dPak is required for integrity of the leading edge cytoskeleton during Drosophila dorsal closure but does not signal through the JNK cascade

The Pak kinases are effectors for the small GTPases Rac and Cdc42 and are divided into two subfamilies. Group I Paks possess an autoinhibitory domain that can suppress their kinase activity in trans. In Drosophila, two Group I kinases have been identified, dPak and Pak3. Rac and Cdc42 participate in dorsal closure of the embryo, a process in which a hole in the dorsal epidermis is sealed through migration of the epidermal flanks over a tissue called the amnioserosa. Dorsal closure is driven in part by an actomyosin contractile apparatus at the leading edge of the epidermis, and is regulated by a Jun amino terminal kinase (JNK) cascade. Impairment of dPak function using either loss-of-function mutations or expression of a transgene encoding the autoinhibitory domain of dPak led to disruption of the leading edge cytoskeleton and defects in dorsal closure but did not affect the JNK cascade. Group I Pak kinase activity in the amnioserosa is required for correct morphogenesis of the epidermis, and may be a component of the signaling known to occur between these two tissues. We conclude that dorsal closure requires Group I Pak function in both the amnioserosa and the epidermis.     

A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures.

Changes in cell morphology are essential in the development of a multicellular organism. The regulation of the cytoskeleton by the Rho subfamily of small GTP-binding proteins is an important determinant of cell shape. The Rho subfamily has been shown to participate in a variety of morphogenetic processes during Drosophila melanogaster development. We describe here a Drosophila homolog, DPAK, of the serine/threonine kinase PAK, a protein which is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK bound to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK was elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of the embryo. We have previously shown that the accumulation of cytoskeletal elements initiating cell shape changes in these cells could be inhibited by expression of a dominant-negative DRacA transgene. We show that leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. We propose that DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure.     

Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.     

Epidermal growth factor stimulation of the ACK1/Dbl pathway in a Cdc42 and Grb2-dependent manner

The tyrosine kinase ACK1 phosphorylates and activates the guanine nucleotide exchange factor Dbl, which in turn directs the Rho family GTP-binding proteins. However, the regulatory mechanism of ACK1/Dbl signaling in response to extracellular stimuli remains poorly understood. Here we describe that epidermal growth factor stimulates the ACK1/Dbl pathway, leading to actin cytoskeletal rearrangements. The role of the two ACK1-binding proteins Cdc42 and Grb2 was assessed by overexpression of the Cdc42/Rac interactive binding domain and a dominant-negative Grb2 mutant, respectively. Specific inhibition of the interaction of ACK1 with Cdc42 or Grb2 by the use of these constructs diminished tyrosine phosphorylation of both ACK1 and Dbl in response to EGF. Therefore, the activation of ACK1 and subsequent downstream signaling require both Cdc42-dependent and Grb2-dependent processes within the cell. In addition, we show that EGF transiently induces formation of the focal complex and stress fibers when ACK1 was ectopically expressed. The induction of these structures was totally sensitive to the action of botulinum toxin C from Clostridium botulinum, suggesting a pivotal role of Rho. These results provide evidence that ACK1 acts as a mediator of EGF signals to Rho family GTP-binding proteins through phosphorylation and activation of GEFs such as Dbl.     

Identification of the region in Cdc42 that confers the binding specificity to activated Cdc42-associated kinase

The Rho family small G-protein Cdc42 has been implicated in a diversity of biological functions. Multiple downstream effectors have been identified. How Cdc42 discriminates the interaction with its multiple downstream effectors is not known. Activated Cdc42-associated tyrosine kinase (ACK) is a very specific effector of Cdc42. To delineate the Cdc42 signaling pathway mediated by ACK, we set about to identify the specific ACK-binding region in Cdc42. We utilized TC10, another member of the Rho family of G-proteins that is 66.7% identical to Cdc42, to construct TC10/Cdc42 chimeras for screening the specific ACK-binding region in Cdc42. A region between switch I and switch II has been identified as the specific ACK-binding (AB) region. The replacement of the AB region with the corresponding region in TC10 resulted in the complete loss of ACK-binding ability but did not affect the binding to WASP, suggesting that the AB region confers the binding specificity to ACK. On the other hand, replacement of the corresponding region of TC10 with the AB region enabled TC10 to acquire ACK-binding ability. Eight residues are different between the AB region and the corresponding region of TC10. The mutational analysis indicated that all eight residues contribute to the binding to ACK2. The assays for the Cdc42-mediated activation of ACK2 indicated that the AB region is essential for Cdc42 to activate ACK2 in cells. Thus, our studies have defined a specific ACK-binding region in Cdc42 and have provided a molecular basis for generating ACK binding-defective mutants of Cdc42 to delineate ACK-mediated signaling pathway.     

