InteractiveFly: GeneBrief

Activated Cdc42 kinase: Biological Overview | References

Activated Cdc42 kinases (Acks) are evolutionarily conserved non-receptor tyrosine kinases. Activating somatic mutations and increased ACK1 protein levels have been found in many types of human cancers and correlate with a poor prognosis. ACK1 is activated by epidermal growth factor (EGF) receptor signaling and functions to regulate EGF receptor turnover. ACK1 has additionally been found to propagate downstream signals through the phosphorylation of cancer relevant substrates. Using Drosophila as a model organism, it has been determined that Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling. Ack anti-apoptotic signaling does not function through enhancement of EGF stimulated MAP kinase signaling, suggesting that it must function through phosphorylation of some unknown effector. Several putative Drosophila Ack interacting proteins were isolated, many being orthologs of previously identified human ACK1 interacting proteins. Two of these interacting proteins, Drk and Yorkie, were found to influence Ack signaling. Drk is the Drosophila homolog of GRB2, which is required to couple ACK1 binding to receptor tyrosine kinases. Drk knockdown blocks Ack survival activity, suggesting that Ack localization is important for its pro-survival activity. Yorkie is a transcriptional co-activator that is downstream of the Salvador-Hippo-Warts pathway and promotes transcription of proliferative and anti-apoptotic genes. yorkie and Ack were found to synergistically interact to produce tissue overgrowth, and yorkie loss of function interferes with Ack anti-apoptotic signaling. These results demonstrate how increased Ack signaling could contribute to cancer when coupled to proliferative signals (Schoenherr, 2012).

The founding member of tha ACK family is human ACK1, which was identified as a protein that binds to CDC42 in its active GTP bound form (Manser, 1993). Since this discovery Ack homologs have been found in chordates, arthropods and nematodes. Ack family members can be divided into three structural categories based on the presence or absence of four conserved domain motifs. All Ack family members contain an amino-terminal tyrosine kinase domain that is flanked by a sterile alpha motif (SAM) and a Src homology 3 (SH3) domain. The carboxy-terminal half of these kinases contains short proline rich sequences, but lacks any identifiable domains, with the exception of two tandemly repeated ubiquitin-associated (UBA) domains located at the extreme carboxy-terminus. ACK1 UBA domains have been shown to interact with both mono and poly-ubiquitinated proteins and are thought to play a role in ACK1 protein turnover (Chan, 2009). The Caenorhabditis elegans Ack homolog, Ark-1, contains no UBA domains, placing it in a class by itself. The other two Ack structural classes can be distinguished by the presence or absence of a Cdc42/Rac interactive binding (CRIB) domain. Human ACK1 and Drosophila PR2 are representative members of the CRIB domain containing structural class, while human TNK1 and Drosophila Ack are members of the structural class lacking a conserved CRIB domain. Variants containing a CRIB domain bind GTP liganded CDC42, but this interaction does not appear to directly influence Ack activity in vitro (Schoenherr, 2012).

Human ACK1 is the most well characterized member of the Ack family. Early studies uncovered a role for ACK1 in the promotion of internalization and down-regulation of activated epidermal growth factor (EGF) receptor. ACK1 tyrosine phosphorylation is enhanced and ACK1 is co-localized with EGF receptor after EGF stimulation (Yang, 2001a; Yang, 2001b). Knockdown of ACK1 reduces the rate of EGF receptor degradation following EGF stimulation (Shen, 2007). While on the surface these data suggest that ACK1 merely serves as a negative regulator of growth factor signaling, ACK1 activation may additionally propagate downstream signaling. Recent studies support this latter alternative by uncovering a role for ACK1 as a positive transducer of cell surface receptor signaling that promotes growth and survival by ACK1 mediated phosphorylation and activation of downstream components, including AKT (Mahajan, 2010) and the androgen receptor (Mahajan, 2007; Schoenherr, 2012 and references therein).

A pro-survival role for Ack function is consistent with reported links between activation of Ack family members and cancer genesis and metastasis. Several somatic missense mutations have been identified in ACK1 from cancer tissue samples that increase ACK1 autophosphorylation and promote cellular proliferation and migration. Amplification of the ACK1 gene in tumors correlates with a poor prognosis, and ACK1 overexpression in cancer cell lines increases invasiveness in a mouse metastasis model, while knockdown of ACK1 reduces the migration of human breast cancer cells (Schoenherr, 2012).

