Protein Interactions

Cdc42 and junctions in Drosophila

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. A biochemical and functional link between these two complexes has been discovered 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. These data highlight a previously unrecognized link between protein complexes that are essential for epithelial polarity and formation of tight junctions (Hurd, 2003).

How epithelial cells subdivide their plasma membrane into an apical and a basolateral domain is largely unclear. In Drosophila embryos, epithelial cells are generated from a syncytium during cellularization. Polarity is established shortly after cellularization when Par-6 and the atypical protein kinase C concentrate on the apical side of the newly formed cells. Apical localization of Par-6 requires its interaction with activated Cdc42 and dominant-active or dominant-negative Cdc42 disrupt epithelial polarity, suggesting that activation of this GTPase is crucial for the establishment of epithelial polarity. Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Genetic and biochemical experiments suggest that phosphorylation by aPKC inactivates Lgl on the apical side. On the basolateral side, Lgl is active and excludes Par-6 from the cell cortex, suggesting that complementary cortical domains are maintained by mutual inhibition of aPKC and Lgl on opposite sides of an epithelial cell (Hutterer, 2004).

These results describe the first steps of a molecular pathway that leads to the establishment of polarity in epithelial cells of the Drosophila ectoderm. The Par-6 protein localizes to the apical cell cortex by binding to Cdc42. Par-6 recruits Bazooka and aPKC and is essential for establishment of the apical domain. Maintenance of Par-6 localization requires Lgl, a substrate of aPKC. Phosphorylation by aPKC inactivates Lgl at the apical cell cortex and restricts Lgl to the basolateral cortex to establish the basolateral domain (Hutterer, 2004).

Apical localization of Par-6 is a key event in the establishment of epithelial polarity. How is Par-6 recruited to the apical cell cortex? In C. elegans, the proteins Par-3, Par-6, and aPKC are localized to the anterior cell cortex before and during the first cell division. Their asymmetric localization is initiated by interaction of the sperm aster with the overlying cell cortex that excludes Par-6 from the posterior cell cortex. During Drosophila cellularization, centrosomes are located apically and it is therefore unlikely that a similar cortical microtubule interaction is responsible for the apical localization of Par-6 (Hutterer, 2004).

Although a distinct apical domain with sharp boundaries is established in epithelial cells only after cellularization, elegant membrane tracer experiments have revealed a subdivision of the plasma membrane into distinct regions already during cellularization. Are these membrane compartments prefiguring the future apical and basolateral domains and is Par-6 localizing apically by recognizing a preformed membrane domain? The first membrane domain is the furrow canal at the tip of the ingrowing cellularization front that is marked by Patj. This domain disintegrates after cellularization and is therefore unlikely to participate in Par-6 localization. During later stages, new membrane is preferentially inserted apically, then apicolaterally. At these stages, newly inserted membrane displaces the pre-existing membrane toward both the apical and basolateral side, indicating that a distinct apical membrane compartment is not established by the end of cellularization. It is therefore unlikely that Par-6 recognizes a preformed apical membrane compartment although these experiments do not rule out a more general role of the vesicle transport machinery in Par-6 localization (Hutterer, 2004).

The results indicate that Par-6 needs to bind to activated Cdc42 in order to localize apically. Since cdc42 mutants cannot be analyzed at this stage, a conserved proline in the CRIB domain was mutated to generate a Par-6 version that no longer binds Cdc42. The structure of the Par-6 Cdc42 complex shows that this residue comes to lie in a hydrophobic groove of the Cdc42 molecule. This may explain why it can be replaced by alanine without affecting Cdc42 binding. When it is deleted, however, one of the adjacent highly charged amino acids will occupy the position of the proline. This could strongly inhibit interaction with the hydrophobic pocket and eliminate binding to Cdc42 both in vertebrates and in flies. Since both Lgl and aPKC still bind Par-6-DeltaP and the protein is expressed at almost wild-type levels from the endogenous promoter in an otherwise null mutant background, par-6-DeltaP embryos are specifically defective in binding of Cdc42 to the Par-6/aPKC complex (Hutterer, 2004).

How does activated Cdc42 localize Par-6? Cdc42 might be required for association of an unidentified Par-6 binding partner that is essential for apical localization of the protein. The conformation of Par-6 changes upon binding to Cdc42, and this could affect interactions with other proteins. However, aPKC and Lgl are the only proteins identified in the Par-6 complex, and their interaction does not depend upon Cdc42 binding. In vertebrates, Par-6 interacts with the Stardust homolog Pals1, and this interaction is regulated by Cdc42. Stardust acts together with its binding partner Crumbs, but apical protein localization is initiated correctly in crumbs mutants. Therefore, it is unlikely that Stardust binding to Par-6 is critical for the initial apical localization of Par-6. It is more likely that Cdc42 activation provides an instructive cue for Par-6 localization. Cdc42 could be preferentially activated on the apical side, for example by localization of an exchange factor, and this could recruit Par-6 to the apical cell cortex. This hypothesis is supported by the ectopic patches of Par-6, which are observed after overexpression of constitutively active Cdc42. Asymmetric activation of Cdc42 is known to polarize other cell types. In yeast, the exchange factor Cdc24 is localized to the incipient bud site. This locally activates Cdc42 and polarizes the actin cytoskeleton toward the site. In migrating neutrophils, Cdc42 is locally activated in response to a chemoattractant gradient by the exchange factor PIXalpha. A clear Drosophila ortholog of PIXalpha exists, but whether it is involved in epithelial polarity remains to be determined (Hutterer, 2004).

Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Lgl acts at the basolateral cortex where it inhibits cortical localization of Par-6. How Lgl excludes Par-6 from the cortex is unclear, but it is remarkable that in other tissues, Lgl actually promotes cortical protein localization. In MDCK cells, Lgl was suggested to regulate basolateral exocytosis and it could recruit a Par-6 antagonizing factor to the basolateral plasma membrane. Since Lgl and Bazooka binding to Par-6 seem to be mutually exclusive, Lgl could also inactivate the Par protein complex by displacing Bazooka. To perform its role in epithelial polarity, Lgl needs to be phosphorylated by aPKC. This modification has been shown to inactivate the protein and release it from its association with membranes and the cytoskeleton. These results suggest that in epithelial cells, apically localized aPKC phosphorylates Lgl to displace the protein from the apical cell cortex. A simple model is proposed in which mutual inhibition between Par-6/aPKC on the apical and Lgl on the basolateral cell cortex maintains epithelial polarity. This model is in agreement with previous studies that demonstrate negative genetic interactions between lgl and proteins that localize to the apical domain. Furthermore, it provides a molecular explanation for the recently described suppression of the lgl mutant epithelial polarity phenotype by reduction of aPKC levels. Negative interactions between the apical and basolateral domains of epithelial cells have been described before. In the Drosophila follicular epithelium, Bazooka is phosphorylated and inhibited by Par-1, a protein kinase located on the basolateral domain, thus restricting the Par protein complex to the apical domain (Hutterer, 2004).

The proteins Par-6, Bazooka, and aPKC localize to the apical cell cortex of both neuroblasts and epithelial cells, but the mechanism of apical localization seems to be different in the two cell types. In epithelial cells, Lgl is required for maintaining Par proteins at the apical cell cortex, while Par protein localization in neuroblasts is Lgl independent. Expression of nonphosphorylatable Lgl disrupts asymmetric cell division in neuroblasts but is without effect in epithelial cells. In addition, overexpression of dominant-active or -negative Cdc42 disrupts epithelial polarity but has no effect on neuroblast division. What is the basis for these differences (Hutterer, 2004)?

