PAK-kinase

DEVELOPMENTAL BIOLOGY

Embryonic

Drosophila PAK-kinase transcripts are detected through embryonic development, apparently ubiquitously. However, PAK-kinase RNA levels are elevated in particular cells. The first obvious elevation is seen in stage 11, in the epidermal cells immediately flanking the amnioserosa. This elevation is particularly pronounced in stage 14 embryos, around the time of dorsal closure initiation. Dorsal epidermal cells in the first row of cells adjacent to the amnioserosa show an increased PAK-kinase mRNA level relative to the rest of the epidermis, with at least two cells flanking each segment border having particularly high levels. Expression is greater in the abdominal segments than in the thorax, but in late embryos increased RNA levels are seen in the thorax and in more ventrally located epidermal cells flanking the segment borders. Following the completion of dorsal closure, an elevation in PAK-kinase transcript levels is found in the dorsal vessel, in the central nervous system, and in the epidermal cells at three thoracic muscle attachment sites in the epidermis. At the end of embryogenesis, elevated PAK-kinase transcript levels are seen in muscle attachment sites throughout the epidermis. PAK-kinase protein distribution is similar to that of the mRNA. The similarities between PAK-kinase distribution and those of integrins and ECM molecules suggest that PAK-kinase may associate with integrin molecules as part of a complex. Antibody to phosphotyrosine reveals a colocalization of phosphotyrosine and PAK-kinase. Phosphotyrosine and PAK-kinase colocalize at cell boundaries along the leading edge during dorsal closure. Heat shock induction of dominant-negative Rac during dorsal closure causes the loss of PAK-kinase and phosphotyrosine from the leading edge, which is likely to be due to the loss of Rac-dependent focal complexes (Harden, 1996).

The Rho GTPases Rac1 and Cdc42 have been implicated in the regulation of axon outgrowth and guidance. However, the downstream effector pathways through which these GTPases exert their effects on axon development are not well characterized. Axon outgrowth defects within specific subsets of motoneurons expressing constitutively active Drosophila Rac1 largely persist even with the addition of an effector-loop mutation to Rac1 that disrupts its ability to bind to p21-activated kinase (Pak) and other Cdc42/Rac1 interactive-binding (CRIB)-motif effector proteins. While hyperactivation of Pak itself does not lead to axon outgrowth defects as when Rac1 is constitutively activated, live analysis reveals that Pak can alter filopodial activity within specific subsets of neurons similar to constitutive activation of Cdc42. Moreover, the axon guidance defects induced by constitutive activation of Cdc42 persist even in the absence of Pak activity. These results suggest that (1) Rac1 controls axon outgrowth through downstream effector pathways distinct from Pak, (2) Cdc42 controls axon guidance through both Pak and other CRIB effectors, and (3) Pak's primary contribution to in vivo axon development is to regulate filopodial dynamics that influence growth cone guidance (Kim, 2003).

These studies support the idea that Rac1 mediates axon outgrowth through downstream effector pathways that are distinct from those that mediate Cdc42-dependent guidance. This is based on the following: (1) by using mutants of Rac1 and Cdc42 that render them constitutively active, the downstream cellular events that each is responsible for during axon development can be effectively isolated. Rac1 and Cdc42 likely work through different effector pathways, since constitutive activation of each leads to distinct phenotypes. (2) An effector-loop mutation that disrupts Cdc42's ability to bind CRIB-motif proteins can effectively suppress axonal defects resulting from constitutively active Cdc42. In contrast, the parallel mutation in Rac1 only partially suppresses defects resulting from constitutive activation of Rac1. Finally, activation of Pak, a CRIB effector for both Rac1 and Cdc42, leads to guidance defects and increased filopodial activities similar to those seen in activated Cdc42 mutants, but not activated Rac1 mutants (Kim, 2003).

A model is favored in which Rac1 mediates axon outgrowth mainly through effector proteins that do not possess the CRIB-motif, while Cdc42 mediates growth cone guidance primarily through CRIB proteins. Since activation of the CRIB protein Pak does not lead to axon outgrowth, but rather, guidance defects, Pak does not seem to play a direct role in Rac1-dependent outgrowth. This model is consistent with reports demonstrating that Pak is not required for axon outgrowth, at least during its initial phase, but is required during proper guidance and targeting of axons in a later phase. This, however, does not necessarily exclude Pak from playing a significant role in other Rac signaling pathways that mediate axon development (Kim, 2003).

Closer examination of growth cone behavior begins to reveal nonlinear signaling events. The ability of activated Pak to enhance filopodial activity similar to what activated Cdc42 achieves suggests that Pak likely plays a role in Cdc42-mediated growth cone guidance that depends on filopodial activity control. In this context, however, Pak's contribution to filopodial regulation is thought necessary, but not entirely sufficient, to dictate proper guidance. Also, Pak may not be the sole effector responsible for Cdc42-dependent filopodial activity. CRIB proteins affected by the Y40C mutation in constitutively active Cdc42 do not play a role in filopodial regulation, suggesting that other effectors are likely playing redundant roles in mediating filopodial activity. In addition, the persistence of guidance defects in motoneurons expressing Dcdc42V12 in a Pak null genetic background further suggests that Cdc42 is likely working through other CRIB effectors, in addition to Pak, to mediate its effects on growth cone behavior and guidance (Kim, 2003).

By isolating cellular events downstream of Rac1 and Cdc42 through the use of constitutively active mutants, the effector pathways responsible for axon development have become amenable to dissection. Since constitutively active mutants lock the GTPases in an active GTP-bound state, downstream events can be examined irrespective of GTPase activation. Furthermore, by coupling effector-loop mutations with the constitutively active mutation, a role for specific effectors becomes more evident. More studies that isolate effector pathways are necessary to complement existing studies (Kim, 2003).

In conclusion, studies using in situ analysis support the general view that cytoskeletal dynamics, outgrowth, and guidance are not necessarily coupled directly under the activation of Rac1 or Cdc42. Instead, these studies imply that a complex repertoire of growth cone behaviors are being mediated in parallel as a meshwork of events, and that the workings of Pak and other Rac1 and Cdc42 effector proteins, many of which are yet to be examined in situ, collectively contribute to proper axon development (Kim, 2003).