Activated ACK1 has been detected in advanced human prostate cancers where it has been shown to phosphorylate three cancer relevant substrates in prostate cancer cell lines: WWOX, AKT, and androgen receptor. WWOX spans the FRA16D chromosomal fragile site that is frequently disrupted in human cancers. While the molecular function of WWOX is not known, it has been shown that the growth of tumor cells lacking WWOX is strongly inhibited by restoring WWOX expression. ACK1 phosphorylation of WWOX leads to the polyubiquitination and degradation of WWOX, which correlates with a tumorigenic role. AKT is a serine/threonine kinase whose activity promotes cell survival and proliferation, while deregulation of the AKT signaling pathway is commonly associated with cancer. ACK1 activation results in tyrosine phosphorylation and apparent activation of AKT in a PI3K independent mechanism. Finally, the activity of the androgen receptor is required for growth of prostate cells. In advanced stages of prostate cancer, these cells lose their dependence on androgens for activation of this receptor to become androgen independent prostate cancer. ACK1 has been found to phosphorylate the androgen receptor, promote androgen independent growth of prostate cells, and activate transcription of androgen inducible genes in the absence of androgen (Schoenherr, 2012).

Less is known about the function of Ack family members lacking CRIB domains, and published studies on TNK1 describe conflicting functions. TNK1 overexpression in cell culture lines inhibits cell growth in a kinase dependent manner. Mutant mice having deletions in the kinase domain of TNK1 develop spontaneous tumors at a high frequency, which is thought to originate from hyperactivation of Ras signaling and suggests that TNK1 functions as a tumor suppressor. In contrast to this function, TNK1 was identified as a potentially oncogenic tyrosine kinase in a mutagenesis screen and activated TNK1 was found in Hodgkin's lymphoma. It is possible that these conflicting findings reflect tissue specific responses or complex dosage sensitivity to TNK1 loss and gain of function (Schoenherr, 2012 and references therein).

In order to better understand the physiological role of Ack family members and determine how Ack might contribute to cancer, genetic and biochemical experiments were conducted in the model organism Drosophila melanogaster. These studies focus on Drosophila Ack, which has a domain structure resembling human TNK1, but shares significantly higher sequence identity with ACK1 in all conserved domains including the kinase domain activation loop. It was found that Drosophila Ack possesses potent anti-apoptotic properties that function downstream of EGF receptor signaling through an unknown mechanism. This activity is dependent on Ack kinase function and can be further stimulated by increased Ras signaling. A protein interaction study was conducted, and it was found that many of the same proteins that associate with human ACK1 also bind to fly Ack. The influence of these proteins on Ack anti-apoptotic activity was tested, and it was determined that the adapter protein Drk (GRB2) is required for this activity, while the transcriptional co-activator protein Yki (YAP) functions synergistically with Ack to promote cell survival and massive tissue overgrowth. These findings support both anti-apoptotic and proliferative roles for Ack family members, which may contribute to cancer genesis and progression (Schoenherr, 2012).

Drosophila has two Ack family members: Ack and PR2. Ack possesses anti-apoptotic properties, while PR2 either does not possess anti-apoptotic properties or requires activators not present in the assay system. While Ack may appear to be more closely related to vertebrate TNK1 because both proteins lack a CRIB domain, Ack is most similar to vertebrate ACK1 based on sequence identity of all shared protein domains. Additionally it was found that many of the proteins that interact with ACK1 also interact with fly Ack. Therefore, the conclusions drawn in this study will likely be applicable to vertebrate ACK1 function (Schoenherr, 2012).