Epithelial cells rely on adherens junctions for maintaining distinct membrane compartments. Such junctions are absent from neuroblasts, and in fact, distinct membrane compartments do not seem to exist. Instead, Par protein localization in neuroblasts requires a protein called Inscuteable that is recruited apically by binding to Bazooka and aPKC and activates heterotrimeric G proteins through an adaptor molecule called Pins. Both Inscuteable and G proteins are essential for maintaining Par protein localization in neuroblasts but not epithelial cells. It is possible that a feedback loop operates downstream of the G proteins to maintain polarity in the absence of diffusion barriers and cellular junctions. Mechanistic differences in the way Par proteins localize are also observed between species. In C. elegans, neither Lgl nor G proteins are required for Par-3 or Par-6 localization. Instead, a Ring finger protein called Par-2 maintains Par-3 and Par-6 at the anterior pole. Cdc42 plays a role, but only in maintenance and not establishment of polarity. Clearly, key players are missing that might help in an understanding of these mechanistic differences (Hutterer, 2004).

Cdc42 binds vertebrate Par-6. Both proteins are implicated in polarizing vertebrate epithelial cells, and their conserved interaction suggests that they achieve this via a conserved mechanism. Although in vertebrates both proteins primarily act on tight junctions, the role of Cdc42 in localizing the Par proteins seems conserved since overexpression of an activated form inhibits the localization of Par-3 to tight junctions in MDCK cells. However, current experiments do not confirm a previously demonstrated role of Cdc42 in activating Par-6-associated aPKC in vitro. Unlike in vertebrates, aPKC is shown to be equally active - at least toward Lgl - when bound to a form of Par-6 that does not interact with Cdc42. Whether species-specific differences or the different experimental setups are responsible for this apparent discrepancy remains unclear. Besides their function in polarity, the Par proteins are involved in proliferation control of vertebrate epithelial cells. Par-6 cooperates with Cdc42 in transforming cells, suggesting a role in oncogenic transformation. In Drosophila, Cdc42, Lgl, and Bazooka were shown to cooperate with activated ras in the formation of metastatic tumors. It can be anticipated that the powerful tool of Drosophila genetics will help to identify other components of this pathway that might clarify its role in carcinogenesis (Hutterer, 2004).

Mbt as a downstream effector of Cdc42

The gene mushroom bodies tiny (mbt) encodes a putative p21-activated kinase (PAK), a family of proteins that has been implicated in a multitude of cellular processes including regulation of the cytoskeleton, cell polarization, control of MAPK signalling cascades and apoptosis. The mutant phenotype of mbt is characterized by fewer neurones in the brain and the eye, indicating a role for the protein in cell proliferation, differentiation or survival. Mutations in mbt interfere with photoreceptor cell morphogenesis. Mbt specifically localizes at adherens junctions (AJs) of the developing photoreceptor cells, and Cdc42, an Mbt interacting protein, is responsible for localization of Mbt to AJs. A structure-function analysis of the Mbt protein in vitro and in vivo revealed that the Mbt kinase domain and the GTPase binding domain, which specifically interacts with GTP-loaded Cdc42, are important for Mbt function. A role for Mbt is proposed as a downstream effector of Cdc42 in photoreceptor cell morphogenesis (Schneeberger, 2003).

Based on their distinct molecular structure, mammalian PAK 1-3 and Drosophila PAK are classed together as the group I PAKs, whereas mammalian PAK4-6 and Mbt constitute the group II PAKs. All PAK proteins share a C-terminal kinase domain and a N-terminal binding domain for proteins of the Rho family of small GTPases (p21-binding domain, PBD). The group I PAKs show some additional structural features that are missing in group II PAKs. Most importantly, the PBD of group I PAKs is C-terminally flanked by the kinase inhibitory domain (KID), which negatively regulates kinase activity through interaction with the kinase domain. Binding of GTP-bound forms of Cdc42 or Rac releases this intramolecular association, resulting in autophosphorylation and full activation of the kinase. Group I PAKs also possess several proline-rich sequences that bind to SH3 domain-containing proteins. Interaction with the SH2/SH3 domain adaptor proteins Nck and the corresponding Drosophila homolog Dock provides a link to cell-surface receptors. SH3 domain-mediated binding to Cool/PIX proteins can positively or negatively regulate PAK kinase activity (Schneeberger, 2003 and references therein).

Mbt mutant flies display a rough eye phenotype (Melzig, 1998). Tangential sections through adult eyes reveal the absence of a variable number of photoreceptor cells in many ommatidia. The rhabdomeres of the remaining photoreceptor cells show morphological defects. The cross-section profiles of the rhabdomeres are enlarged and neighbouring rhabdomeres often contact each other, a condition not seen in the wild type. Longitudinal sections reveal that the rhabdomeres are twisted, fragmented and do not extend from the pseudocone to the floor of the retina. These phenotypes suggested that Mbt is required for recruitment and/or proper differentiation of photoreceptor cells (Schneeberger, 2003).

To analyse the function of Mbt during eye development, a polyclonal antiserum was generated and eye-antennal imaginal discs from third instar larvae and pupae were stained. Differentiation of the cells that comprise the single eye units (ommatidia) occurs in a step-wise fashion and is initiated in the morphogenetic furrow, which moves across the eye disc from posterior to anterior. Staining of third-instar eye-imaginal discs with the Mbt antiserum revealed an accumulation of the Mbt protein at apical membrane sites of the photoreceptor cells as soon as they become recruited to the ommatidial clusters and initiate differentiation. Low levels of Mbt protein were detected at the membranes of undifferentiated cells. Staining was completely absent in eye discs derived from mbtP1 mutant larvae, demonstrating the specificity of the antiserum and confirming the notion that mbtP1 is a complete loss-of-function allele (Melzig, 1998). To determine the subcellular localization of Mbt more precisely, eye discs were co-stained with anti-Armadillo (Arm, Drosophila ß-Catenin) antibodies, a marker for adherens junctions (AJs). Staining for both largely overlaps in the photoreceptor cells. From apical to basal cross sections it became evident that Mbt is less abundant in the most apical domain of Arm expression in the photoreceptor cells (Schneeberger, 2003).

The final architecture of the ommatidia is established during pupal development and is accompanied by major morphological changes. At 37% of pupal development (p.d.), the apical domains of the photoreceptor cells have involuted. Thus, the apical domains of the photoreceptors point toward the center of the ommatidial cluster. After involution, the apical membranes of the photoreceptor cells start to expand to form the rhabdomeres. Each rhabdomere is surrounded by the stalk membrane, which connects it to the zonula adherens. As shown by anti-Arm staining, the AJs span the whole proximal-to-distal length of the photoreceptors at 50% p.d. Mbt remains colocalized with Arm at AJs of pupal photoreceptor cells at different stages of their development. Higher levels of Mbt expression can also be seen in the future bristle cells, whereas cone and pigment cells express low levels of Mbt. A 3D reconstruction of a wild-type ommatidial cluster stained with anti-Arm and anti-Mbt antibodies shows the colocalization of both proteins at AJs of the photoreceptor cells along the whole proximodistal length. In summary, these data provide evidence that Mbt is localized at AJs of photoreceptor cells from the initial recruitment to their final differentiation (Schneeberger, 2003).