Group I PAKs function downstream of Rac to promote podosome invasion during myoblast fusion in vivo

The p21-activated kinases (PAKs) play essential roles in diverse cellular processes and are required for cell proliferation, apoptosis, polarity establishment, migration, and cell shape changes. This study has identified a novel function for the group I PAKs in cell-cell fusion. The two Drosophila group I PAKs, DPak3 and DPak1, have partially redundant functions in myoblast fusion in vivo, with DPak3 playing a major role. DPak3 is enriched at the site of fusion colocalizing with the F-actin focus within a podosome-like structure (PLS), and promotes actin filament assembly during PLS invasion. Although the small GTPase Rac is involved in DPak3 activation and recruitment to the PLS, the kinase activity of DPak3 is required for effective PLS invasion. A model is proposed whereby group I PAKs act downstream of Rac to organize the actin filaments within the PLS into a dense focus, which in turn promotes PLS invasion and fusion pore initiation during myoblast fusion (Duan, 2012).

The PAK family of Ser/Thr kinases have been implicated in many biological processes, including cell migration, invasion, proliferation, and survival, as well as regulation of neuronal outgrowth, hormone signaling, and gene transcription. However, a role for PAKs in muscle development and cell-cell fusion has not been previously uncovered. This study reveals an essential function for Drosophila group I PAKs during myoblast fusion in vivo. Specifically, it was shown that the two group I PAKs in Drosophila, DPak3 (a close homologue of mammalian PAK2) and DPak1 (a close homologue of mammalian PAK1), have partially redundant functions in myoblast fusion, based on the following lines of evidence. First, double and single mutants of dpak3 and dpak1 exhibited a range of fusion defects, dependent on the residual endogenous protein level. Clearly, DPak3 plays a more significant role than DPak1, and the minor role of DPak1 can only be revealed in the context of the dpak1,dpak3 double mutant. Second, DPak3 is enriched in the F-actin foci in wild-type embryos. In contrast, DPak1 only accumulates in the F-actin foci in the absence of DPak3, consistent with its compensatory function in the fusion process. Third, overexpression of DPak1 in the dpak3^zyg mutant leads to a slight but reproducible rescue of fusion. Finally, overexpression of a kinase-inactive form of DPak3 (DPak3K322A) in dpak3^zyg mutant embryos significantly enhances the fusion defect, presumably by forming nonproductive DPak3K322A-substrate complexes that exclude DPak1 (Duan, 2012).

What accounts for the differential effects of DPak3 and DPak1 in myoblast fusion? One possibility is that DPak3 is recruited to the PLS at a higher level than DPak1 in wild-type embryos. However, the different recruitment levels cannot solely account for the differential effects of these two proteins because DPak1 overexpression in dpak3^zyg mutant embryos does not completely rescue the fusion defect. A second possibility is that DPak3 and DPak1 may have different interacting partner(s) in the PLS, and thus may respond differently to upstream Rac signaling and/or transduce different downstream signals. In this regard, it has been reported that human PAK2, but not PAK1, can interact with MYO18A, which is involved in actin filament organization and cell migration. A third possibility is that these two kinases may have intrinsic differences in substrate binding affinity and/or kinase activity. For example, DPak3 may preferentially bind and activate specific substrates in wild-type embryos and DPak1 could only access and/or inefficiently activate these substrates in the absence of DPak3. In support of this hypothesis, expressing the kinase-inactive from of DPak3 (DPak3K322A) in the dpak3^zyg mutant abolishes the functional compensation by DPak1, suggesting that DPak3K322A may efficiently compete with DPak1 for substrate binding by forming high-affinity DPak3K322A-substrate complexes. Obviously, identification of the preferred substrates of these group I PAKs in vivo will be required to further test this hypothesis (Duan, 2012).

Previous studies have shown that the activity of group I PAKs is regulated by the small GTPases Rac/Cdc42. The subcellular localization of group I PAKs, on the other hand, is thought to be controlled by SH2-SH3 domain-containing small adaptor proteins Nck and Grb. Although the expression of a dominant-negative form of Rac resulted in a loss of DPak1 localization at the leading edge during dorsal closure in Drosophila embryos, it was unclear if Rac directly regulates DPak1 recruitment to the leading edge. This study provides evidence that the localization of DPak3 to a specific subcellular structure, the F-actin focus within the podosome-like structure (PLS), is directly controlled by Rac. First, Rac colocalizes with DPak3 within the F-actin foci during myoblast fusion. Second, DPak3 is no longer localized to the F-actin foci in rac1,rac2 double mutant embryos. Third, DPak3 carrying mutations in the Rac-binding domain (DPak3H29,31L) fails to localize to the F-actin foci or rescue the dpak3^zyg mutant phenotype, despite its constitutive kinase activity (Duan, 2012).

It is noted that although the subcellular localization of group II PAKs has been shown to be controlled by Cdc42 in cultured mammalian cells and in Drosophila photoreceptor cells, this study reveals, for the first time, such a localization mechanism for a group I PAK. Moreover, this study has positioned group I PAKs in a new signaling branch downstream of the Rac GTPase during myoblast fusion, in addition to the previously known branch involving the Scar complex (Duan, 2012).

Mammalian group I PAKs have been implicated in regulating podosome formation, size, and number in cultured cells. However, the function of PAKs in individual podosomes, especially in intact organisms, remained completely unknown. This study demonstrates that group I PAKs are required for regulating the invasive behavior of individual podosomes in an intact organism. DPak3 is required specifically in the FCMs and colocalizes with the F-actin foci within the PLS. It was also shown also show that in dpak3 mutants, the F-actin foci persisting to late developmental stages appear dispersed and fail to invade into the apposing founder cells/myotubes. As a result, fusion pores fail to form between these defective FCMs and their apposing founder cells/myotubes. Thus, the current study not only strongly supports the model that PLS invasion is required for fusion pore formation, but also reveals, for the first time, that group I PAKs are important regulators of podosome invasion in vivo (Duan, 2012).

