BIOLOGICAL OVERVIEW

dreadlocks (dock), the Drosophila homolog of the mammalian oncogene Nck, was identified in a screen for P element insertion lines defective for photoreceptor R cell axon projections. dreadlocks (named for the appearance of the photoreceptor cell axon projection pattern in the mutant) is required for R cell growth cone guidance and targeting. The axons of photoreceptor cells grow toward the optic lobe of the brain, terminating in the lamina, the most superficial layer of neurons in the optic lobe. In most dock mutants, axon bundles fan out unevenly as they exit the optic stalk en route to the developing lamina. Fibers pathfind abnormally in this region with evidence of crossing over, abnormal fasciculation, and gross alterations in retinotopy. Frequently observed are clumps of R cell growth cones, terminating in the lamina and separated by gaps. Thicker bundles project through these clumps into the medulla (an adjacent and more internal layer of the optic lobe), resulting in hyperinnervated regions of the medulla separated by uninnervated regions. In many cases, R cell axons terminate at different levels within the lamina, giving rise to an uneven lamina neuropil. In addition, some R1-R6 growth cones fail to terminate in the lamina and instead innervate the medulla terminal field. Thus dock mutants show defects in R cell fasciculation, targeting and retinotopy (Garrity, 1996).

Dock function is not exclusively confined to retinal axon pathfinding. Dock is widely expressed in neurons and at muscle attachment sites in the embryo, and this expression pattern has both maternal and zygotic components. In motoneurons, Dock is concentrated in growth cones. Loss of zygotic dock function causes a selective delay in synapse formation during axonogenesis. Absence of RP3 synapses is due to a defect in terminal guidance and/or differentiation of the RP3 growth cones, rather than to alterations in their axonal outgrowth from the CNS. The innervation of muscles 7 and 6 by RP3 is apparently normal later in development, in dock mutant third-instar larvae. This indicates that RP3 can always form synapses in dock mutants and that synapse formation is delayed so that synapses have all developed by hatching or shortly thereafter. Maturation of the growth cone into a synapse involves extensive cytoplasmic rearrangements, including the formation of focal attachments to the muscle fibers. The involvement of Dock in these complex processes could explain the delay in RP3 synaptogenesis observed in mutant embryos (Desai, 1999).

One of the reasons for a growing interest in Dock is its homology to Nck, a mammalian oncogene with many interactive partners. Dock belongs to a growing family of adapters consisting exclusively of src homology 2 (SH2) and SH3 domains. Other family members include Grb2/Drk/Sem-5, Crk, and SLAP. Dock protein contains three SH3 domains and a single SH2 domain and is highly related to mammalian Nck. SH2 and SH3 domains are found in a wide variety of intracellular signaling proteins and mediate specific protein-protein interactions. SH2 domains bind to specific phosphotyrosine-containing peptide motifs on receptor tyrosine kinases, whereas SH3 domains bind to consensus PXXP sites. Grb2/Drk/Sem-5 has been shown to play an essential role in cell growth and differentiation by linking upstream tyrosine kinase signaling events via SH2 binding to specific phosphotyrosines on activated receptors and SH3 binding to distinct polyproline regions in the downstream effector Sos, leading to the activation of Ras. Nck, an SH2/SH3 adapter protein, plays a complex role in signal transduction, transducing signals from a multitude of receptors to downstream effectors. Nck has an effect on the cytoskeletal dynamics as well as on nuclear gene expression. Among its many functions Nck acts in vertebrates in axon guidance, suggesting that Dock function in the eye of Drosophila and during CNS development has a counterpart in vertebrates. Among its many functions Nck is an important intermediary linking EphB1 (a receptor tyrosine kinase involved in axonal guidance) to activation of Jun N-terminal kinase, an activator of the transcription factor Jun (Stein, 1998).

Since it is likely that Dock mediates growth-cone guidance by transmitting upstream tyrosine phosphorylation signals through its SH2 domain to changes in the actin-based cytoskeleton via its SH3 domains, altered forms of Dock were tested for rescue of photoreceptor growth cone defects in dock mutants. It has been demonstrated that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor growth cones, and that Dock displays different domain requirements in different neurons (Rao, 1998).

