some have clear homologs in metazoa and/or fungi. Among the (in many cases putative) effectors found in Dictyostelium are the CRIB domain proteins (WASP and two related proteins, eight PAK kinases and a novel gelsolin-related protein), components of the Scar/WAVE complex, 10 formins, four IQGAPs, two members of the PCH family, numerous lipid kinases and phospholipases, and components of the NADPH oxidase and the exocyst complexes. In general, the repertoire of Rho signaling components of Dictyostelium is similar to that of metazoa and fungi.

Growth cone steering by a physiological electric field requires dynamic microtubules, microfilaments and Rac-mediated filopodial asymmetry

Electric fields (EFs) resembling those in the developing and regenerating nervous systems steer growth cones towards the cathode. Requirements for actin microfilaments, microtubules and their interactions during EF growth cone steering have been presumed, but remain unproven. Here, we demonstrate essential roles for dynamic microfilaments and microtubules in cathode-directed migration. Cathodal turning of growth cones on cultured Xenopus embryonic spinal neurons was attenuated significantly by nanomolar concentrations of the microfilament inhibitor latrunculin, the microtubule-stabilising drug taxol, or the microtubule-destabilising drugs vinblastine or nocodazole. Dynamically, the cathodal bias of filopodia preceded cathodal turning of the growth cone, suggesting an instructive role in EF-induced steering. Lamellipodial asymmetry accompanied turning. Filopodia and lamellipodia are regulated by the GTPases Cdc42 and Rac, respectively, and, as shown in the companion paper in this issue, peptides that selectively prevented effector binding to the CRIB domains of Cdc42 or Rac abolished cathodal growth cone turning during 3 hours of EF exposure. Here, the Rac peptide suppressed lamellipodium formation, increased the number of filopodia, abolished cathodal filopodial orientation, and prevented cathodal steering. The Cdc42 peptide suppressed filopodium formation, increased lamellipodial area and prevented cathodal steering. The cathodal bias of lamellipodia was independent of Cdc42 CRIB activity and was not sufficient for cathodal steering in the absence of filopodia, but the cathodal bias of filopodia through Rac CRIB activity was necessary for cathodal turning. Understanding the mechanism for cathodal growth cone guidance will enhance the emerging clinical effort to stimulate human spinal cord regeneration through EF application.

The Presumptive Phosphatidylserine Receptor Is Dispensable for Innate Anti-inflammatory Recognition and Clearance of Apoptotic Cells

The role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells. Taking advantage of this property of fibroblasts, we generated, PSR(+/+), PSR(+/-), and PSR(-/-) fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells.

The regulation of mDia1 by autoinhibition and its release by Rho•GTP

Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin-homology domains 1 and 2. Diaphanous-related formins (Drfs) are regulated by a RhoGTPase-binding domain situated in the amino-terminal (N-terminal) region and a carboxy-terminal Diaphanous-autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N-terminus of mDia1 (mDia(N)) and its release by Rho*GTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDia(N) construct and DAD by X-ray crystallography. The core DAD region is an alpha-helical peptide, which binds in the most highly conserved region of mDia(N) using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby Rho*GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho*GTP accelerates the dissociation of DAD from the mDia(N)*DAD complex.

Small GTPase Proteins Rin and Rit Bind to PAR6 GTP-dependently and Regulate Cell Transformation

The novel small GTPases Rin and Rit are close relatives of Ras, and recent studies show that they play a role in mediating neuronal differentiation. However, the direct effectors of Rin and Rit have yet to be fully characterized. Here we showed that Rin and Rit directly bind to the PDZ domain of PAR6, a cell polarity-regulating protein, in a GTP-dependent manner both in vivo and in vitro. Moreover, Rin and Rit can form a ternary complex consisting of PAR6 and Rac/Cdc42, members of the Rho family of small GTPases modulating cell growth and polarity. This ternary complex synergistically potentiates cell transformation in NIH3T3 cells, and the interaction between Rin/Rit and the PDZ domain of PAR6 is important for this effect. These results suggest that the Rin/Rit-PAR6-Rac/Cdc42 ternary complex may work physiologically in the cells, such as in tumorigenesis.