How do group I PAKs regulate PLS invasion? The dispersed morphology of the F-actin foci in dpak3 mutants suggests that group I PAKs may be involved in organizing branched actin filaments into a dense focal structure within the PLS. Because the kinase activity of DPak3 is required for its function during myoblast fusion, DPak3 may regulate actin cytoskeletal remodeling by phosphorylating downstream substrates associated with the actin cytoskeleton, such as regulators of actin polymerization, depolymerization, and/or actin filament bundling/cross-linking. Genetic and immunohistochemical analyses suggest that DPak3 is unlikely to promote actin polymerization via the Arp2/3 NPFs WASP and Scar, because DPak3 functions in parallel with the WASP and Scar complexes and the amount of F-actin in each PLS is not markedly reduced in dpak3^zyg mutant embryos. In addition, DPak3 is unlikely to suppress actin depolymerization via PAK’s well-characterized substrate, LIM kinase (LIMK), because loss-of-function mutants of LIMK and its substrate, the actin depolymerization factor cofilin, did not have a myoblast fusion defect, and DPak3 did not show genetic interactions with LIMK or cofilin during myoblast fusion. Therefore, it is conceivable that the group I PAKs may regulate actin bundling and/or cross-linking proteins, which, in turn, organize the assembly of branched actin filaments into tightly packed bundles to promote PLS invasion. In this regard, it has been shown that a tight intermolecular packing of the actin filaments mediated by actin cross-linkers leads to the formation of highly stiff actin bundles that exert large protrusive forces against the cell membrane. Future experiments are required to identify the bona fide downstream substrate(s) of DPak3 in regulating PLS invasion during myoblast fusion (Duan, 2012).

Interestingly, mammalian group I PAKs have been associated with cellular invasion of other cell types, such as cancer cells during metastasis. Elevated expression and hyperactivity of PAK1 and PAK2 are seen in several types of tumors. Overexpression of constitutively active PAK1 promotes cancer cell migration and invasion, whereas inhibiting PAK1 suppresses these phenotypes. It is well known that cancer cell invasion is mediated by invadopodia, which are podosome-like structures with larger F-actin-enriched cores and less dynamic actin polymerization. A role of PAK1 and PAK2 in invadopodia formation in an invasive metastatic human melanoma cell line has been revealed. Thus, further studies of PAK function in podosome invasion in Drosophila myoblast fusion will not only provide additional insights into muscle differentiation, but also cancer cell invasion during tumorigenesis (Duan, 2012).

Effects of Mutation or Deletion

Pak-kinase mutants are lethal. Flies homozygous or transheterozygous for Pak mutations die as pharate adults, although occasional adult escapers are seen. They are uncoordinated and have crumpled wings but are otherwise wild-type in appearance. In Pak strong loss-of-function mutants, photoreceptor R cell axons extend into the brain normally. However, these fibers do not spread evenly within the lamina and medulla. As a result, some regions are hyperinnervated while others lack innervation. In the medulla neuropil, R cell axons fail to find their proper targets but instead, terminate as thick, blunt-ended fascicles. Hence, in contrast to wild type, Pak mutant R cells do not elaborate a smooth topographic map in the lamina and medulla neuropils (Hing, 1999).

Dscam, a Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dreadlocks (Dock), an SH3/SH2 adaptor protein required for axon guidance. Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, which contains a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable immunoglobulin (Ig) and transmembrane (TM) domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity is likely to contribute to the specificity of neuronal connectivity (Schmucker, 2000).

To gain insight into the mechanisms by which growth cones integrate guidance cues, a combined biochemical and genetic analysis of the Dock signal transduction pathway has been pursued. Dock is an adaptor protein containing 3 SH3 domains and a single SH2 domain, and is closely related to mammalian Nck. dock mutants show defects in axon guidance in the adult fly visual system and in the embryonic nervous system. Based on the role of the adaptor protein Grb-2 in linking receptor tyrosine kinases to Ras, it is proposed that Dock links guidance receptors to downstream regulators of the actin cytoskeleton. Pak, a p21-activated serine/threonine kinase, acts downstream of Dock in adult photoreceptor neurons. Dock binds through its second SH3 domain to Pak and Pak binds directly to Rho family GTPases, evolutionarily conserved regulators of the actin-based cytoskeleton. Genetic studies reveal that both Pak's kinase activity and its interaction with Rho family GTPases are essential for axon guidance (Schmucker, 2000 and references therein).

Dscam binds directly to multiple domains of Dock and is widely expressed on axons in the embryonic nervous system. Dscam is required for recognition of an intermediate targeting determinant for Bolwig's nerve: Dock and Pak are required for this step, and Dscam shows dosage-sensitive interactions with both dock and Pak. Based on these studies, it is proposed that Dscam recognizes a guidance signal(s) and translates it into changes in the actin-based cytoskeleton through Dock and Pak (Schmucker, 2000).

Dscam RNA is expressed in Bolwig's organ as well as more generally within the CNS and PNS. The protein product is exclusively expressed on axon processes. To assess whether selective expression of Dscam in Bolwig's nerve is sufficient to rescue the mutant, a transgene encoding full-length Dscam driven by the GMR promoter (a strong transcriptional driver providing Bolwig's organ-specific expression) was constructed and it was introduced into the germline by P element DNA transformation. Two independent insertions were characterized. In a wild-type (or a Dscam mutant) background, 100% of the embryos carrying one or two copies of GMR-Dscam exhibit strong axon guidance phenotypes. Individual axons project in abnormal directions over the surface of the optic lobe and rarely contact P2. It is unclear whether this reflects the sensitivity of Bolwig's nerve guidance to increased levels of Dscam or misexpression in Bolwig's organ of an inappropriate isoform, or both. Due to the large size of the Dscam locus (61 kb), whether or not the wild-type gene rescues the mutant phenotype could not be assessed. In any case, the dominant phenotype precludes assessing transgene rescue of the mutant phenotype. In contrast, GMR-dock rescues 85% of dock mutant embryos (Schmucker, 2000).