Drosophila Dok is required for embryonic dorsal closure

Embryonic dorsal closure (DC) in Drosophila is a series of morphogenetic movements involving the bilateral dorsal movement of the epidermis (cell stretching) and dorsal suturing of the leading edge (LE) cells to enclose the viscera. The Syk family tyrosine kinase Shark plays a crucial role in this Jun amino-terminal kinase (JNK)-dependent process, where it acts upstream of JNK in LE cells. Using a yeast two-hybrid screen, the unique Drosophila homolog of the downstream of kinase (Dok) family, Ddok, was identified by its ability to bind Shark SH2 domains in a tyrosine phosphorylation-dependent fashion. In cultured S2 embryonic cells, Ddok tyrosine phosphorylation is Src dependent; Shark associates with Ddok and Ddok localizes at the cell cortex, together with a portion of the Shark protein. The embryonic expression pattern of Ddok resembles the expression pattern of Shark. Ddok loss-of-function mutant (Ddok(PG155)) germ-line clones possess DC defects, including the loss of JNK-dependent expression of dpp mRNA in LE cells, and decreased epidermal F-actin staining and LE actin cable formation. Epistatic analysis indicates that Ddok functions upstream of shark to activate JNK signaling during DC. Consistent with these observations, Ddok mutant embryos exhibit decreased levels of tyrosine phosphorylated Shark at the cell periphery of LE and epidermal cells. As there are six mammalian Dok family members that exhibit some functional redundancy, analysis of the regulation of DC by Ddok is expected to provide novel insights into the function of the Dok adapter proteins.     

Epidermal growth factor-dependent regulation of Cdc42 is mediated by the Src tyrosine kinase

Treatment of cells with epidermal growth factor (EGF) promotes the activation of the small GTP-binding protein Cdc42, as well as its phosphorylation in cells. The EGF-dependent phosphorylation of Cdc42 occurs at tyrosine 64 in the Switch II domain and appears to be mediated through the Src tyrosine kinase, because both the expression of a dominant-negative Src mutant (mouse Src(K297R)) and treatment of cells with the Src kinase inhibitor PP2 blocks the EGF-stimulated phosphorylation of Cdc42, whereas expression of an activated Src mutant (Src(Y529F)) promotes phosphorylation in the absence of EGF treatment. The EGF-stimulated phosphorylation of Cdc42 is not required for its activation, nor does it directly affect the interactions of activated Cdc42 with target/effector proteins including PAK, ACK, WASP, or IQGAP. However, the EGF-stimulated phosphorylation of Cdc42 is accompanied by an enhancement in the interaction of Cdc42 with the Rho-GDP dissociation inhibitor (RhoGDI). The EGF-stimulated activation of Cdc42 does require activated Src, as well as the Vav2 protein, a member of the Dbl family of guanine nucleotide exchange factors. Src catalyzes the tyrosine phosphorylation of Vav2, and overexpression of Vav2 together with activated Src (Src(Y529F)) can completely bypass the need for EGF to promote the activation of Cdc42. Thus, EGF signaling through Src appears to have dual regulatory effects on Cdc42: 1). it leads to the activation of Cdc42 as mediated by the Vav2 guanine nucleotide exchange factor, and 2). it results in the phosphorylation of Cdc42, which stimulates the binding of RhoGDI, perhaps to direct the movement of Cdc42 to a specific cellular site to trigger a signaling response, because Cdc42-RhoGDI interactions are essential for Cdc42-induced cellular transformation.     

Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila

Src42A is one of the two Src homologs in Drosophila. Src42A protein accumulates at sites of cell-cell or cell-matrix adhesion. Anti-Engrailed antibody staining of Src42A protein-null mutant embryos indicated that Src42A is essential for proper cell-cell matching during dorsal closure. Src42A, which is functionally redundant to Src64, was found to interact genetically with shotgun, a gene encoding E-cadherin, and armadillo, a Drosophila beta-catenin. Immunoprecipitation and a pull-down assay indicated that Src42A forms a ternary complex with E-cadherin and Armadillo, and that Src42A binds to Armadillo repeats via a 14 amino acid region, which contains the major autophosphorylation site. The leading edge of Src mutant embryos exhibiting the dorsal open phenotype was frequently kinked and associated with significant reduction in E-cadherin, Armadillo and F-actin accumulation, suggesting that not only Src signaling but also Src-dependent adherens-junction stabilization would appear likely to be essential for normal dorsal closure. Src42A and Src64 were required for Armadillo tyrosine residue phosphorylation but Src activity may not be directly involved in Armadillo tyrosine residue phosphorylation at the adherens junction.     