Interaction with the SH3 Domain Protein Bem1 Regulates Signaling by the Saccharomyces cerevisiae p21-Activated Kinase Ste20

The Saccharomyces cerevisiae PAK (p21-activated kinase) family kinase Ste20 functions in several signal transduction pathways, including pheromone response, filamentous growth, and hyperosmotic resistance. The GTPase Cdc42 localizes and activates Ste20 by binding to an autoinhibitory motif within Ste20 called the CRIB domain. Another factor that functions with Ste20 and Cdc42 is the protein Bem1. Bem1 has two SH3 domains, but target ligands for these domains have not been described. Here we identify an evolutionarily conserved binding site for Bem1 between the CRIB and kinase domains of Ste20. Mutation of tandem proline-rich (PxxP) motifs in this region disrupts Bem1 binding, suggesting that it serves as a ligand for a Bem1 SH3 domain. These PxxP motif mutations affect signaling additively with CRIB domain mutations, indicating that Bem1 and Cdc42 make separable contributions to Ste20 function, which cooperate to promote optimal signaling. This PxxP region also binds another SH3 domain protein, Nbp2, but analysis of bem1Delta versus nbp2Delta strains shows that the signaling defects of PxxP mutants result from impaired binding to Bem1 rather than from impaired binding to Nbp2. Finally, the PxxP mutations also reduce signaling by constitutively active Ste20, suggesting that postactivation functions of PAKs can be promoted by SH3 domain proteins, possibly by colocalizing PAKs with their substrates. The overall results also illustrate how the final signaling function of a protein can be governed by combinatorial addition of multiple, independent protein-protein interaction modules.

Serotonin-Induced Regulation of the Actin Network for Learning-Related Synaptic Growth Requires Cdc42, N-WASP, and PAK in Aplysia Sensory Neurons

Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). We have isolated cDNAs encoding Aplysia Rho, Rac, and Cdc42 and found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities.

Regulation of H2O2 generation in thyroid cells does not involve Rac1 activation

The H2O2 generating system of the thyrocyte and the O2- generating system of macrophages and leukocytes present numerous functional analogies. The main constituent enzymes belong to the NADPH oxidase (NOX) family (Duox/ThOX for the thyroid and NOX2 /gp91phox for the leukocytes and macrophages), and in both cell types, H2O2 generation is activated by the intracellular generation of Ca2+ and diacylglycerol signals. Nevertheless, although the controls involved in these two systems are similar, their mechanisms are different. The main factors controlling O2- production by NOX2 are the cytosolic proteins p67phox and p47phox, and Rac, a small GTP-binding protein. We have previously reported that there is no expression of p67phox and p47phox in thyrocytes. Here, we investigated whether Rac1 is an actor in the thyroid H2O2-generating system. Ionomycin- and carbamylcholine-stimulated H2O2 generation was measured in dog thyroid cells pretreated with the Clostridium difficile toxin B, which inhibits Rac proteins. Activation of Rac1 was measured in response to agents stimulating H2O2 production, using the CRIB domain of PAK1 as a probe in a glutathione S-transferase (GST) pull-down assay. Among the various agents inducing H2O2 generation in dog thyrocytes, carbamylcholine is the only one which activates Rac1, whereas phorbol ester and calcium increase alone have no effect, and cAMP inactivates it. Moreover, whereas toxin B inhibits the stimulation of O2- generation by phorbol ester in leukocytes, it does not inhibit H2O2 generation induced by carbamylcholine and ionomycin in dog thyrocytes. Unlike in leukocytes, Rac proteins do not play a role in H2O2 generation in thyroid cells. A different regulatory cascade for the control of H2O2 generation remains to be defined.

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.

Cellular localization of activated N-WASP using a conformation-sensitive antibody.