Dock can be ectopically expressed in any organ using various GAL4 drivers. In this type of experiment, a transcriptional activator (GAL4) is expressed under the control of a selected promoter, in this case derived from the gene elav. The dock coding sequence is placed adjacent to a UAS type promoter, the target of GAL4. Expression of UAS wild-type dock driven by elav-GAL4 assures that expression of dock is neuron-specific. Neuron-specific expression of dock rescues the dock mutant phenotype in the visual system. This finding establishes that dock is required in postmitotic neurons consistent with its role in guidance. This demonstrates that dock is not required for earlier stages of development (i.e., proliferation and cell fate determination) (Rao, 1998).

dock mutant transgenes containing point mutations in SH3 and SH2 domains were tested for their ability to restore the normal pattern of R cell axonal projections in dock null mutants. Each SH3 domain binding pocket was disrupted by substituting lysine for a conserved tryptophan residue: the first SH3 domain, SH3-1, was disrupted by a W48K mutation; the SH3-2 domain was disrupted by a W151K mutation, and the SH3-3 domain was disrupted by a W225K mutation. This amino acid substitution inhibits binding of Nck SH3 domains to polyproline-containing polypeptides in vitro. The notion that the binding properties of mammalian Nck and Dock are likely to be very similar gains support with the finding that neuron-specific expression of human Nck rescues the dock mutant phenotype. SH2 function was disrupted by substituting a glutamine for an invariant arginine at a position (R336Q) deep within the phosphotyrosine binding pocket. This mutation, placed into the SH2 domain of PI3 kinase, more efficiently inhibits binding to phosphotyrosine-containing polypeptides than the more commonly used lysine substitution. All mutant transgenes were expressed in postmitotic neurons by using the UAS-Gal4 system with elav-GAL4 as the driver. Immunohistological studies have also demonstrated that the mutant proteins are made in R cells, and transported to the growth cone (Rao, 1998).

The requirement of each domain was assessed by testing the ability of singly mutant transgenes to rescue the dock phenotype. Of the three SH3 domains, only SH3-2 was found to be essential for the restoration of normal connectivity. Not only do the SH3-1 and SH3-3 mutants fully rescue the dock mutant defects, even doubly mutant transgenes in which both SH3-1 and SH3-3 are inactivated efficiently rescue the R cell guidance defects (Rao, 1998).

The SH2 domain is not required for the R cell innervation pattern. Surprisingly, the dock transgene containing the R336Q mutation in the SH2 domain fully rescues the R cell innervation pattern in dock mutants. To assess whether rescue reflects residual binding of the mutated SH2 domain to its cognate ligand, a dock transgene in which the SH2 domain was deleted in its entirety was introduced into dock mutants. This construct largely, but not completely, rescues the mutant phenotype. These findings are in marked contrast to the essential requirement of the SH2 domain of the C. elegans and Drosophila homologs of the Grb2 adapter, Drk and Sem-5, respectively. It is suggested that because the SH2 point-mutant and deletion transgenes do not rescue lethality, the SH2 domain is required in other neurons (Rao, 1998 and references).

To determine whether these domain requirements apply to all neurons, the ability of mutant transgenes to rescue neuronal connectivity defects in the inner optic ganglia of dock mutants was assessed. There are four highly ordered neuropil regions in the optic lobe: the lamina, medulla, lobula, and lobula plate. R cell axon innervation induces the development of the outer optic lobe (i.e., lamina and outer medulla), but not the inner optic lobe (i.e., inner medulla, lobula, and lobula plate). In dock mutants all neuropil structures are completely disrupted. This phenotype can be rescued in its entirety by expression of wild-type dock specifically in postmitotic neurons. Consistent with a direct role in the guidance of these neurons, their growth cones and axons are seen to be highly enriched with Dock protein. However, because of the cellular complexity of the inner optic ganglion and the lack of cell-type specific markers in this region, a role for Dock in other aspects of postmitotic neuronal differentiation cannot be ruled out. As seen for the R cells, the SH3-2 domain is essential for the formation of fiber patterns in the inner optic ganglia and transgenes singly or doubly mutant for SH3-1 and SH3-3 rescue the phenotype. In contrast to R cells, however, the SH2 domain is essential for the fiber patterns in the inner optic ganglia. Hence, Dock couples guidance and/or differentiation signals in different ways in different neurons (Rao, 1998).