The observed phenotypes in mbtP1 eyes could result from a defect in cell proliferation, photoreceptor cell recruitment or differentiation. To determine whether mbt mutations affect recruitment or early neuronal differentiation of photoreceptor cells, wild-type and mbtP1 third instar larval eye discs were stained with an antibody against the neuronal differentiation marker Elav. Only rarely did mbtP1 ommatidia contain fewer Elav-positive cells than wild-type clusters. This result was confirmed by using HRP as an independent differentiation marker. This suggests that a failure in recruitment of photoreceptor cells is not the major cause of the mbt phenotype (Schneeberger, 2003).

The specific localization of Mbt at AJs prompted a look for AJ defects in mbtP1 third instar and pupal eye imaginal discs with an anti-Arm antibody. In third instar eye discs, the AJs of the developing photoreceptor cells appear disorganized. Frequently, the AJs extend laterally. The AJ defects become much more pronounced at pupal stages. At 37% p.d., AJs fail to extend in proximodistal direction. At 50% p.d., AJs are fragmented and form patchy and disorganized structures. To verify these results and to exclude the possibility that mbtP1 disturbs only Arm localization without affecting AJs, mbtP1 eye imaginal discs were co-stained with anti-Canoe antibodies as an independent AJ marker. Canoe and Arm remain colocalized in mbtP1 eye discs. In addition, wild-type and mbtP1 pupal eye discs were stained with antibodies against the apical determinant Crumbs (Crb) and the Discs large (Dlg) protein, which is a marker for septate junctions in epithelial cells. Crumbs is essential to maintain AJ integrity during photoreceptor cell morphogenesis and is localized at the stalk membrane between AJs and the rhabdomeres. Compared with wild-type ommatidia, Crb and Dlg are de-localized in mbtP1 mutant cells. In summary these data suggest that Mbt function is required in the developing photoreceptor cells to undergo their morphological changes (Schneeberger, 2003).

To gain insight into the molecular mechanisms that control Mbt function, the binding of Mbt to Rho-type GTPases was tested. Group I PAKs have been shown to interact via the p21-binding domain (PBD) with GTP-loaded Rac and Cdc42 but not with Rho, whereas the group II PAK proteins PAK4 and PAK5 preferentially bind to GTP-bound Cdc42 (Abo, 1998; Pandey, 2002). Myc-tagged versions of the Drosophila homologs of Cdc42, Rac1 and Rho1 were co-expressed with HA-tagged Mbt in HEK293 cells. Co-immunoprecipitation experiments revealed a nearly exclusive binding of Cdc42 to Mbt. Rac1 showed only a very weak interaction whereas no binding of Rho1 to Mbt was detected. The specificity of the interaction between Cdc42 and Mbt was tested by mutation of two conserved histidine residues in the PBD to leucine (MbtH19,22L). The mutant Mbt protein was unable to bind to Cdc42. Thus, the interaction between Cdc42 and Mbt is indeed mediated by the PBD. To determine whether activation of Rho-type GTPases influences binding to Mbt in vivo, the constitutively activated variants Cdc42G12V, Rac1G12V and RhoG14V were co-expressed with Mbt in HEK293 cells. Cdc42G12V showed an enhanced interaction with Mbt when compared to wild-type Cdc42. This result indicates that only the active, GTP-bound form of Cdc42 binds to Mbt. To verify this result, Cdc42 was expressed as a GST-fusion protein in bacteria and used in pull-down experiments upon loading with GDP or GTP. Mbt selectively binds to GTP-loaded Cdc42 but not to unloaded or GDP-loaded Cdc42. Thus, the preference for binding GTP-bound Cdc42 appears to be a common feature among group II PAKs (Schneeberger, 2003).

The PBD of group I PAKs is C-terminally flanked by the kinase inhibitory domain (KID). Binding of activated Cdc42 and Rac relieves the inhibitory influence of the KID on PAK kinase activity. In addition, group II PAKs share significant sequence homology C-terminal to the PBD, but the sequences differ significantly from the group I PAK KID (Schneeberger, 2003).

To analyse the influence of Cdc42 binding on Mbt kinase activity the Cdc42 binding-deficient MbtH19,22L construct was used. A second Mbt construct used in this study bears a mutation in the kinase domain. This mutation (T525A), located in the linker region between subdomains VII and VIII, corresponds to the T777A mutation in the Saccharomyces cerevisiae PAK protein Ste20p and has been found to disrupt autophosphorylation and catalytic activity of Ste20p. HEK293 cells were transfected with HA-tagged wild-type or the presumptive kinase-dead version of Mbt and the immunopurified protein complexes were incubated with kinase buffer and gamma-32P]ATP together with myelin basic protein (MBP) as a substrate. Compared with wild type Mbt, the T525A mutation strongly reduced autophosphorylation and substrate phosphorylation. Co-expression of Cdc42 with Mbt did not increase autophosphorylation or MBP phosphorylation when compared with cells transfected with Mbt alone. Importantly, co-expression of Mbt and the constitutively activated Cdc42G12V construct slightly reduced rather than enhanced the ability of wild-type Mbt to phosphorylate MBP. Conversely, the Cdc42-binding defective

MbtH19,22L protein showed a moderate increase of MBP phosphorylation independent of co-expression with Cdc42 or Cdc42G12V. Autophosphorylation was not affected by removal of the Cdc42-binding site. These results fit with previous observations that kinase activity of PAK4, 5 and 6 is not upregulated upon Cdc42 binding, whereas deletion of the PBD can lead to enhanced kinase activity. Thus, group II PAKs appear to differ from group I PAKs in their mechanism to regulate kinase activity (Schneeberger, 2003).

In order to test the requirement of the Cdc42-binding domain and the kinase domain for Mbt function in vivo, wild-type or mutated Mbt proteins were expressed during eye development in the absence of the endogenous Mbt protein. Northern blot analysis and antibody staining indicates that mbtP1, which carries a P-element insertion in the protein encoding sequence, is a complete loss-of function allele. Gal4:238Y-driven expression of a mbt cDNA in the brain is sufficient to rescue the mbtP1 brain phenotype (Tettamanti, 1997; Melzig, 1998). Gal4:238Y is also expressed in the eye-antennal imaginal disc in a manner that closely resembles the expression pattern of the endogenous Mbt protein. Consistent with this observation, the eye phenotype of mbtP1 flies was completely rescued by Gal4:238Y-driven expression of the wild-type mbt cDNA. By contrast, the Cdc42-binding deficient MbtH19,22L protein was unable to rescue the mbtP1 eye phenotype, whereas the kinase-defective MbtT525A construct partially rescues the mbtP1 eye phenotype (Schneeberger, 2003).

In summary, these experiments have verified the importance of the Cdc42-binding domain and the kinase domain for the in vivo function of Mbt during eye development. The partial rescue ability of the MbtT525A construct indicates that some functions of Mbt are independent of kinase activity. The differences observed in the rescue ability of the kinase defective MbtT525A and the Cdc42 binding-deficient MbtH19,22L proteins also suggests that Cdc42 binding to Mbt influences Mbt function in a kinase-independent manner. One possibility that was investigated was the proper localization of the Mbt protein to AJs (Schneeberger, 2003).