Whether dock and Pak are functional components of a Dscam guidance pathway was assessed through genetic analysis. Mistargeting defects of Bolwig's nerve were observed in some 44% of the embryos heterozygous for both Dscam and dock. In contrast, only 4%-6% and 10%-13% of embryos heterozygous for either dock or Dscam, respectively, show defects. Similarly, whereas some 38% of embryos heterozygous for both Dscam and Pak have an abnormal Bolwig's nerve, only 5% were defective in embryos heterozygous for Pak. The synergistic interactions between Dscam, dock, and Pak, the similarity of complete loss-of-function phenotypes, and the physical interactions between these proteins are consistent with their acting together to mediate recognition between the Bolwig's nerve growth cones and P2 (Schmucker, 2000).

Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. The guanine nucleotide exchange factor (GEF) Trio has been identified as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. It is proposed that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension (Newsome, 2000).

Dock and Pak regulate olfactory axon pathfinding in Drosophila

The convergence of olfactory axons expressing particular odorant receptor (Or) genes on spatially invariant glomeruli in the brain is one of the most dramatic examples of precise axon targeting in developmental neurobiology. The cellular and molecular mechanisms by which olfactory axons pathfind to their targets are poorly understood. The SH2/SH3 adapter Dock and the serine/threonine kinase Pak are necessary for the precise guidance of olfactory axons. Using antibody localization, mosaic analyses and cell-type specific rescue, it is observed that Dock and Pak are expressed in olfactory axons and function autonomously in olfactory neurons to regulate the precise wiring of the olfactory map. Detailed analyses of the mutant phenotypes in whole mutants and in small multicellular clones indicate that Dock and Pak do not control olfactory neuron (ON) differentiation, but specifically regulate multiple aspects of axon trajectories to guide them to their cognate glomeruli. Structure/function studies show that Dock and Pak form a signaling pathway that mediates the response of olfactory axons to guidance cues in the developing antennal lobe (AL). These findings therefore identify a central signaling module that is used by ONs to project to their cognate glomeruli (Ang, 2003).

ONs of the antennae and maxillary palps undergo terminal differentiation during early metamorphosis and become predestined to express particular Or genes and synapse in specific glomeruli. Between 20 and ~50 hAPF, their axons leave the nascent antenna in fascicles and enter the AL in search of their targets. Projection neurons (PNs) the targets of the incoming axons acquire their cell fates, which predetermine their glomerular choice, during larval development. During early pupal development their dendrites enter the AL and become precisely paired with ON axons in specific glomeruli. Thus, ONs expressing a given Or gene rendezvous with PNs of a particular identity within a topographically defined glomerulus in the AL (Ang, 2003).

In wild type flies, olfactory axons take stereotyped paths on the surface of the AL to converge on their cognate glomeruli. Detailed characterization of the axon trajectories, using Gal4 drivers expressed in different subclasses of ONs shows that, upon arrival at the anterolateral point of the AL, afferents project directly, with little sidetracking to their postsynaptic targets. As in the mouse and moth, these axon pathways are bilaterally symmetric and invariant from AL to AL. How is this precise wiring pattern formed during development? In one model, each ON initially sends collaterals to multiple glomeruli and then withdraws the inappropriate branches in a process requiring odorant-evoked activity. Alternatively, the invariant pattern of connections is the result of directed axon migrations in response to spatially restricted pathfinding cues in the developing AL. A definitive answer to this question will require developmental study or direct observation of the extending axons. However, at least two observations are consistent with the notion that olfactory axons navigate directly to their cognate glomeruli. (1) A temporal lag between early axon pathfinding and subsequent Or gene expression indicates that an odorant-evoked activity is unlikely to play an important role. Indeed, activity is neither required for formation nor maintenance of the olfactory map in mouse and moth. (2) Importantly, the finding that the growth cone guidance genes, dock and Pak, are needed for development of the olfactory map, provides strong evidence that directed axon migration plays a key role in the matching of ON axons with their correct glomeruli. Directed navigation of olfactory axons to their targets is also observed in zebrafish and moth (Ang, 2003).

In dock and Pak mutants, the stereotyped connectivity of AL neuropil is severely disrupted, leading to an aglomerular phenotype. Three pieces of evidence are presented indicating that dock and Pak function in ONs: (1) antibody staining shows that Dock and Pak proteins are expressed in antennal axons during the period in which they are projecting to the brain; (2) consistent with their requirements in ONs, removal of dock and Pak activities from only the antennae results in ectopic targeting of olfactory axons, and (3) expression of dock and Pak cDNAs specifically in ONs in otherwise mutant animals leads to strong rescue of the mutant AL phenotype. Although numerous glomeruli were restored upon the expression of the wild-type cDNAs, some glomeruli were not. The incomplete rescue is thought to be due to the expression of SG18.1-Gal4 in only a subset of all the ONs. However, it is also possible that the partial rescue reflects an additional requirement of dock and Pak functions in the brain. A recent study indicates that ONs may be divided into different classes based on the timing of their projections. It was not determined further whether dock and Pak are required in all ONs or in only a specific subset. Although dock and Pak are specifically required in ONs, finding of nonautonomous effects on the morphogenetic changes of the PNs and AL glia is in accord with earlier studies in which ONs were physically or genetically ablated. The data therefore show that proper termination of ON axons is also an important step in the sculpting of the AL neuropil into distinct glomeruli (Ang, 2003).