The analysis of single-domain mutants has established that SH3-1, SH3-3, and SH2 domains are not essential for Dock function in R cells. Hence, either the SH3-2 domain is sufficient for Dock function in these neurons or the loss of the SH2 domain is compensated by the SH3-1 and SH3-3 domains. To assess this possibility, a set of doubly and triply mutant forms of dock was tested for phenotypic rescue. Double mutants containing the SH2 point mutation in combination with mutations in either SH3-1 or SH3-3 show only weak rescue in contrast to the near-complete rescue of the R cell connection defects observed with the singly mutant transgenes. A construct triply mutant for SH3-1, SH3-3, and SH2 does not rescue, and R cell projections in larvae carrying this construct are indistinguishable from dock mutants, or dock mutants carrying a rescue construct in which the SH3-2 domain is inactivated. These data demonstrate that the SH3-1 and SH3-3 domains are functionally redundant with the SH2 domain in R cells; that is, Dock function in these neurons requires either functional SH3-1 and SH3-3 domains or a functional SH2 domain (Rao, 1998).

These data establish that multiple, but redundant, domains are essential for Dock function. In the inner optic ganglia, neurons require both the SH3-2 and the SH2 domains. In contrast, Dock can function in R cells either with the SH3-2 and SH2 domains, or with all three SH3 domains in the absence of the SH2 domain. Dock may function as an adapter by binding signaling molecules together, or alternatively, each domain in Dock may function independently. To distinguish between these possibilities, the ability of mutant transgenes to complement one another was assessed. Two mutant transgenes, one carrying the SH3-2 mutation and the other carrying mutations in SH3-1, SH3-3, and SH2 were introduced together into a homozygous dock mutant background. No rescue activity was detected. These mutant transgenes do not function in a dominant negative fashion either alone or in combination. Hence, Dock requires multiple domains acting in cis. This is consistent with the proposal that Dock functions as an adapter to link signaling molecules together in growth cones (Rao, 1998).

Studies of mammalian Nck have suggested possible biochemical functions for Dock. That Nck can couple directly to receptor tyrosine kinases has been demonstrated in several mammalian tissue culture systems. Most interestingly, activation of the c-met receptor, a multifunctional receptor recently shown to regulate motoneuron guidance decisions in vertebrates, leads to recruitment of Nck into a receptor-containing complex. Several proteins implicated in the control of the actin-based cytoskeleton also bind to Nck: these include WASp, the product of the Wiskott-Aldrich syndrome gene, and PAK (see Drosophila PAK-kinase) and PRK2, both members of the p21-activated protein kinase family. All three proteins also bind to Rho-family GTPases, which have been shown to regulate the structure of the actin-based cytoskeleton. Whereas WASp binds to SH3-3, both PRK2 and PAK bind to SH3-2. Induction of cytoskeletal changes in cultured cells with activated PAK requires the presence of a Nck binding site and is correlated with increased Nck binding. Interestingly, Dock binds strongly through its second SH3 domain to DPAK (J. Xiao and S. L. Zipursky, unpublished data cited in Rao, 1998), a Drosophila homolog of PAK, which is expressed in the nervous system and colocalizes to both tyrosine-phosphorylated proteins and F-actin. Like mammalian PAK, DPAK also binds to activated Rho-family GTPases Rac and Cdc42. These studies raise the intriguing possibility that the physical interactions between Dock, DPAK, and Cdc42 may regulate growth-cone motility in Drosophila (Rao, 1998 and references).

Slit stimulation recruits Dock and Pak to the Roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline

Drosophila Roundabout is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Evidence is presented that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance (Fan, 2003).

Strong defects in embryonic axon guidance are observed only when both the maternal and zygotic components of dock function are removed. In these maternal minus dock mutants (dock^mat), phenotypes reminiscent of loss of robo function can often be seen. dock^mat embryos examined with an antibody that labels all axons frequently show thickening of commissural axon bundles and a commensurate reduction in the thickness of longitudinal axon bundles. Staining these embryos with an antibody that selectively labels noncrossing axons (anti-fasII) reveals a significant degree of ectopic midline crossing. These phenotypes are similar to, but considerably less severe than, those observed in robo mutants. The similarity in mutant phenotypes that is observed provides genetic support for the idea that dock could contribute to Robo repulsion (Fan, 2003).