Group II PAKs lack the N-terminal binding site for the Nck/Dock adaptor protein, which could provide a link to membrane-bound proteins. To investigate whether the Cdc42-binding domain is responsible for the observed localization of Mbt to AJs, wild-type and mutated mbt cDNAs were expressed with the Gal4:238Y driver line in a mbtP1 mutant background and the subcellular localization of the corresponding Mbt proteins were analysed in pupal eye discs. The expression pattern of the transgenic, non-mutated Mbt protein in the eye imaginal disc closely resembles the expression pattern of the endogenous Mbt protein. High levels of transgenic Mbt accumulate at the AJs of the developing photoreceptor cells. Consistent with the complete rescue of the adult mbtP1 eye phenotype, no morphological abnormalities were observed when pupal eye discs were stained with anti-Mbt or anti-Arm antibodies. An identical localization pattern was observed when the kinase-defective MbtT525A protein was expressed in the mbtP1 mutant background, indicating that eliminating kinase activity does not influence the subcellular distribution of the Mbt protein. However, as revealed by co-staining with an anti-Arm antibody, the transgenic MbtT525A protein only partially rescued the AJ defects in mbtP1 animals. The AJs extend to some degree in proximal-to-distal direction but still do not have a regular architecture. This result correlates with the partial rescue observed in adult eyes. By contrast, the Cdc42 binding-deficient MbtH19,22L protein did not accumulate at AJs but instead was distributed within the cytoplasm. Anti-Arm staining revealed that the MbtH19,22L protein is unable to rescue the AJs defects seen in mbtP1 mutant eye discs. Thus, there is an absolute requirement of the Cdc42 binding domain for localization and function of the Mbt protein during eye development. To exclude the possibility that the failure of the MbtH19,22L protein to localize at AJs is not a secondary effect of the mbtP1 phenotype itself, the MbtH19,22L protein was also expressed in a wild-type background. Although endogenous and transgenic Mbt protein cannot be distinguished in this case, two observations were made: (1) the MbtH19,22L protein did not cause any obvious AJs defects when expressed in a wild-type background; (2) Mbt protein was found at AJs and in the cytoplasm. Because no cytoplasmic Mbt protein was detected upon expression of the non-mutated Mbt protein in a wild-type background, it is concluded that the MbtH19,22L protein localizes in the cytoplasm (Schneeberger, 2003).

To show that Cdc42 and not another protein bound to the PBD of Mbt is responsible for localization of Mbt to AJs, animals that either lacked Cdc42 function or ectopically expressed mutated versions of the Cdc42 protein were examined. Because removal of Cdc42 function causes lethality (Genova, 2000), homozygous mutant Cdc423 or Cdc424 cell clones were generated using the MARCM system. Only those cells that are homozygous for the Cdc42 mutation express the membrane localized mCD8 marker. Most of the Cdc423 or Cdc424 clones obtained in the eye disc contain only a few mCD8-positive cells. Consistent with findings that Cdc42 mutant cells can initiate their differentiation into photoreceptor cells (Genova, 2000), the majority of mCD8-positive (Cdc42 mutant) photoreceptor cells analysed extend an axon. From single apical sections and apical-to-basal cross-sections it is evident that Mbt is localized at the apical side of photoreceptors, whereas the mCD8 marker labels the whole cell surface of the Cdc42 mutant photoreceptor cell, including the axonal projection. Loss of Cdc42 function is accompanied by the loss of apical Mbt protein (Schneeberger, 2003).

The influence of the constitutively activated (GTP-loaded) Cdc42G12V and of the dominant-negative (GDP-loaded) Cdc42T17N protein on Mbt localization was examined. Because expression of these constructs with the Gal4:238Y driver line results in embryonic lethality, the eye-specific GMR-Gal4 driver line was used. Consistent with the finding that Mbt only binds to GTP-loaded Cdc42, expression of Cdc42T17N has only minor effects on Mbt localization and the AJs morphology. By contrast, Cdc42G12V causes a dramatic change in the Mbt and Arm expression pattern. Mbt accumulates at membrane sites of all cells. Arm expression can only be seen at early stages of photoreceptor cell recruitment, indicating that Cdc42G12V completely disrupts the integrity of AJs in the developing photoreceptor cells (Schneeberger, 2003). One major difference between group I and group II PAKs is the regulation of kinase activity. For group I PAK proteins, binding of GTP-bound Cdc42 or Rac releases the inhibitory effect of the KID on catalytic activity. The lack of an obvious KID in group II PAKs is reflected by their distinct biochemical properties. In contrast to group I PAKs, a slightly reduced rather than enhanced kinase activity is observed upon co-expression of Mbt and a constitutively active variant of Cdc42 in serum starved cells. A Cdc42 binding-deficient Mbt protein showed enhanced kinase activity in vitro. Similar results have been reported for other group II PAKs. Kinase activity of PAK4 was not further enhanced upon co-transfection of activated Cdc42 but deletion or mutation of the PBD of PAK4 and PAK6 results in enhanced kinase activity (Schneeberger, 2003).

From these data, the question remains of what role activated Cdc42 plays in regulating the functions of group II PAKs. Genetic studies have verified the importance of the kinase domain and the PBD for the in vivo function of Mbt. Despite increased kinase activity in vitro, a construct lacking the PBD is unable to rescue the mbtP1 mutant phenotype in the eye. However, a kinase-dead Mbt protein partially rescued the mbtP1 phenotype. This indicates that Cdc42 binding to Mbt fulfils some additional essential functions that are independent of kinase activity. Localization studies show that one major function of the PBD is to recruit Mbt specifically to adherens junctions. These data are also supported by the observation that localization of a PBD-deficient Mbt protein to the cellular membrane, by fusing it to a general membrane targeting sequence, is not sufficient to restore the wild-type function of the protein. It is therefore proposed that Cdc42 has a dual function: specific recruitment of Mbt to AJs and regulation of the catalytic activity of Mbt. The importance of proper targeting of PAK proteins to distinct subcellular compartments for their in vivo function is also evident from other studies. PAK4 recruitment to Golgi membranes by activated Cdc42 is dependent on an intact PBD (Abo, 1998). Activated Rac and Cdc42 also promote the relocalization of a recently described group II PAK protein in Xenopus laevis, X-PAK5, from microtubule networks to actin-rich regions (Cau, 2001). In the case of group I PAKs, autophosphorylation of the Nck and PIX SH3 domain binding sites has been suggested as a mechanism to control cycling between different cellular compartments. Also, D-PAK function in the photoreceptor axons and growth cones is dependent on the interaction with the Drosophila Nck homolog dreadlocks (Dock), which binds to the tyrosine phosphorylated axon guidance receptor DSCAM through its SH2 domain (Schneeberger, 2003).

Rho GTPases are important regulators of the actin cytoskeleton and are involved in many developmental processes that require morphological changes of epithelial and neuronal cells. Each GTPase regulates a diverse range of effector molecules and thus induces multiple defects when misregulated. For this reason, it is difficult to reconcile all the data obtained with the loss-of-function Cdc42 alleles and the various ectopically expressed Cdc42 variants (Eaton, 1995; Genova, 2000; Harden, 1999; Luo, 1994; Riesgo-Escovar, 1996), but a number of conclusions can be drawn with respect to the function of Mbt and D-PAK. (1) Activated Cdc42 has an effect on the levels of D-PAK accumulating at the dorsal most ends (leading edge) of epidermal cells flanking the amnioserosa (Harden, 1999). This is consistent with the result that overexpression of activated Cdc42 in the eye disc leads to accumulation of Mbt at the membrane. (2) The mbt and the Cdc42 mutant phenotypes in the eye display some similarities. In the eye disc, cells devoid of endogenous Cdc42 or Mbt function can initiate their differentiation into photoreceptor cells (Genova, 2000). In the adult eye, loss of Cdc42 function also causes the loss of photoreceptor cells and defects in rhabdomere morphology of the remaining photoreceptor cells. At first sight the similarities in the loss-of-function phenotypes contradict the biochemical data, implying a negative role for Cdc42 in kinase activation. (3) The Cdc42 binding-deficient Mbt protein, despite enhanced kinase activity in vitro, does not cause visible phenotypes in the eye even when expressed in a wild-type background. There are several possibilities to reconcile the data. In all cases, Mbt is either not present or not localized to AJs. Some functions of Mbt might be independent of kinase activity. Alternatively, the moderate increase in kinase activity of the MbtH19,22L protein might not be sufficient to induce dominant phenotypes. A more detailed analysis of Mbt kinase activity in vivo requires the identification of physiological substrates (Schneeberger, 2003).