Evidence is provided that the disruption in AL development in dock and Pak mutants is not an indirect effect of aberrant cell-fate determination or axonogenesis. By contrast, the precise targeting of ON axons is severely disrupted in dock and Pak mutants. To identify the cause of the mistargeting, the axon pathways of individual ON classes (Or47a, and Or47b) were examined at the single-cell level. Although an additional short branch was observed in 9% of dock mutant neurons, the most striking defect observed in single-cell clones was the chaotic trajectories exhibited by both the ipsilateral and contralateral axons of the ONs. It is concluded that the primary function of dock and Pak in ONs is axon pathfinding, to steer ON axons precisely to their target glomeruli. In mouse, mutations in the odorant receptor genes abolish the ability of olfactory axons to pathfind in the anteroposterior axis without affecting their migration in the dorsoventral axis, leading to the proposal that odorant receptors participate in the recognition of only anteroposterior guidance cues. However, after examining several hundred ALs for each dock and Pak mutant, no consistent patterns were observed in the mistargeting of ON axons. The ON classes are affected to different degrees by the loss of dock and Pak activities. Although Or22a and Or47a axons terminate in numerous ectopic glomeruli, Or47b axons terminate in a single glomerulus, albeit mis-shapen, in the approximate position of the wild-type VA1lm. The reason for the differential sensitivity of the ON subtypes to the loss of dock and Pak functions is not known. One possibility is that Or47b axons, which are among the first axons to enter the AL, are confronted with fewer developing glomeruli and hence fewer guidance choices than Or22a and Or47a axons that enter the AL later. Alternatively, Or47b axons may have less need for dock- and Pak-mediated navigational functions because VA1lm is located near the nerve entry point. Indeed, while the Or47b ipsilateral axons frequently terminate accurately on VA1lm, the contralateral axons, which have to project across the entire AL surface, are often misrouted. In contrast to the severe projection defects in the AL, the migration of dock and Pak mutant axons through the antennal nerve takes place normally. It is possible that the lack of requirement of dock and Pak functions during this phase of axon growth reflects a different guidance mechanism in the antennal nerve (Ang, 2003).

The observation that the ON axon trajectories are severely disrupted in dock and Pak mutants suggests that the genes may mediate the detection or response of the growth cones to guidance cues in the environment. The results indicate that in these events, dock and Pak are very likely to act in a signaling pathway: (1) loss of either dock or Pak functions results in olfactory connectivity phenotypes that are indistinguishable; (2) both dock or Pak function autonomously in ONs; (3) mutations that disrupt the domains of Dock (second SH3 domain) and Pak (N-terminal PXXP domain; Pak⁴) that mediate interaction between the two proteins, disrupt ON axon targeting. It is therefore proposed that Dock and Pak are part of a signal transduction cascade that allows ONs to find and precisely pair with the correct postsynaptic partners. Although severely disrupted, the guidance of ON axons in dock and Pak mutants is not completely abolished, indicating that other genes function to steer ON axons to their targets as well (Ang, 2003).

Rac. acting through Pak, promotes epithelial cell rearrangement during tracheal tubulogenesis in Drosophila

Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. The tracheal system of Drosophila was used to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. A reduced Rac activity leads to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibits dynamic epithelial cell rearrangement. In contrast, hyperactivation of Rac inhibits assembly of newly synthesized E-Cadherin into cell junctions and causes loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules (Chihara, 2003).

p21-activated kinase (Pak) is known as a mediator of the activity of Rac GTPase. Tracheal defects similar to those of Rac1, 2 mutants are found in pak mutants. Furthermore, Rac1, 2 and Pak mutations synergistically enhance tracheal defects. Such results suggest that Rac and Pak are required for directed movement of tracheal branches (Chihara, 2003).

The loss of Rac activity also causes a defect in cell differentiation. Tips of dorsal branch 1-9 are normally capped with terminal cells that extend terminal branch in the ventral direction. In Rac 1, 2 mutant embryos, the loss of terminal branches was observed with high penetrance. Consistently, serum response factor (SRF), a marker protein for the terminal cell, also disappears, suggesting that terminal cell differentiation does not occur (Chihara, 2003).

Since directed cell migration and terminal cell differentiation are processes requiring FGF signaling, it was asked whether Rac is involved in FGF signaling (a strong genetic interaction). Although tracheal patterning is only mildly affected by half dose reductions of bnl (ligand), btl (receptor) and dof (intracellular effector), the phenotype is strongly enhanced by introducing one copy of Rac1, 2 mutant chromosome from mothers. A similar genetic interaction was found between pak and bnl. These genetic interactions suggest that Rac and Pak are required for the migration of tracheal branches in response to FGF signaling (Chihara, 2003).

To determine the epistatic relationship between Rac and FGF signaling, the effect of constitutive activation of Rac was tested in btl mutants. In the btl mutant, tracheal branching does not proceed beyond the invagination at stage 11, and MAP kinase activation is absent (Chihara, 2003).

Expression of Rac1V12 partially restores the movement of tracheal cells, and activates MAP kinase, as revealed by staining with the antibody against the diphosphorylated form of MAP kinase (dp-MAPK). These results suggest that Rac activation is an essential downstream event of tracheal cell motility induced by FGF signaling (Chihara, 2003).

Extracellular signals that promote tracheal branching are good candidates for regulators of Rac in tracheal cells. In this regard, the strong genetic interaction between Rac and FGF signaling components observed suggests an intriguing possibility that FGF signaling activates Rac within tracheal cells to promote both cell motility and cell rearrangement. In support of this idea, it was found that activated Rac 1 partially rescues tracheal cell motility and MAP kinase activation in btl mutants. Involvement of Rac in FGF-dependent events may not be limited to cell motility. Expression of SRF, the product of one of the target genes activated by FGF signaling in the tracheal system, is lost in the mutant trachea with reduced Rac activity because of Rac 1, 2 mutation or Rac 1N17. This result suggests that Rac also regulates transcription (Chihara, 2003).

Several lines of evidence suggest that FGF signaling is activated locally at the tip of branches, and activation of FGF signaling in all tracheal cells prevents branching, suggesting that localized activation of FGF signaling is essential for branching. Therefore the proposed function of Rac in transducing FGF signaling must be localized at the tip of branches. How does the proposed function of Rac in transducing FGF signaling relate to the Rac function in regulating cell rearrangement? Since the effect of Rac 1N17 is most clearly observed in cells destined to become tracheal stalk cells, the location of tracheal cells requiring two of the Rac functions appears to be different. One idea is that FGF signaling activated at the tracheal tip is transmitted to tracheal stalk cells by a secondary signal that activates Rac to promote cell rearrangement. It will be important to identify the upstream signal regulating Rac in stalk cells (Chihara, 2003).