Ack function can suppress programmed cell death induced by Hid or Rpr expression in the developing eye and wing discs. Overexpression studies reveal that Ack kinase activity is required for suppression of apoptosis induced by hid but is unnecessary for suppression of rpr-induced apoptosis. It was further shown that Ack loss of function enhances cell death induced by expression of both of these genes, and it was determined that Ack is critically required for the survival of rpr expressing eye tissue. The molecular mechanisms underlying these differential requirements are not known. hid and rpr are known to function in a multimeric protein complex on the mitochondria outer membrane to promote apoptosis. Both hid and rpr are able to stimulate apoptosis by competing with initiator and effector caspases for DIAP binding, but rpr additionally induces DIAP auto-ubiquitination leading to DIAP degradation. While Ack kinase activity may be important for aspects of hid complex regulation, it is tempting to speculate that the UBA domains of Ack may play a critical role in the modulation of DIAP or Rpr ubiquitination and stability (Schoenherr, 2012).

EGF receptor signaling has been shown to activate ACK1 in vertebrates, and EGF signaling was found to enhance the anti-apoptotic function of Ack in Drosophila. ACK1 negatively regulates EGF receptor signaling by stimulating endocytosis of activated receptor complexes. The evidence supports the idea that Drosophila Ack, in conjunction with SH3PX1, functions in a similar manner, which will be described elsewhere. In Drosophila, EGF signaling is anti-apoptotic through the activation of MAPK, which phosphorylates and inactivates Hid. If Ack affected apoptosis exclusively through attenuation of EGF signaling, then it would be expected that Ack loss of function would be anti-apoptotic while gain of function would be pro-apoptotic, which is opposite to what was observed. By using the hidAla5 mutant, it was demonstratde that Ack does not modulate programmed cell death through activation of MAPK (Schoenherr, 2012).

The anti-apoptotic function of Ack is surprisingly robust compared to other proteins that were have tested. The studies show that activity of the kinase domain contributes to Ack anti-apoptotic function. Based on this, it is concluded that Ack propagates anti-apoptotic signals by phosphorylating downstream targets. Several ACK1 substrates have been identified that are attractive candidates for the regulation of programmed cell death: the putative tumor suppressor WWOX, the apoptosis inhibiting protein kinase AKT and the caspase-cleaved ubiquitin E3 ligase NEDD4. Akt1 loss and gain of function alleles and Nedd4 RNAi fail to significantly modify hid induced small eye phenotypes. Wwox RNAi is able to suppress hid induced apoptosis but not nearly as robustly as Ack expression. Since ACK1 mediated phosphorylation of WWOX leads to WWOX destruction, it would be predicted that Wwox RNAi would phenocopy Ack expression in the assay system, but it is unable to reproduce the magnitude of Ack anti-apoptotic function. This does not rule out Wwox as an anti-apoptotic substrate target of Ack, but it demonstrates that Wwox is not the only cell death relevant substrate of Ack. It is worth noting that hid and rpr act fairly late within the programmed cell death pathway, being just a step upstream of initiator caspase activation. Therefore, Ack must target substrates that have activities influencing hid, rpr or events at the level of caspase activation (Schoenherr, 2012).

Several Ack physically interacting proteins were identified using a tandem affinity purification strategy. Many of these have vertebrate homologs that have been previously determined to interact with ACK1. Drk and Yki have the most pronounced effect on Ack's anti-apoptotic properties, and their contribution to Ack signaling was further characterized. Drk is the fly ortholog of vertebrate GRB2, which has previously been described as an ACK1 and TNK1 interacting protein. In the case of TNK1, GRB2 is tyrosine phosphorylated by TNK1, which disrupts the ability of GRB2/SOS complexes to activate Ras. This does not appear to be the case for Drosophila Ack, because even though Drk forms a complex with tyrosine phosphorylated Ack, no evidence of tyrosine phosphorylation on Drk was found. Rather, the data support that Drk association with Ack is required for Ack anti-apoptotic properties. It is proposed that Drk SH3 domains likely interact with PXXP motifs in the C-terminal half of Ack similar to the interaction described in vertebrates (Galisteo, 2006). This interaction could then lead to the recruitment of Ack into protein complexes required for Ack activation (Schoenherr, 2012).