The described roles of PAK proteins and RhoGTPases in regulating the actin cytoskeleton and the results presented in this study imply that Mbt localized at AJs mediates signals to the cytoskeleton to ensure proper photoreceptor cell morphogenesis. Because Mbt interacts only with GTP-loaded Cdc42, recruitment of Mbt to AJs would require localized Cdc42 activation. Although there is no direct evidence so far for a selective Cdc42 activation at AJs in photoreceptor cells, studies in mammalian epithelial cell lines have demonstrated Cdc42 activation by the AJ protein E-cadherin (S. H. Kim, 2000). In addition, the molecular link between Mbt and the actin cytoskeleton remains to be defined. As has been reported for PAK4, the Drosophila homolog of LIMK could provide a link between Mbt and the actin cytoskeleton (Dan, 2001; Ohashi, 2000). Based on the similar mutant phenotype, the PDZ domain protein Canoe could be another interaction partner of Mbt. Canoe is localized at AJs and the mammalian ortholog Afidin has been shown to bind to actin filaments. Unravelling the precise molecular functions of Mbt in cell morphogenesis awaits the identification of interaction partners and physiological substrates (Schneeberger, 2003).

ACK family tyrosine kinase activity is a component of Cdc42 signaling during dorsal closure

Drosophila Ack is one of two members of the ACK family of nonreceptor tyrosine kinases in Drosophila. The ACKs are likely effectors for the small GTPase Cdc42, but signaling by these proteins remains poorly defined. ACK family tyrosine kinase activity functions downstream of Drosophila Cdc42 during dorsal closure of the embryo; overexpression of Ack can rescue the dorsal closure defects caused by dominant-negative Cdc42. Similar to known participants in dorsal closure, Ack is enriched in the leading edge cells of the advancing epidermis, but it does not signal through activation of the Jun amino-terminal kinase cascade operating in these cells. Transcription of Ack is responsive to changes in Cdc42 signaling specifically at the leading edge and in the amnioserosa, two tissues involved in dorsal closure. Unlike other members of the ACK family, Ack does not contain a conserved Cdc42-binding motif, and transcriptional regulation may be one route by which Dcdc42 can affect Ack function. Expression of wild-type and kinase-dead Ack transgenes in embryos, and in the developing wing and eye, reveals that ACK family tyrosine kinase activity is involved in a range of developmental events similar to that of Cdc42 (Sem, 2002).

The predicted protein is most similar to murine ACK (GenBank accession no. NP_058068), with the two proteins showing 68% identity in their tyrosine kinase domains. The next closest matches were with human ACK-1 and bovine ACK-2, with their tyrosine kinase domains showing 67% identity to Ack. Ack is a component of signaling by the adaptor protein Dock (Clemens, 2000). Another ACK-like tyrosine kinase has been described in Drosophila. DPR2 (Fak-like tyrosine kinase) encodes predicted proteins of 1,274 and 1,356 amino acids that differ in their N termini but have identical tyrosine kinase domains. The DPR2 tyrosine kinase domain has 44% identity with that of Ack, and it is significantly more divergent from the mammalian ACKs than Ack, showing only 44% identity with ACK-1 in the tyrosine kinase domain. The tyrosine kinase domain of DPR2 is most similar to that of ARK-1, a Caenorhabditis elegans member of the ACK family. Members of the ACK family share conserved motifs in addition to their tyrosine kinase domains. All have a conserved stretch of sequence N terminal to the tyrosine kinase domain and all have an SH3 domain on the C-terminal side. With the exception of Ack and the human protein TNK1, other members of the family have a CRIB (Cdc42/Rac interactive binding) domain next to the SH3 domain. The CRIB domain has been found in a wide range of proteins and mediates binding to the Rho family members Cdc42 and Rac. With regard to the ACK family, the CRIB domains of ACK-1 and DPR2 have been shown to bind Cdc42. Finally, members of the family have proline-rich C termini containing copies of the minimal SH3-binding motif PXXP (Sem, 2002).

An attempt to inhibit Ack function by using RNAi yielded no obvious phenotypic effects, suggesting that loss of Ack is nonlethal. A likely possibility is that Ack shares target proteins with the other ACK family tyrosine kinase in Drosophila, DPR2. Expression of Ack transgenes during development did produce phenotypic effects, presumably by affecting Ack and/or DPR2 signaling pathways. Expression of KD-Ack during embryogenesis, wing development, and eye development results in a range of phenotypic effects similar to those caused by loss-of-function mutations in Cdc42 or by expression of Cdc42N17. More importantly, overexpression of wild-type Ack can suppress dorsal closure defects caused by Cdc42N17 expression. The extensive rescue of Cdc42N17-induced dorsal closure failures by Ack overexpression indicates that ACK family tyrosine kinase activity is a major route for Cdc42 signaling during dorsal closure. Overexpression of Ack does not trigger ectopic activation of the JNK cascade, in contrast to other findings that constitutive activation of Cdc42 signaling using Dcdc42V12 induces this pathway. Furthermore, the JNK cascade is not disrupted by either impairment of ACK family tyrosine kinase function through expression of KD-Ack or by loss of zygotic Ack through a deficiency removing the Ack gene. These results suggest that the JNK cascade does not lie downstream of ACK family tyrosine kinase activity in Cdc42 signaling. The JNK cascade does not drive expression of Ack. This work is consistent with analysis of loss-of-function alleles of Cdc42, which indicates that the JNK cascade is not a major component of Dcdc42 signaling. Cdc42 may normally make a minor contribution to the activation of the JNK cascade that could be greatly amplified by expression of Dcdc42V12 (Sem, 2002).

The possibility that the ACK family tyrosine kinase activity acting downstream of Cdc42 during dorsal closure is provided entirely by DPR2 cannot be excluded. However, the leading-edge enrichment of Ack and the alterations in Ack transcription in the leading edge and amnioserosa in response to Cdc42 transgene expression are indications that Ack has a role in Cdc42 signaling during dorsal closure. The transcriptional regulation of Ack does not appear to be a simple homeostatic response, since it is tissue specific and works in opposite directions in two tissues, i.e., dominant-negative Cdc42 causes upregulation of Ack transcripts at the leading edge, whereas constitutively active Cdc42 causes upregulation of transcription in the amnioserosa. The relevance of this transcriptional regulation of Ack remains unknown, but it may provide a route for Cdc42 to regulate Ack function during dorsal closure. The serine/threonine kinase PAK, a likely downstream effector for Rac1 and Cdc42, also responds transcriptionally to a change in Cdc42 signaling in the amnioserosa but, interestingly, in the opposite direction from Ack, in that it is dominant-negative Cdc42 that induces upregulation of PAK transcription in this tissue (Sem, 2002).

Cdc42 might also regulate Ack through its GTPase activity. Although Cdc42 does not appear to bind Ack directly, it could possibly influence Ack function indirectly in a signaling complex. An indirect mode of activation of ACK proteins by Cdc42 proteins is consistent with the finding that constitutively active Cdc42 fails to activate ACK-2 in vitro but can promote activation when cotransfected with ACK-2 in vivo (Sem, 2002 and references therein).