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 (DC) 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. DC is driven in part by an actomyosin contractile apparatus at the leading edge (LE) 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 (AID) of dPak leads to disruption of the LE cytoskeleton and defects in DC does 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. It is concluded that DC requires Group I Pak function in both the amnioserosa and the epidermis (Conder, 2004).

The first accumulations of LE actin at the onset of DC occur at the phosphotyrosine-rich adherens junctions, forming puncta termed actin-nucleating centers (ANCs) that extend apically in the dorsal-most epidermal cells (DMEs). Disruption of the LE contractile apparatus either through loss of maternal and zygotic dPak, or expression of dPak-AID in DME cells is accompanied by loss of phosphotyrosine nodes marking adherens junctions. dPak may be functioning in the assembly and/or regulation of the adherens junctions/ANCs at the LE and could control the LE cytoskeleton in this manner. dPak accumulates at the LE in response to Cdc42 signaling, and the ability of Cdc42 to drive accumulation of phosphotyrosine at the LE is blocked in dpak⁶ mutant embryos. The Drosophila Group II Pak Mbt is recruited to adherens junctions by Cdc42 in developing photoreceptor cells (Schneeberger, 2003). In mbt mutants, photoreceptor morphogenesis is disrupted and adherens junctions become patchy and disorganized. In both DC and developing photoreceptors, localization of Pak kinases to adherens junctions may be controlled by localized Cdc42 activation (Schneeberger, 2003). Cdc42 has been shown to be activated in live cells by E-cadherin, an adherens junction protein. E-cadherin is a component of the phosphotyrosine-rich adherens junctions at the LE regulated by Group I Pak kinase activity. In addition to adherens junction proteins, βPS integrin and talin, components of integrin-mediated adhesions, accumulate at the LE during DC. Work in cultured cells indicates that one means by which Pak1 can be localized to the cell periphery is through recruitment to integrin-based focal adhesions/complexes by interaction with Pak-interacting exchange factor (PIX). The PIX binding site of Pak1 is conserved in dPak, and a Drosophila PIX homologue, dPix, is required for dPak localization at the NMJ. It is interesting to note that the truncated protein encoded by the dpak⁶ allele, which fails to localize properly at cell peripheries, including the LE of the DMEs, is missing the PIX binding site (Conder, 2004).

Another potential route for dPak regulation of the LE cytoskeleton is through phosphorylation of proteins known to regulate actin cytoskeletal dynamics such as LIM kinase, Filamin A and p41-Arc, which have been shown to be substrates for mammalian Pak1. Yet another possibility is that dPak regulates the LE cytoskeleton through the JNK cascade. Loss of phosphotyrosine nodes and the contractile apparatus at the LE is seen with impairment of the JNK pathway in the DME cells. Group I Paks have been shown to be activators of MAPK cascades such as the JNK pathway, operating, at least in some cases, as MAP4Ks. This study is the first characterization of Group I Pak function in JNK cascade activation using loss-of-function mutations. The JNK cascade in the DME cells during DC represents the JNK pathway most thoroughly studied in vivo. This pathway is still active in the DME cells in embryos lacking maternal and zygotic dPak, and in embryos expressing dPak-AID, indicating that dPak plays little or no role in activating the DC JNK pathway. Another Ste20-related kinase, Misshapen, a member of the GCK family, has been identified as a likely MAP4K acting upstream of the JNK cascade in the DME cells (Conder, 2004).

Although dPak is required for integrity of the LE cytoskeleton, the DME cells are still capable of elongating significantly in the D-V direction when dPak is absent. Furthermore, dPak does not become enriched in the DME cells until after DC has commenced. These results suggest that dPak does not contribute to the initial DME cell shape changes, and may not be required for the establishment of the LE cytoskeleton but rather its maintenance (Conder, 2004).

Embryos expressing dPak-AID in the amnioserosa show defects in head involution and DC. These embryos are considerably more disrupted than embryos devoid of maternal and zygotic dPak, indicating that dPak-AID affects more than dPak in this tissue. A likely explanation is that Pak3 activity in the amnioserosa is also being disrupted by dPak-AID. The results suggest that Group I Pak kinase activity in the amnioserosa is a component of signaling from the amnioserosa required for epidermal morphogenesis. DME cell fate is dependent on communication between the amnioserosa and the dorsal epidermis. Down-regulation of JNK signaling in the amnioserosa, mediated by Hnt and Puc, is required for proper assembly of specialized adherens junctions and F-actin in the DME cells. Furthermore, a single row of DME cells is always specified at the interface between amnioserosa and dorsal epidermis. The inhibition of Group I Pak kinase activity in the amnioserosa does not completely disrupt the DME cell fate, however, because DME cells still assemble LE adherens junctions and show elongation. Nevertheless, the clear defects in DC indicate that Group I Pak kinase activity in the amnioserosa contributes to migration of the lateral epidermis (Conder, 2004).

Three results indicate that Group I Pak kinase activity in the amnioserosa does not make a major contribution to amnioserosa morphogenesis: (1) amnioserosa cell shape change still occurs in embryos lacking maternal and zygotic dPak; (2) inhibition of Group I Pak kinase activity in the amnioserosa does not prevent apical constriction of amnioserosa cells; (3) Rac1V12 is still capable of inducing excessive contraction of the amnioserosa in a dpak⁶ mutant background. Presumably, Rac uses cytoskeletal effectors other than dPak to drive cell shape change in the amnioserosa (Conder, 2004).