Yki is a transcriptional co-activator that regulates expression of genes with proliferative and anti-apoptotic functions. The vertebrate homolog of Yki is Yes Associated Protein (YAP), which has not previously been identified as an ACK1 or TNK1 interacting protein. Yki and YAP studies have focused primarily on the pathways that regulate their function as transcription factors. Given the role of Yki and YAP in transcriptional control of proliferative and anti-apoptotic genes, it would seem likely that Ack activity leads to enhancement of Yki function. However, this does not appear to be the case because Ack overexpression does not lead to increased Yki nuclear localization or increased expression of yki target genes. Rather, the data indicate that Yki directly interacts with Ack in the cytoplasm and functions to regulate Ack activity. In support of this, it was found that Ack colocalizes with Yki, and yki dosage reduction suppresses Ack anti-apoptotic function. Yki contains two WW domains, which may interact with conserved PPXY motifs that are present in the region flanked by the SH3 and UBA domains of Ack family members. In vertebrates, these PPXY motifs have been shown to interact with WWOX, which also contains two WW domains. Further studies are required to define how Yki and Ack interact (Schoenherr, 2012).

Yki expression in the fly eye produces an overgrowth phenotype that is indicative of its role in regulating proliferation. Ack overexpression produces a slightly larger eye due to inhibition of apoptotic events that occur during normal eye development. Simultaneous expression of yki and Ack results in a synergistic effect that produces enormous eyes. These results reveal that in addition to anti-apoptotic function, Ack can also enhance proliferation. This illustrates how increased Ack signaling could contribute to cancer when coupled to proliferative signals. Indeed, the results are consistent with recent reports of ACK1 activating somatic mutations and gene amplification being associated with human cancers. At present the key anti-apoptotic substrates of Ack and their mechanisms of action remain to be determined. With their discovery will come a better understanding of Ack signaling and potentially new targets for cancer interventions (Schoenherr, 2012).

Leading edge-secreted Dpp cooperates with ACK-dependent signaling from the amnioserosa to regulate myosin levels during dorsal closure

Dorsal closure of the Drosophila embryo is an epithelial fusion in which the epidermal flanks migrate to close a hole in the epidermis occupied by the amnioserosa, a process driven in part by myosin-dependent cell shape change. Dpp signaling is required for the morphogenesis of both tissues, where it promotes transcription of myosin from the zipper (zip) gene. Drosophila has two members of the Activated Cdc42-associated Kinase (ACK) family: DACK and PR2. Overexpression of DACK in embryos deficient in Dpp signaling can restore zip expression and suppress dorsal closure defects, while reducing the levels of DACK and PR2 simultaneously using mutations or amnioserosa-specific knock down by RNAi results in loss of zip expression. ACK function in the amnioserosa may generate a signal cooperating with Dpp secreted from the epidermis in driving zip expression in these two tissues, ensuring that cell shape changes in dorsal closure occur in a coordinated manner (Zahedi, 2008).

The results on the regulation of zip expression by Dpp are consistent with the model of Fernandez and colleagues in which two rounds of dpp expression in the DME cells regulate dorsal closure (Fernandez, 2007). In the first round of dpp expression, before completion of germband retraction, Dpp signals from the DME cells to the amnioserosa. This signaling can be visualized by pMad staining in the amnioserosa, which is obvious during germband retraction but fades away by the beginning of dorsal closure, a pattern paralleled by zip expression. In the second round of signaling, occurring during dorsal closure, Dpp signals to the dorsal epidermis, as demonstrated by robust pMad in this tissue and high zip levels in the DME cells. Dpp signaling and ACK function are both necessary but not sufficient for zip expression in the embryo during dorsal closure. Activation of either Dpp signaling or ACK function in prd stripes does not lead to ectopic zip expression, indicating that in each case additional inputs are required. DACK is able to elevate zip expression only on the dorsal side of the embryo in regions where zip is normally expressed, indicating that required additional inputs are present; it is proposed that one such input is Dpp signaling. When DACK is overexpressed in the amnioserosa, either in prd stripes or throughout the whole tissue, its effects on zip expression are non–cell-autonomous, leading to up-regulation of zip expression throughout the dorsal side of the embryo. With regard to the zip expression pattern, seen with prd-Gal4-driven DACK transgenes, a diffusible signal emitted from prd stripes in the amnioserosa could attain a fairly uniform distribution over the dorsal side of the embryo. It is proposed that Dpp secreted from the DME cells cooperates with a diffusible signal from the amnioserosa (regulated by ACK in a kinase-independent manner) to drive coordinated zip expression in these two tissues. ACK appears to make the larger input into zip expression as DACK overexpression results in a clear elevation in zip levels on the dorsal side of the embryo, but this is not seen with excessive Dpp signaling. Simultaneously activating Dpp signaling and overexpressing DACK with prd-Gal4 is not sufficient to promote ectopic zip expression (for example in the ventral epidermis), indicating that other components are required for zip expression, consistent with DACK operating through downstream signaling events (Zahedi, 2008).