The high level of Ack protein seen in mitotic domains is of interest, since Cdc42 is involved in yeast budding and cytokinesis in Xenopus laevis embryos. To date, no defects in Drosophila cytokinesis have been seen with impaired Cdc42 function, although constitutively active Cdc42 disrupts cellularization of the embryo, a specialized form of cytokinesis (Sem, 2002).

The wing blisters induced by expression of KD-Ack are reminiscent of those found in wings bearing clones homozygous for loss-of-function mutations in the genes encoding the Drosophila integrins alphaPS1, alphaPS2, and ßPS. There is evidence that the mammalian ACKs function in integrin signaling, and the Drosophila wing may provide a useful model to genetically dissect this role for the ACK family (Sem, 2002).

Despite being among the first-described potential effectors for Cdc42, the ACKs remain poorly characterized in terms of the signaling they participate in. The strong eye phenotypes generated by Ack transgene expression should provide a particularly good system for investigating signaling pathways involving the Drosophila ACK family proteins. The rough eye phenotypes generated by Rho family transgene expression in Drosophila have been used to identify second site mutations in genes encoding components of Rho family signal transduction, and deficiencies suppressing the rough eye phenotype induced by overexpression of wild-type ACK have been identified (Sem, 2002).

Drosophila WASp interacts with Cdc42

Wiskott-Aldrich Syndrome proteins (WASp) serve as important regulators of cytoskeletal organization and function. These modular proteins, which are well-conserved among eukaryotic species, act to promote actin filament assembly in response to cues from various signal transduction pathways. Genetic analysis has revealed a requirement for the single Drosophila homolog, WASp, in cell-fate decisions governing specific neuronal lineages. This unique developmental context was used to assess the contributions of established signaling and cytoskeletal partners of WASp. Biochemical and genetic evidence is presented that, as expected, Drosophila WASp performs its developmental role via the Arp2/3 complex, indicating conservation of the cytoskeletal aspect of WASp function in vivo. In contrast, association with the key signaling molecules CDC42 and PIP2 is not an essential requirement, implying that activation of WASp function in vivo depends on additional or alternative signaling pathways (Tal, 2002).

Evidence presented in this study suggests that the role of WASp in cell fate determination in neural lineages involves established cytoskeletal partners of WASp, and in particular, the Arp2/3 protein complex. Binding studies demonstrate a capacity for WASp to directly associate with monomeric actin via WA, the C-terminal cytoskeleton-interacting domain present in all WASp and WASp-related proteins. In parallel, the WA domain of WASp is shown to interact with components of the Arp2/3 complex, the primary downstream target of signal transduction pathways operating through WASp family proteins. The in vivo significance of these associations, which are characteristic of WASp elements in general, is demonstrated by a dual genetic approach. The final 30 residues at the C-terminal end of the WA domain of WASp prove necessary for rescue of WASp mutant phenotypes, while mutations in the Arp2/3 complex subunit Arpc1 lead to cell-fate transformations and neuronal excess during sensory organ development, a distinct, WASp-like phenotype. Taken together with the binding studies, these genetic observations imply that engagement of the cytoskeletal machinery via the C-terminal WA domain is an essential aspect of WASp function in vivo (Tal, 2002).

Several additional inferences can be drawn from the reported results, regarding the mechanism by which the cytoskeleton-interacting domain of WASp operates. Significant function is retained after removal of the extreme C-terminal 15 residues, corresponding to the A (acidic) portion of the WA domain. This observation suggests that the remaining WA sequences, comprising the so-called central (C) domain, contribute significantly to the functional interaction with Arp2/3, and is in good keeping with a recent study highlighting the importance of the C domain in WASp-based activation of the actin-nucleating activity of Arp2/3. A second noteworthy aspect is the inability of the full WASp WA domain to rescue WASp mutant phenotypes on its own, even though biochemical studies have repeatedly demonstrated constitutive Arp2/3 activation by the isolated WA domains of various WASp and WASp-related proteins. This observation implies that N-terminal regions absent from the truncated protein play important in vivo roles, beyond their established capacity to relieve a self-inhibitory conformation. These may include proper localization of the activated protein to specific cellular sites of function (Tal, 2002).

The cellular mechanism by which WASp influences lineage decisions during Drosophila development remains unknown. The data presented here strongly suggest that the functional requirement for WASp is mediated via the Arp2/3 complex, thereby implying that WASp-dependent cell-fate specification involves reorganization of the actin-based cytoskeleton. It remains to be seen just how this intriguing connection between the cytoskeletal machinery and a key developmental mechanism is carried out (Tal, 2002).

In contrast to the demonstration of a functional connection in vivo between WASp and the established cytoskeletal partners of WASp proteins in general, the data suggest that association with the major established activators of WASp, the small GTPase CDC42 and the phosphoinositide PIP2, is not essential for the developmental roles carried out by the Drosophila WASp homolog. Characterization of the prototype WASp as a CDC42-binding protein is a longstanding observation, and a functional connection between CDC42 activation of WASp elements and reorganization of the actin cytoskeleton via Arp2/3 has been firmly established. Association with PIP2 has gained prominence as an alternative activating mechanism of WASp, while optimal activation is achieved by the combined action of both signaling molecules. The Drosophila WASp protein interacts with both CDC42 (in its activated state) and PIP2, and this association maps to the well-conserved domains identified and characterized as the CDC42 and PIP2 binding sites in WASp. Elimination of these sites, however, does not interfere with the ability of WASp transgenic constructs to rescue WASp mutant phenotypes, suggesting that association with these elements, either separately or in combination, is not an essential aspect of WASp function during Drosophila development (Tal, 2002).

Several issues are raised by these unexpected observations and warrant further discussion. One issue is the basis for evolutionary conservation of the activator binding sites, despite the absence of an essential developmental role. This situation is not without precedence, and may indicate that the conserved sites function in a relatively subtle context, which the phenotypic studies have failed to identify. A second, cardinal issue is the implications these findings have for understanding the WASp molecular pathway. In particular, the possibility that elements other than CDC42 and PIP2 contribute significantly to WASp activation must be considered. Elements of tyrosine-kinase signaling pathways constitute possible alternative candidates, since several such molecules have been shown to associate with and activate WASp. This contribution could act in concert with the functions performed by CDC42 and PIP2, but may well provide the primary activating signal in this particular in vivo setting. These observations thus suggest caution in drawing inferences from in vitro studies, and underscore the need for further work, with particular emphasis on genetic screens designed to identify additional, physiological activators of the WASp pathway (Tal, 2002).

Genghis khan interacts with Cdc42

Genghis khan was isolated in a search for proteins that physically interact with the Drosophila small GTPases Rac1 and Cdc42. Gek does not bind to Cdc42N17, a dominant negative mutant that preferentially stays in the GDP-bound state. The ability of Gek to bind Cdc42 in its GTP-bound form (but not in its GDP-bound form) suggests that Gek is an effector of Cdc42. A mutation in the Cdc42 effector domain (A35), which is important for signaling to downstream targets, eliminates Gek binding. Gek does not bind to Drosophila Rac. Deletion of three residues in Gek, which correspond to three conserved residues of the Cdc42/Rac interactive binding (CRIB) domain, disrupt Gek's binding to Cdc42. Gek exhibits kinase activity using histone as a substrate (Luo, 1997).