The tyrosine kinase ACK1 associates with clathrin-coated vesicles through a binding motif shared by arrestin and other adaptors

One target for the small GTPase Cdc42 is the nonreceptor tyrosine kinase activated Cdc42-associated kinase (ACK), which binds selectively to Cdc42.GTP. We report that ACK1 can associate directly with the heavy chain of clathrin. A central region in ACK1 containing a conserved motif behaves as a clathrin adaptor and competes with beta-arrestin for a common binding site on the clathrin N-terminal head domain. Overexpressed ACK1 perturbs clathrin distribution, an activity dependent on the presence of C-terminal "adaptor" sequences that are also present in the related nonkinase gene 33. ACK1 interacts with the adaptor Nck via SH3 interactions but does not form a trimeric complex with p21-activated serine/threonine kinase, which also binds Nck. Stable low level expression of green fluorescent protein-ACK1 in NIH 3T3 cells has been used to localize ACK1 to clathrin-containing vesicles. The co-localization of ACK1 in vivo with clathrin and AP-2 indicates that it participates in trafficking, underlying an ability to increase receptor-mediated transferrin uptake.     

Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1

Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.     

Phosphorylation of WASP by the Cdc42-associated kinase ACK1: dual hydroxyamino acid specificity in a tyrosine kinase

ACK1 is a nonreceptor tyrosine kinase that associates specifically with Cdc42. Relatively few ACK1 substrates and interacting proteins have been identified. In this study, we demonstrated that ACK1 phosphorylates the Wiskott-Aldrich syndrome protein (WASP), a Cdc42 effector that plays an important role in the formation of new actin filaments. ACK1 and WASP interact in intact cells, and overexpression of ACK1 promotes WASP phosphorylation. Phosphorylation of WASP in vitro was enhanced by the addition of Cdc42 or phosphatidylinositol 4,5-biphosphate, presumably due to release of the autoinhibitory interactions in WASP. Surprisingly, when we mapped the sites of WASP phosphorylation, we found that ACK1 possesses significant serine kinase activity toward WASP (directed at Ser-242), as well as tyrosine kinase activity directed at Tyr-256. A serine peptide derived from the Ser-242 WASP phosphorylation site is also a substrate for ACK1. ACK1 expressed in bacteria retained its serine kinase activity, eliminating the possibility of contamination with a copurifying kinase. Serine phosphorylation of WASP enhanced the ability of WASP to stimulate actin polymerization in mammalian cell lysates. Thus, the tyrosine kinase ACK1 acts as a dual specificity kinase toward this substrate. In contrast to other dual specificity kinases that more closely resemble Ser/Thr kinases, ACK1 is a tyrosine kinase with an active site that can accommodate both types of hydroxyamino acids in substrates.     

Activation of the guanine nucleotide exchange factor Dbl following ACK1-dependent tyrosine phosphorylation

Signals triggered by diverse receptors modulate the activity of Rho family proteins, although the regulatory mechanism remains largely unknown. On the basis of their biochemical activity as guanine nucleotide exchange factors (GEFs), Dbl family proteins are believed to be implicated in the regulation of Rho family GTP-binding proteins in response to a variety of extracellular stimuli. Here we show that GEF activity of full-length proto-Dbl is enhanced upon tyrosine phosphorylation. When transiently coexpressed with the activated form of the non-receptor tyrosine kinase ACK1, a downstream target of Cdc42, Dbl became tyrosine-phosphorylated. In vitro GEF activity of Dbl toward Rho and Cdc42 was augmented following tyrosine phosphorylation. Moreover, accumulation of the GTP-bound form of Rho and Rac within the cell paralleled ACK-1-dependent tyrosine phosphorylation of Dbl. Consistently, activation of c-Jun N-terminal kinase downstream of Rho family GTP-binding proteins was also enhanced when Dbl was tyrosine-phosphorylated. Collectively, these findings suggest that the tyrosine kinase ACK1 may act as a regulator of Dbl, which in turn activates Rho family proteins.     