It is well established that communication between the amnioserosa and the epidermis is critical for embryonic morphogenesis, and this study has identified the zip locus as one target of such crosstalk, with zip transcription in both tissues dependent on signals secreted by both tissues. A diffusible signal from the amnioserosa to the epidermis has been proposed in the regulation of germband retraction by Hindsight, a transcription factor that is member of the U-shaped group of genes expressed in the amnioserosa, and preliminary results indicate that the U-shaped group is involved in the regulation of zip expression in both the amnioserosa and the epidermis. How could ACK tie into transcriptional regulation of a diffusible signal from the amnioserosa to the epidermis? There is evidence that ACK functions in clathrin-mediated receptor endocytosis in a kinase-independent manner (Hopper, 2000; Teo, 2001; Yang, 2001a; Yeow-Fong, 2005), and is possible that ACK regulates by receptor endocytosis a pathway in the amnioserosa that leads to a transcriptional response. These data suggest that the kinase activity of DACK may actually impair its ability to drive zip expression, as KD-DACK promoted higher zip levels than wild-type DACK. Interestingly the binding of mammalian ACK1 to SNX9/SH3PX1, a member of the sorting nexin family of proteins involved in the sorting of proteins in the endosomal pathway, is inhibited by ACK1 kinase activity (Yeow-Fong, 2005). The interaction of ACK with a sorting nexin is conserved in flies, where Drosophila SH3PX1 binds to DACK (Worby, 2002; Zahedi, 2008).

In addition to the transcription of myosin in the DME cells being dependent on input from the amnioserosa, assembly of the actomyosin contractile apparatus in the DME cells requires an expression border for the adhesion molecule Echinoid between the amnioserosa and the epidermis. The juxtaposition in epithelia of cells expressing Ed with those not expressing Ed triggers actomyosin cable assembly. Ed is expressed in the epidermis but not the amnioserosa and this provides a means, in addition to ACK-mediated signaling, by which the amnioserosa 'communicates' with the epidermis in regulating the actomyosin contractile apparatus (Zahedi, 2008).

Previous studies have found that global activation of Cdc42 signaling in tkv mutant embryos could suppress the dorsal closure defects caused by a reduction in Dpp signaling, and the present results indicate a major route of action for this suppression is ACK (Ricos, 1999). The activation of Cdc42 throughout the embryo leads to increased expression of DACK specifically in the amnioserosa, and this study has shown that overexpressing DACK in this tissue can suppress tkv dorsal closure defects (Sem, 2002). The results indicate a tissue-specific regulation of DACK levels by Cdc42 that may be part of a sophisticated signaling network enabling the coordinated morphogenesis of tissues in the embryo. DACK does not bind Cdc42 but PR2 does and Cdc42 may also regulate ACK function during dorsal closure through direct interaction with PR2 (Burbelo, 1995; Sem, 2002). The serine/threonine kinase dPak, an effector for Rac/Cdc42, may also be a component of Cdc42-mediated communication between the amnioserosa and the epidermis during dorsal closure. It has been shown that dPak expression in the amnioserosa is regulated by Cdcd42, but in the opposite direction from DACK in that impairment of Cdc42 signaling leads to elevated dpak transcription in this tissue (Sem, 2002). Impairing dPak kinase activity through amnioserosa-specific expression of the dPak autoinhibitory domain leads to defects in head involution and dorsal closure (Zahedi, 2008).