The finding that expression of dPak-AID with either the LE or amnioserosa GAL4 drivers causes a high frequency of failures to form cuticle is curious. These GAL4 drivers do not show expression in the lateral epidermis with the exception of GAL4^332.3, which shows scattered epidermal expression after completion of dorsal closure. One possibility is that many embryos are dying in late embryogenesis before completion of cuticle secretion. Another, perhaps less likely explanation, is that Group I Pak kinase activity in the amnioserosa and DME cells is required non-autonomously for cuticle secretion in the epidermis through cell-cell signaling. Whatever the mechanism, a further indication that alterations in Rho family small GTPase/Pak signaling have consequences with regard to cuticle secretion is demonstrated by the finding that the disruption of cuticle secretion by Rac1V12 expression is suppressed by zygotic loss of dPak (Conder, 2004).

The polarized accumulation of Pak proteins in cells at sites of dynamic actin regulation has been described in a variety of different contexts, including accumulation at the LE of motile cells, similar to what is seen in DC. For example, during closure of a fibroblast monolayer wound, activated Pak1 accumulates at the LE of migrating fibroblasts. Pak1 is also enriched at the LE of migrating MCF-7 human breast cancer cells, where it participates in reorganization of the cortical actin cytoskeleton. DC, a well characterized process in which loss-of-function mutations are available in the genes encoding many of the participants, should provide a convenient system for the genetic dissection of Pak-mediated signaling regulating the LE cytoskeleton during cell migration (Conder, 2004).

Lim kinase functions dowstream of Pak to regulate the development of olfactory and neuromuscular synapses

Lim Kinase (Limk) belongs to a phylogenetically conserved family of serine/threonine kinases, which have been shown to be potent regulators of the actin cytoskeleton. Despite accumulating evidence of its biochemical actions, its in vivo function has remained poorly understood. The association of the Limk1 gene with Williams Syndrome indicates that proteins of this family play a role in the nervous system. To unravel the cellular and molecular functions of Limk, the Limk gene in Drosophila has been either knocked out or activated. At the neuromuscular junction, loss of Limk leads to enlarged terminals, while increasing the activity of Limk leads to stunted terminals with fewer synaptic boutons. In the antennal lobe, loss of Limk abolishes the ability of p21-activated kinase (Pak) to alter glomerular development. In contrast, increase in Limk function leads to ectopic glomeruli, a phenotype suppressible by the coexpression of a hyperactive Cofilin gene. These results establish Limk as a critical regulator of Cofilin function and synapse development, and a downstream effector of Pak in vivo (Ang, 2006).

In vitro experiments with the human proteins show that Pak1 phosphorylates and activates Limk1. It was hypothesized that, in the fly, Pak acts upstream of Limk to regulate glomerular development in the ALs. In the Pak⁴/Pak⁶ null mutant, the ALs are smaller compared with those of wild type. Staining with the nc82 mAb shows that the boundaries surrounding individual glomeruli are frequently missing, resulting in loss in distinctiveness of most of the marker glomeruli. Thus, Pak activity is necessary for glomerular development. To determine if elevated Pak is sufficient to alter AL development, the hyperactive Pak^myr molecule was expressed under the control of SG18.1-Gal4. Expression of Pak^myr in the ORNs results in strong disruption in the anatomy of the ALs. Although the neuropil is partitioned, the compartments correspond poorly to known glomeruli. Consequently, the marker glomeruli are identifiable in only 0% to 79% of ALs. The abnormal structures of the glomeruli are reflected in the DM2 glomerulus (labeled with the Or22a-Nsyb::GFP transgene). Unlike the wild type where it is distinct and symmetrically positioned, in the Pak^myr animal, it is poorly demarcated from the surrounding neuropil and asymmetrically positioned. It is possible that adjacent glomeruli are fused to form larger structures in the Pak^myr-expressing animal. Thus, increased presynaptic Pak activity is sufficient to disrupt glomerular morphogenesis. To determine if Limk mediates Pak function, UAS-Pak^myr was expressed in the Limk⁵ null mutant background. In the Limk⁵/Y; SG18.1-Gal4/UAS-Pak^myr animal, the AL neuropil is partitioned into compartments that correspond well to known glomeruli. Marker glomeruli can now be identified in 79% to 100% of the ALs. The DM2 glomeruli are morphologically distinct and symmetrically positioned. Thus, mutation in Limk blocks the ability of Pak^myr to interfere with glomerular development. This result supports the idea that Limk functions downstream of Pak to regulate glomerular development (Ang, 2006).

Genetic evidence for antagonism between Pak protein kinase and Rho1 small GTPase signaling in regulation of the actin cytoskeleton during Drosophila oogenesis

During Drosophila oogenesis, basally localized F-actin bundles in the follicle cells covering the egg chamber drive its elongation along the anterior-posterior axis. The basal F-actin of the follicle cell is an attractive system for the genetic analysis of the regulation of the actin cytoskeleton, and results obtained in this system are likely to be broadly applicable in understanding tissue remodeling. Mutations in a number of genes, including that encoding the p21-activated kinase Pak, have been shown to disrupt organization of the basal F-actin and in turn affect egg chamber elongation. pak mutant egg chambers have disorganized F-actin distribution and remain spherical due to a failure to elongate. In a genetic screen to identify modifiers of the pak rounded egg chamber phenotype several second chromosome deficiencies were identified as suppressors. One suppressing deficiency removes the rho1 locus, and using several rho1 alleles it was determined that removal of a single copy of rho1 can suppress the pak phenotype. Reduction of any component of the Rho1-activated actomyosin contractility pathway suppresses pak oogenesis defects, suggesting that Pak counteracts Rho1 signaling. There is ectopic myosin light chain phosphorylation in pak mutant follicle cell clones in elongating egg chambers, probably due at least in part to mislocalization of RhoGEF2, an activator of the Rho1 pathway. In early egg chambers, pak mutant follicle cells have reduced levels of myosin phosphorylation and it is concluded that Pak both promotes and restricts myosin light chain phosphorylation in a temporally distinct manner during oogenesis (Vlachos, 2011).