Crystal structures of the phosphorylated and unphosphorylated kinase domains of the Cdc42-associated tyrosine kinase ACK1

ACK1 is a multidomain non-receptor tyrosine kinase that is an effector of the Cdc42 GTPase. Members of the ACK family have a unique domain ordering and are the only tyrosine kinases known to interact with Cdc42. In contrast with many protein kinases, ACK1 has only a modest increase in activity upon phosphorylation. We have solved the crystal structures of the human ACK1 kinase domain in both the unphosphorylated and phosphorylated states. Comparison of these structures reveals that ACK1 adopts an activated conformation independent of phosphorylation. Furthermore, the unphosphorylated activation loop is structured, and its conformation resembles that seen in activated tyrosine kinases. In addition to the apo structure, complexes are also presented with a non-hydrolyzable nucleotide analog (adenosine 5'-(beta,gamma-methylenetriphosphate)) and with the natural product debromohymenialdisine, a general inhibitor of many protein kinases. Analysis of these structures reveals a typical kinase fold, a pre-organization into the activated conformation, and an unusual substrate-binding cleft.     

The Rac GTPase-activating protein RotundRacGAP interferes with Drac1 and Dcdc42 signalling in Drosophila melanogaster

RhoGTPases are negatively regulated by GTPase-activating proteins (GAPs). Here we demonstrate that Drosophila RotundRacGAP is active in vitro on Drac1 and Dcdc42 but not Drho1. Similarly, in yeast, RotundRacGAP interacts specifically with Drac1 and Dcdc42, as well as with their activated V12 forms, showing a particularly strong interaction with Dcdc42V12. In the fly, lowering RotundRacGAP dosage specifically modifies eye defects induced by expressing Drac1 or Dcdc42 but not Drho1, confirming that Drac1 and Dcdc42 are indeed in vivo targets of RotundRacGAP. Furthermore, embryonic-directed expression of either RotundRacGAP, or dominant negative Drac1N17, transgenes induces similar defects in dorsal closure and inhibits Drac1-dependent cytoskeleton assembly at the leading edge. Expression of truncated forms of RotundRacGAP shows that the GAP domain of RotundRacGAP is essential for its function. Unexpectedly, transgenes encoding Drac1N17, Dcdc42N17, or RotundRacGAP do not affect the c-Jun N-terminal kinase-dependent gene expression of decapentaplegic and puckered, indicating that another Drac1-independent signal redundantly activates this pathway. Finally, in a situation where Drac1 is constitutively activated, RotundRacGAP greatly reduces the ectopic expression of decapentaplegic, possibly by negatively regulating Dcdc42.     

Activated Cdc42-associated kinase 1 is a component of EGF receptor signaling complex and regulates EGF receptor degradation

Cdc42-associated tyrosine kinase 1 (ACK1) is a specific down-stream effector of Cdc42, a Rho family small G-protein. Previous studies have shown that ACK1 interacts with clathrin heavy chain and is involved in clathrin-coated vesicle endocytosis. Here we report that ACK1 interacted with epidermal growth factor receptor (EGFR) upon EGF stimulation via a region at carboxy terminus that is highly homologous to Gene-33/Mig-6/RALT. The interaction of ACK1 with EGFR was dependent on the kinase activity or tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 was colocalized with EGFR on EEA-1 positive vesicles upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNAi inhibited ligand-induced degradation of EGFR upon EGF stimulation, suggesting that ACK1 plays an important role in regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin-association (Uba) domain at the carboxy terminus, ACK1 binds to both poly- and mono-ubiquitin. Overexpression of the Uba domain-deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Taken together, our studies suggest that ACK1 senses signal of EGF and regulates ligand-induced degradation of EGFR.     

The Fes/Fer non-receptor tyrosine kinase cooperates with Src42A to regulate dorsal closure in Drosophila

Fes/Fer non-receptor tyrosine kinases regulate cell adhesion and cytoskeletal reorganisation through the modification of adherens junctions. Unregulated Fes/Fer kinase activity has been shown to lead to tumours in vivo. Here, we show that Drosophila Fer localises to adherens junctions in the dorsal epidermis and regulates a major morphological event, dorsal closure. Mutations in Src42A cause defects in dorsal closure similar to those seen in dfer mutant embryos. Furthermore, Src42A mutations enhance the dfer mutant phenotype, suggesting that Src42A and DFer act in the same cellular process. We show that DFer is required for the formation of the actin cable in leading edge cells and for normal rates of dorsal closure. We have isolated a gain-of-function mutation in dfer (dfergof) that expresses an N-terminally fused form of the protein, similar to oncogenic forms of vertebrate Fer. dfergof blocks dorsal closure and causes axon misrouting. We find that in dfer loss-of-function mutants beta-catenin is hypophosphorylated, whereas in dfergof beta-catenin is hyperphosphorylated. Phosphorylated beta-catenin is removed from adherens junctions and degraded, thus implicating DFer in the regulation of adherens junctions.     

CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium

Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.