Novel guanine nucleotide exchange factor GEFmeso of Drosophila melanogaster interacts with Ral and Rho GTPase Cdc42

A DBL-like guanine nucleotide exchange factor (GEF) in Drosophila, called GEFmeso has been identified as a novel binding target of the Ras-like GTPase Ral. Previous studies suggested that some aspects of Ral activity, which is involved in multiple cellular processes, are mediated through regulation of Rho GTPases. This study shows in vitro association of GEFmeso with the GTP-bound active form of Ral and the nucleotide-free form of the Rho GTPase Cdc42. GEFmeso fails to bind to other Rho GTPases, showing that Cdc42 is a specific interaction partner of this GEF. Unlike Ral and Cdc42, which are ubiquitously expressed, GEFmeso exerts distinct spatio-temporal expression patterns during embryonic development, suggesting a tissue-restricted function of the GEF in vivo. Based on previous observations that mutations in Cdc42 or overexpression of mutant alleles of Cdc42 lead to distinct effects on wing development, the effects of overexpression of dominant-negative and activated versions of Ral on wing development were analyzed. In addition, GEFmeso overexpression studies as well as RNAi experiments were performed. The results suggest that Ral, GEFmeso and Cdc42 act in the same developmental pathway and that GEFmeso mediates activation of Cdc42 in response to activated Ral in the context of Drosophila wing development (Blanke, 2006).

GEFmeso is expressed in spatially restricted patterns during embryogenesis and imaginal disc development. GEFmeso encodes at least two transcripts of different sizes. The longer transcript contains a N-terminal DH and PH domain in an arrangement that is characteristic for GEFs of the DBL family. GEFmeso is conserved in other insects such as Anopheles, but no direct vertebrate homologue could be identified. It interacts with constitutively active DRalG20V protein and, to a lower degree, with the dominant-negative mutant DRalS25N. Deletion analysis indicates that the Pro707-Ser830 interval of GEFmeso is the core region, which mediates DRal binding. This sequence interval lacks similarity to the Ral binding regions of previously identified mammalian Ral binding proteins such as RLIP76. It is noteworthy that these proteins also fail to have a common sequence motif for Ral binding (Blanke, 2006).

Ral has been implicated in the regulation of the cytoskeleton reorganization required for cell migration and cell shape changes. Both processes are also dependent on Rho GTPase activities. Furthermore, the Ral effector protein RLIP76/RalBP1/RIP1 functions as a GAP for Cdc42 and Rac, respectively. These results imply that the biological response to Ral activity could be mediated by these Rho GTPase activities. The current results provide additional evidence for a molecular link between DRal and Rho GTPase activity. GEFmeso binds to activated DRal and specifically associates with only one of the Rho-like GTPases of Drosophila, i.e., the nucleotide-depleted DCdc42. The finding that both DRal and DCdc42 are in vitro binding targets of GEFmeso and the fact that loss-of-function and gain-of-function experiments with DRal and GEFmeso transgenes in the wing result in DCdc42-like wing phenotypes suggest that both DRal and GEFmeso may act either in the same or a parallel genetic circuitry as DCdc42 (Blanke, 2006).

The expression of GEFmeso is restricted to distinct regions of the developing embryo including the prospective mesodermal region on the ventral side of the blastoderm embryo and a stripe pattern in the developing mesoderm as well as growing imaginal discs of the larvae. In contrast DRal and DCdc42 are ubiquitously expressed, suggesting that DRal and DCdc42 functions are spatio-temporally regulated by restricted expression of mediators like GEFmeso. In this context it is noteworthy to mention that the number of both GEFs and possible Rho GTPase substrates are increased during evolution, and that individual GEFs become more specialized in higher eukaryotes. For example, many of the mammalian DBL family members exert tissue- and cell type-specific expression patterns and can act on distinct subsets of Rho GTPases or on a single specific target Rho GTPase. This evolutionary mechanism allows further diversification of even those signaling pathways that are based on ubiquitously expressed key signaling molecules like, for example, small GTPases, which are in this way involved in diverse cellular processes (Blanke, 2006).

During gastrulation GEFmeso is likely to be required for the mesoderm invagination process. This conclusion is based on the finding that ubiquitous GEFmeso activity prior to and during gastrulation of the embryo impairs this process severely. Although it is not known whether this effect involves DRal and/or DCdc42 activities, the result underscores the need for a strictly regulated expression of GEFmeso and argues for a function of GEFmeso as a signaling component exerting spatio-temporal restricted activation of target proteins (Blanke, 2006).

The combined results are consistent with the proposal that GEFmeso acts as a spatio-temporal restricted signaling component that mediates DRal activity and provides a direct link between activated DRal and a downstream DCdc42-dependent developmental process. It is not known yet whether the DRal-GEFmeso-DCdc42 pathway is also linked to the previously established Ras-RalGDS-Ral or Rap-RGL-Ral signaling pathways. Nevertheless, the DRal-GEFmeso-DCdc42 cascade provides another example of a signaling pathway, where multiple small GTPases are linked by GEFs within one signaling cascade (Blanke, 2006).

Drosophila Cip4 and WASp define a branch of the Cdc42-Par6-aPKC pathway regulating E-Cadherin endocytosis

Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. This study shows that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. The Drosophila Cdc42-interacting protein 4 (Cip4) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. Altogether These results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue (Leibfried, 2008).

Cdc42 has been implicated in the regulation of polarity establishment in the early Drosophila embryo. The function was shown to be dependent upon the interaction of Cdc42 with the Baz-Par6-aPKC complex that promotes the exclusion of Lgl through Lgl phosphorylation by aPKC. However, the role of Cdc42 in epithelial tissue is unlikely to depend only on its regulation of aPKC because aPKC was shown to be dispensable for apico-basal polarity establishment in the Drosophila embryo. The role of Cdc42 in mammalian epithelial cells has so far been examined by the expression of constitutively active and dominant-negative forms of Cdc42, and such an examination has led to conflicting results in establishing the exact role of Cdc42 in apico-basal polarity maintenance. Nonetheless, they point toward an important role of Cdc42 in the regulation of polarized trafficking. The possible role of Cdc42 in polarized trafficking in epithelial cells was further strengthened by the identification of Cdc42 and the Par complex as regulators of endocytosis in both mammalian cells and C. elegans. Nevertheless, the precise role of Cdc42 and the Par complex in the regulation of endocytosis has remained poorly understood except in migrating cells in which the Par complex was shown to inhibit integrin endocytosis via Numb (Leibfried, 2008).

Cdc42 and its effector Drosophila Cip4 have been found to regulate E-Cad endocytosis and that their loss of function is associated with the formation of long tubular endocytic structures similar to what is observed upon blocking Dynamin function. It is therefore proposed that in Drosophila epithelial cells, Cdc42 controls the early steps of E-Cad endocytosis via Cip4. Because Cdc42, aPKC, and Par6 loss of function are associated with similar defects in E-Cad and Cip4 localization, a simple model is favored, in which the loss of aPKC or Par6 activity disrupts Cdc42 localization or activity and in turn prevents Cip4 function (Leibfried, 2008).

The identified role of PCH family of protein stems in part from the biochemical analysis of Toca-1 as a regulator of actin polymerization. Toca-1 is necessary to activate actin polymerization and actin comet formation downstream of PIP2 and Cdc42 in a WASp-dependent manner (Ho, 2004). On the basis of elegant biochemical assays, Toca-1 was further shown to be necessary to alleviate the WIP inhibitory activity on WASp, in order to allow efficient Arp2/3 activation by WASp (Ho, 2004). Toca-1 was proposed to play an essential role in the fine spatial and temporal regulation of actin polymerization in both cell migration and vesicle movement. Cip4 has been implicated in microtubule organizing center (MTOC) polarization in immune natural killer cells (Banerjee, 2007), a process in which Cdc42 and the Par complex are also involved. Importantly, because Cip4 was shown to bind microtubules, the interaction between Cdc42 and Cip4 might indicate that Cip4 might also be an effector of Cdc42-Par complex in the regulation of MTOC polarization (Leibfried, 2008).