This study establishes the basal F-actin of the follicular epithelium as an attractive system for the genetic analysis of the signaling pathways regulating the formation of stress fiber-like structures. The actin bundles in the follicle cells appear to be similar to the ventral stress fibers of nonmotile cultured cells, for which one model of stress fiber formation is that it is driven by bundling of actin filaments by actomyosin contractility. Consistent with this model, the results indicate that the major cause of basal F-actin disruption in pak mutant cells is misregulated actomyosin contractility that can be suppressed by reduction of the Rho1 pathway. It was found that Pak regulates pMLC distribution during oogenesis, at first being required for pMLC and later restricting where it is present. Such conflicting roles for Pak have been reported in isolation in mammalian cell culture studies, but these results are the first to show that they can be temporally separated during development of an epithelial cell. Paks from diverse species can function as MLCKs, and such an activity for Pak is indicated in early stage egg chambers, where Pak's MLCK function is opposed by the Flap wing (Flw) MLC phosphatase. Later in oogenesis, around the time of egg chamber elongation, Pak restricts the distribution of MLC phosphorylation and comes into conflict with the Rho1/Rok pathway (Vlachos, 2011).

There are a number of ways that Pak could impinge on the Rok pathway, with one being at the level of RhoGEF2 at the top of the pathway. Pak is required for the basal localization of RhoGEF2, and the mislocalized RhoGEF2 seen in pak mutant clones could at least in part be responsible for the ectopic pMLC seen in older egg chambers. A protein similar to RhoGEF2 in mammals, P115-RhoGEF, appears to be negatively regulated by Pak binding to its DH-PH domain, but no similar physical interaction was found between Pak and the RhoGEF2 DH-PH, nor has an effect of Pak on Rho1-GTP levels been detected, although it is possible that there could be an effect not detectable by the assays that were used. A recent study showed that the PDZ domain of RhoGEF2 is required for its localization at the furrow canal during cellularization. Furthermore, the novel protein Slam, which complexes with the RhoGEF2 PDZ domain, is required for RhoGEF2 localization during cellularization, and it will be of interest to determine if Pak regulation of RhoGEF2 localization in the follicular epithelium involves the PDZ domain and/or Slam. Another possibility is that Pak regulates RhoGEF2 through a trimeric G-protein interaction. RhoGEF2 is a member of the RGS-containing family of GEFs that interact with the activated G_α subunits of trimeric G proteins through their RGS domain and members of the Pak family bind the G_βγ subunit complex through a motif conserved in Drosophila Pak (Vlachos, 2011).

Another route by which Pak could be restricting pMLC distribution is through regulation of a MLCK cooperating with Rok. Work on mammalian Pak has demonstrated that Pak can negatively regulate the activity of MLCK, thus reducing the level of MLC phosphorylation, and three potential MLCKs were considered as candidate Pak targets. Alleles of these genes did not suppress pak oogenesis defects, suggesting either that they are not regulated by pak during oogenesis or that more than one is being regulated by Pak. Another possibility is that Pak is directly regulating Rok in some manner to restrict the output of this pathway. Interestingly, in the columnar epithelial cells over the occyte in late egg chambers, Pak does not regulate MLC phosphorylation and this may be to allow the extensive actomyosin contractility likely to be required to shape these cells (Vlachos, 2011).

Finally, the possibility that Pak could be regulating pMLC levels simply by controlling the overall amount of MLC has not been eliminated, but this seems unlikely given the considerable evidence that vertebrate Pak regulates MLC phosphorylation (Vlachos, 2011).

The finding that RhoGEF2 is a basally localized regulator of actomyosin contractility in the follicular epithelium is consistent with numerous previous studies indicating that RhoGEF2 is the major activator of Rho1 during epithelial morphogenesis. Two other RhoGEFs known to regulate actin, Pebble and RhoGEF64C, did not affect the pak mutant egg chamber phenotype. Deficiencies and/or alleles disrupting 20 other predicted RhoGEFs were tested for the ability to suppress the dpak mutant egg chamber phenotype and found that none were effective. Similarly, a recent study tested predicted RhoGEFs as Rho1 regulators in driving epithelial morphogenesis during imaginal disc morphogenesis and concluded that RhoGEF2 is a key regulator. Many of the RhoGEFs have not been characterized functionally, although some have been shown to be GEFs for GTPases other than Rho1 and to function in nonepithelial cells such as neurons (Vlachos, 2011).

RhoGEF2 is enriched at the basal end of the follicle cells throughout oogenesis including the points of basal membrane separation between follicle cells that occurs during follicle cell flattening in late stage egg chambers. Recently, it was shown that the Rho1 actomyosin contractility pathway is required for this separation between follicle cells at the basal membrane and presumably this signaling is activated by RhoGEF2 (Vlachos, 2011).

RhoGEF2 alleles are much more effective than alleles of other Rho1 pathway components at extending the life span of pak mutant females, implying that RhoGEF2 may have roles independent of the Rho1 actomyosin contractility pathway that could be regulated by Pak. There is evidence that RhoGEFs have functions distinct from small GTPase activation; for example, Pebble has a Rho1-independent role in mesoderm migration (Vlachos, 2011).

In addition to the Rho pathway, an antagonistic relationship between Pak and the Dpp pathway in the regulation of egg chamber elongation was uncovered. A recent study of the Drosophila wing disc demonstrated that Dpp signaling regulates the subcellular distribution of Rho1 activity and MLC phosphorylation in epithelial cells. If this link between pathways also occurs in the follicular epithelium, it may be that loss of Dpp is suppressing the pak mutant phenotype through disruption of Rho1 signaling. Another possibility is that Dpp regulation of the actin filament cross-linking protein α-actinin in the follicular epithelium is relevant (Vlachos, 2011).

The ability of wun and wun2 alleles to suppress the pak egg chamber elongation defect might also be due to downregulation of the Rho1 pathway, as wun was picked up in an overexpression screen for suppressors of impaired Rho1 signaling. Wun and Wun2 belong to a family of lipid phosphate phosphatases that regulate the levels of lipids involved in signaling including lysophosphatidic acid, which is an important activator of the RhoA pathway (Vlachos, 2011).

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date revised: 23 August 2014

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