Head involution defects and germband retraction failures are seen in ACK-deficient embryos and loss of DACK can suppress the head involution defects and germband retraction failure caused by overexpression of Dpp. These results suggest that Dpp signaling and ACK cooperate in the regulation of these morphogenetic events in addition to dorsal closure. A recent review has highlighted the parallels between dorsal closure and head involution in terms of morphogenetic events and the genes required, with both involving epithelial sheet migration, zip expression and Dpp signaling (VanHook, 2008). DACK overexpression leads to excessive zip levels in the head and it is likely that ACK and Dpp signaling work together to provide myosin for head involution. That signaling from the amnioserosa is involved in regulating head involution is supported by an earlier finding that impairing dPak function in the amnioserosa causes failures in this process (Zahedi, 2008).

Does ACK impact Dpp signaling other than at the level of zip transcription? Homozygosity for a DACK allele suppresses the ectopic wing vein phenotype caused by excessive Dpp signaling. It is likely that this phenotype is caused by something other than misregulation of zip expression, and ACK could be regulating the expression of a subset of Dpp target genes (other than dad or salm) or may be interacting with the Dpp pathway at another level (Zahedi, 2008).

TGF-β family signaling is a central regulator of dorsal closure and other epithelial fusions, but how Dpp controls dorsal closure has not been well-defined. We have shown that regulation of zip expression in cooperation with the Drosophila ACKs constitutes a major route of action of Dpp during dorsal closure. These findings may be relevant to vertebrate wound healing, in which closure of the wound involves both epithelial movement and TGF-β–dependent contraction of connective tissue in the wound (Zahedi, 2008).

Drosophila Ack targets its substrate, the sorting nexin DSH3PX1, to a protein complex involved in axonal guidance

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, the SH2 domain of Dock was used to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which has been shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system. The smallest protein in this complex p63 has now been identified. p63 has been named DSH3PX1 because it appears to be the Drosophila ortholog of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. DSH3PX1 interacts with the Drosophila ortholog of Wasp, a protein component of actin polymerization machinery, and DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking (Worby, 2001).

This study has identified DAck as a member of a complex of proteins involved in axonal guidance via its association with Dock. It is important to note that experiments in fly embryos using dsRNAs directed against Dock and DAck result in similar axonal pathfinding defects as assayed by Bolwig's organ development. Given the ability of DAck to interact with DSH3PX1, a potential sorting nexin that associates with the clathrin-coated adaptor protein 50, and the ability of mammalian ACK1 to interact with clathrin, it is tempting to speculate that DAck is involved in regulating the extracellular presentation of Dscam and/or other Dock-associated receptors by endocytosis via clathrin-coated pits. This speculation is further supported by the role of C. elegans ACK-related tyrosine kinase-1 in down-regulating Let-23, the C. elegans epidermal growth factor receptor orthologue. Furthermore, DSH3PX1 was identified as a substrate for DAck, and it was demonstrated that phosphorylation of DSH3PX1 probably increases its interaction with Dock while decreasing its interaction with WASP. In this scenario, DAck acts as a molecular switch to control DSH3PX1 protein-protein interactions. Nonphosphorylated DSH3PX1 interacts strongly with WASP, a known modulator of the actin cytoskeleton. When phosphorylated, DSH3PX1 interacts preferentially with Dock. In addition, the PX domain of DSH3PX1 interacts with phospholipids, thereby targeting DSH3PX1 to specific cellular membranes. It would be interesting to understand how the Dock SH2 domain chooses among its binding partners, i.e. Dscam versus DAck versus DSH3PX1, and how the resulting protein complexes ultimately influence neurite outgrowth. For now, the complexity of the protein-protein interactions involving Dock preclude directly linking a specific Dock protein complex to specific changes in the actin cytoskeleton. Nevertheless, Dock and the proteins recruited by Dock are clearly instrumental in signaling changes in the actin cytoskeleton that are required for directed axonal growth (Worby, 2001).

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, 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. 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 alpha_PS1, alpha_PS2, and ß_PS. 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).

Search PubMed for articles about Drosophila Ack

Burbelo, P. D., Drechsel, D. and Hall, A. (1995). A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. J. Biol. Chem. 270: 29071–29074. PubMed ID: 7493928

Chan, W., et al. (2009). Down-regulation of Active ACK1 Is Mediated by Association with the E3 Ubiquitin Ligase Nedd4-2. J. Biol. Chem. 284: 8185–8194. PubMed ID: 19144635

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date revised: 11 October 2012

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