In mammalian cells, regulation of endocytic-vesicle formation has been proposed to be dependent upon both branched actin-filament formation and Dynamin. The role of WASp and Arp2/3 in the regulation of E-Cad endocytosis may therefore indicate that Cip4, which is also known to form dimers, can promote vesicle scission by recruiting Dynamin and promoting actin polymerization via WASp. Therefore, it is proposed that Cip4 and WASp act as a link between Cdc42-Par6-aPKC and the early endocytic machinery to regulate E-Cadherin endocytosis in epithelial cells (Leibfried, 2008).

A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A

Epigenetic mechanisms regulate genome activation in diverse events, including normal development and cancerous transformation. Centromeres are epigenetically designated chromosomal regions that maintain genomic stability by directing chromosome segregation during cell division. The histone H3 variant CENP-A resides specifically at centromeres, is fundamental to centromere function and is thought to act as the epigenetic mark defining centromere loci. Mechanisms directing assembly of CENP-A nucleosomes have recently emerged, but how CENP-A is maintained after assembly is unknown. This study shows that a small GTPase switch functions to maintain newly assembled CENP-A nucleosomes. Using functional proteomics, it was found that MgcRacGAP (a Rho family GTPase activating protein) interacts with the CENP-A licensing factor HsKNL2. High-resolution live-cell imaging assays, designed in this study, demonstrated that MgcRacGAP, the Rho family guanine nucleotide exchange factor (GEF) Ect2, and the small GTPases Cdc42 and Rac, are required for stability of newly incorporated CENP-A at centromeres. Thus, a small GTPase switch ensures epigenetic centromere maintenance after loading of new CENP-A (Lagana, 2010).

Epigenetic regulation of genome activity is critical during development and stem cell maintenance, and increasing amounts of evidence highlight its importance in cancers. However, mechanisms controlling epigenetic regulation during a single cell cycle are generally less well understood, compared with those involved in transcriptional programmes. Centromere specification is an epigenetic regulatory event that controls genome activity at singular chromosomal loci and occurs each cell cycle. Nucleosomes that contain CENP-A are thought to epigenetically define centromeres. During DNA replication, centromere identity is maintained by segregating CENP-A equally to the two daughter chromosomes. Before the subsequent S-phase, additional CENP-A must be incorporated at centromeres, thus propagating the centromere epigenetic mark. Critical to this cycle is maintenance of the proper amount of CENP-A; too little or too much CENP-A incorporation could result in either loss of centromere identity or errors in chromosome segregation. This study describes a mechanism to ensure maintenance of the proper CENP-A levels during the cell cycle regulated by a Rho family small GTPase molecular switch (Lagana, 2010).

Proteomics and quantitative imaging assays were used to identify a previously unknown step in centromere maintenance. MgcRacGAP, together with the GEF ECT2, and their cognate small GTPase Cdc42 (or possibly Rac) specifically maintain CENP-A at centromeres. MgcRacGAP localization to centromeres at the end of G1 is incongruous with a role in CENP-A loading and strongly suggests that MgcRacGAP acts in maintenance and not licensing or loading of CENP-A. Pulse-chase analysis revealed that MgcRacGAP is required specifically for maintenance of newly incorporated CENP-A as old CENP-A from the previous cell cycle was present at normal levels at centromeres. Reciprocal immunoprecipitation of MgcRacGAP did not isolate HsKNL2, probably because of a large excess of MgcRacGAP bound to other known interacting proteins in the cytoplasm (data not shown). These results support the conclusion that a minor subset of MgcRacGAP is bound to HsKNL2 for a brief period each cell cycle and imply that non-overlapping MgcRacGAP-containing protein complexes function in cells. Overall, this work defines a new event in epigenetic centromere regulation and reveals its control by a small GTPase molecular switch (Lagana, 2010).

A model is proposed wherein the HsKNL2–Mis18 complex licenses centromeres for loading of new CENP-A by the combined activities of HJURP and CAF1. After loading (approximately 8–12 h after anaphase onset), HsKNL2–Mis18 recruits Cdc42. The activity of Cdc42 is required for preservation of newly incorporated CENP-A and thus finalizes centromere repopulation. Cdc42 activity requires GTPase cycling facilitated by MgcRacGAP and the GEF ECT2. The results predict that newly incorporated CENP-A is distinct from CENP-A remaining from the previous cell cycle and can be recognized and removed. It is proposed that Cdc42 activity modifies (by either adding or removing a mark on) newly incorporated CENP-A, rendering it identical to old CENP-A. The manifestation of this mark could be any distinguishing modification, including but not limited to, recruitment of an additional protein, conformational change of the CENP-A nucleosome, or any of a range of post-translational modifications. New CENP-A that is not modified would be recognized as erroneously incorporated and removed from chromatin during a late-G1 surveillance step, or during DNA replication (Lagana, 2010).

In budding yeast, excess CENP-A (CSE-4) mislocalized to the chromosome arms is removed and selectively degraded through a proteasome-based mechanism. If this mechanism is conserved in human cells, it is expected to be less stringent, as overexpressed CENP-A localizes diffusely to chromosome arms without causing obvious defects in cell division. Alternatively or additionally, centromere maintenance could involve the chromatin remodelling protein RSF-1, which is required for CENP-A nucleosome stability. However, because RSF-1 is proposed to function in mid-G1 before MgcRacGAP and Cdc42 localize to centromeres, it is unlikely to be the downstream target of small GTPase activity at centromeres (Perpelescu, 2009). Regardless of the removal mechanism, it is proposed that a GTPase switch is spatially and temporally restricted through regulated localization to centromeres precisely after CENP-A doubling to promote the removal of spurious CENP-A (either excess at centromeres, or outside true centromere loci). By restricting centromere size, this 'quality control' mechanism helps to ensure proper centromere function and kinetochore assembly, thus preventing aneuploidy. Furthermore, it is possible that this mechanistic theme will apply to other epigenetic events that contribute to genomic regulation (Lagana, 2010).

Zizimin-related (Zir), a Rho guanine nucleotide exchange factor (RhoGEF) homologous to the mammalian Dock-C/Zizimin-related family, was identified in a screen to find new genes involved in the Drosophila cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. RhoGEFs activate Rho-family GTPases, which are known to be central regulators of cell migration, spreading and polarity. When a parasitoid wasp is recognized as foreign, multiple layers of circulating immunosurveillance cells (haemocytes) should attach to the egg. In Zir mutants this process is disrupted and lamellocytes, a haemocyte subtype, fail to properly encapsulate the wasp egg. Furthermore, macrophage-like plasmatocytes exhibit a strong reduction in their ability to phagocytise Escherichia coli and Staphylococcus aureus bacteria. During encapsulation and phagocytosis Zir genetically interacts with two Rho-family GTPases, Rac2 and Cdc42. Finally, Zir is dispensable for the humoral immune response against bacteria. It is proposed that Zir is necessary to activate the Rho-family GTPases Rac2 and Cdc42 during the Drosophila cellular immune response (Sampson, 2012).

Cdc42: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation and Ectopic Expression | References

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