The main regulators of Arp2/3 activity appear to be N-WASP and the other members of the Scar/WAVE family of proteins. We show here that after EGF stimulation, N-WASP is recruited to the nucleation zone of the dynamic leading edge compartment of carcinoma cells, with maximal recruitment of N-WASP within 1 min after EGF stimulation. The timing of N-WASP recruitment mirrors the timing of barbed-end formation at the leading edge. To determine the cellular activation of N-WASP after EGF stimulation, we made a conformation-sensitive antibody (CSA) against the CRIB domain of N-WASP that is predicted to recognize N-WASP in its open, active conformation, but not in its closed, inactive conformation. The ability of CSA to detect only active N-WASP was demonstrated by in vitro experiments using immunoprecipitation of active N-WASP from EGF-stimulated cells and Cdc42 activation of N-WASP activity. In cell staining experiments, N-WASP is maximally accessible to CSA 40 sec after EGF stimulation and this activated N-WASP is in the nucleation zone. These results indicate that active N-WASP is present at the leading edge of lamellipods, an unexpected finding given its reported involvement in filopod formation. This work establishes the feasibility of using antibodies directed against specific conformations or epitopes with changing accessibilities as a window on the status and localization of activity.

Isotype-specific Degradation of Rac Activated by the Cytotoxic Necrotizing Factor 1

The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli activates members of the Rho family by deamidation of glutamine 61/63. Because this amino acid is crucial for GTP hydrolysis, deamidation of glutamine 61/63 results in constitutively active Rho proteins. Recently, it was shown that the level of CNF1-activated Rac is rapidly diminished in CNF1-treated cells by proteolytic degradation. Here, we studied the requirements for CNF1-induced Rac degradation. By overexpressing His-tagged activated Rac mutants we show that constitutive activation is necessary for degradation of Rac. However, permanent activation is not sufficient for degradation, because Rac that is constitutively activated by transamidation at glutamine 61 by the Bordetella dermonecrotic toxin is not degraded. Overexpression of His-tagged Rac mutants deficient in interaction with GTPase-activating protein (Rac(N92D) and Rac(Y64H)) and guanosine nucleotide dissociation inhibitor (Rac(H103E)) were degraded after activation by CNF1, whereas Rac(Y40C), which is not able to interact with CRIB domain effectors or plenty of SH3, was not degraded. Isoprenylation and the presence of a putative mitotic destruction box are essential for CNF-induced degradation. In contrast to Rac1, Rac2, and Rac3 were not degraded following constitutive activation by CNF1. Using site-directed mutagenesis, we defined the polybasic region and amino acids 90, 107, 147, and 151 as responsible for isotype-specific degradation.

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.

Genetic analysis of the interface between Cdc42p and the CRIB domain of Ste20p in Saccharomyces cerevisiae.

Mutagenesis was used to probe the interface between the small GTPase Cdc42p and the CRIB domain motif of Ste20p. Members of a cluster of hydrophobic residues of Cdc42p were changed to alanine and/or arginine. The interaction of the wild-type and mutant proteins was measured using the two-hybrid assay; many, but not all, changes reduced interaction between Cdc42p and the target CRIB domain. Mutations in conserved residues in the CRIB domain were also tested for their importance in the association with Cdc42p. Two conserved CRIB domain histidines were changed to aspartic acid. These mutants reduced mating, as well as responsiveness to pheromone-induced gene expression and cell cycle arrest, but did not reduce in vitro the kinase activity of Ste20p. GFP-tagged mutant proteins were unable to localize to sites of polarized growth. In addition, these point mutants were synthetically lethal with disruption of CLA4 and blocked the Ste20p-Cdc42p two-hybrid interaction. Compensatory mutations in Cdc42p that reestablished the two-hybrid association with the mutant Ste20p CRIB domain baits were identified. These mutations improved the pheromone responsiveness of cells containing the CRIB mutations, but did not rescue the lethality associated with the CRIB mutant CLA4 deletion interaction. These results suggest that the Ste20p-Cdc42p interaction plays a direct role in Ste20p kinase function and that this interaction is required for efficient activity of the pheromone response pathway.

Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1.

The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.

Cdc42 Regulation of Kinase Activity and Signaling by the Yeast p21-Activated Kinase Ste20

The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated G beta gamma complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway.

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.