If dock and robo function together during midline guidance, they should be coexpressed in embryonic axons. This is indeed the case. Double labeling of embryos with antibodies raised against Dock and Robo reveals substantial coexpression of the two proteins. Both Dock and Robo show enriched expression on CNS axons beginning as early as stage 12, corresponding to the time of initial axon outgrowth. At these early stages of axon growth, Dock is detected in the pCC axon, a cell known to express Robo, as revealed by double labeling with FasII. Interestingly, while Robo shows a regionally restricted expression pattern with high levels of expression on longitudinal portions of axons and low levels in commissural axons, Dock is expressed equivalently in both commissural and longitudinal axon segments. This observation raises the possibility that Dock could have additional roles in the guidance of commissural axons not shared by Robo. These observations show that Dock and Robo are both present at the right time and place to function together during midline repulsion (Fan, 2003).

Biochemical data suggests that the interaction between Dock and Robo is an SH3-dependent interaction and that the first two SH3 domains of Dock are most important for mediating Robo binding. Based on the observations that a three-protein interaction can be detected between Robo, Dock, and Pak and that Pak has been shown to interact with the SH3-2 domain of Dock, it is believed that the SH3-1 domain is the most important for Robo and Dock binding. Furthermore, Slit stimulation enhances Dock's ability to bind to Robo, suggesting a ligand-regulated SH3 domain interaction. This represents a different kind of adaptor interaction to many that have been observed previously, where Nck appears to interact with a number of tyrosine-kinase receptors through an SH2 domain/phosphotyrosine interaction. In the latter case, how ligand binding to the receptor regulates the Nck SH2 domain interaction is quite well understood. The observation that the Robo receptor shows a ligand-regulated SH3 domain interaction with Dock/Nck suggests that somehow ligand binding results in an increased availability of the SH3 binding sites in the receptor (Fan, 2003).

The regions of Robo that appear to be most important for the interaction are the proline-rich regions CC2 and CC3. Individual mutations in these motifs strongly reduce the amount of Dock that coimmunoprecipitates with Robo in cell culture, while removing both of these motifs completely abolishes binding. Furthermore, expression of Robo receptors that lack the CC2 and CC3 motifs in transgenic Drosophila disrupt the in vivo function of the receptor. It is important to stress that the CC2 and CC3 sequences are not only involved in Dock binding, but also bind Ena, Abl, and potentially other proteins as well. In addition, CC2 and CC3 are also required for the observed upregulation of Rac activity. The fact that many proteins bind Robo at these sites prevents clear conclusions about why the ΔCC2ΔCC3 mutant receptor is nonfunctional. In the future, more precisely defining the binding requirements of the many proteins that interact with Robo may allow forms of Robo to be created that specifically disrupt the binding of some partners and not others, which in turn should provide insight into the relative roles of different Robo signaling outputs (Fan, 2003).

The implication of Rac in Robo repulsion (dominant negative Rac1 shows a strong enhancement of slit;robo/+ defects) was unexpected in view of the well-established role of Rac as a positive regulator of axon outgrowth. On the surface, this finding appears quite contradictory to the function of Rac to promote actin polymerization at the leading edge of motile cells and axons. One possible explanation of this finding is that perhaps Rac can have different or even opposite effects on the actin cytoskeleton, depending on the molecular context in which it is activated and its overall level of activity. For example, depending on the coordinate local function of other small GTPases and actin regulatory proteins, the consequences of Rac function could be different. It is interesting to note that in addition to a role for Rac, genetic analysis and previously published data also support an important role for Rho in midline repulsion. Furthermore, in addition to strongly stimulating Rac activity, Slit has been shown have a modest stimulatory effect on Rho activity. The implication of both Rac and Rho in mediating repulsive responses has also been suggested to explain the output of the Plexin receptor. It will be interesting in the future to determine the interrelationship between Rac and Rho outputs in the context of Robo repulsion as well as in signaling downstream of other attractive and repulsive axon guidance receptors (Fan, 2003).

As an alternative to the context- and level-dependent explanation of the role of Rac in Robo repulsion, the observed axon steering defects in embryos where both Rac and Slit function are reduced, or in embryos deficient for multiple rac genes, could be explained as a secondary consequence of defects in the rate of axon extension. In this scenario, Rac's role in repulsive axon guidance would be intimately coupled with its role in axon outgrowth. That is to say, that appropriate steering decisions go hand and hand with the appropriate regulation of the rate of axon outgrowth (e.g., you are more likely to miss your exit if you are driving too fast). In this regard, it is important to emphasize that even repulsive cues can have stimulatory effects on axon extension. For example, in addition to repelling Xenopus spinal neurons, Slit also has a stimulatory effect on the rate of axon extension (Fan, 2003 and references therein).

Perhaps the most difficult observation to explain is how reciprocal shifts in Pak levels can lead to similar consequences for Robo repulsion. Since the enhancing effects of Pak overexpression in partial loss-of-function robo backgrounds are more dramatic with the membrane-tethered form of Pak, it is tempting to speculate that in order to signal properly, turning Pak activity on and off needs to be tightly controlled. Little is known about how Pak signaling is terminated and it seems quite possible that the membrane-tethered version of pak is not as effectively regulated as the wild-type form of pak. Interestingly, in genetic backgrounds where robo signaling is specifically compromised in its output, through reduction of rac, introducing the UASPak^Myr transgene can partially suppress the midline crossing defects. Given the clear ability of alterations in pak expression to modulate midline repulsion and the observation that Slit can promote the formation of a Robo, Dock, and Pak protein complex, it is somewhat surprising that complete removal of zygotic pak does not have major consequences for embryonic axon guidance. Indeed, in the absence of clear loss-of-function phenotypes in pak mutants, it is difficult to argue unequivocally for a critical role of endogenous pak in robo function. There are a number of potential explanations for these observations including, but not limited to, maternal pak contribution and the potential redundant function of a second pak-like gene. Future experiments should address these possibilities in order to link pak more firmly to robo (Fan, 2003).

Dock has been suggested to act downstream of the Dscam axon guidance receptor during pathfinding of Bolwig's nerve, and the vertebrate homolog of Dock, Nck, has also been linked to several guidance receptors in vitro, including Eph receptors and c-Met receptors. More recently, Nck has been shown to directly interact with the cytoplasmic domain of the vertebrate attractive Netrin receptor DCC. The Nck and DCC interaction is important for DCC's function to stimulate axon extension in vitro. Together these observations raise the question of whether a similar DCC/Nck interaction occurs in Drosophila, and if so whether the interaction is important for the in vivo function of Drosophila DCC (encoded in the fly by the frazzled gene) to attract commissural axons across the midline. Interestingly, in addition to its substantial overlap in expression with the Robo receptor, Dock protein is also expressed in commissural portions of axons, as is the Frazzled receptor. While the dock mutant phenotype is most consistent with a role in midline repulsion, an additional function in attraction cannot be ruled out. In the future it will be interesting to test for genetic interactions between frazzled, dock, Rac, and pak to determine if this signaling module is also employed during midline axon attraction in Drosophila (Fan, 2003).

The implication of Dock/Nck and Rac in both DCC-mediated attraction and Robo-mediated repulsion raises the obvious question of how the specificity of attraction and repulsion is controlled and argues against a committed role of either of these signaling molecules to either one or the other type of responses. This is perhaps not too surprising, given the fact that Robo and DCC receptors themselves are intimately connected through their ability to form a heteromeric receptor complex with potentially unique signaling properties. Although it remains possible that signaling molecules or adaptors will be identified that can account for the specificity, an alternative possibility is that it is the coordinate regulation, relative activity levels, and combinatorial action of a core group of common signaling molecules that makes the difference in attraction versus repulsion (Fan, 2003).

Biochemical data support the idea that Slit stimulation of Robo can regulate the recruitment of Dock and Pak to the Robo receptor and also trigger an increase in Rac activity. Both of these events are dependent on the CC2 and CC3 sequences in Robo's cytoplasmic domain. Thus, the observations are consistent with either a Dock-dependent or a Dock-independent recruitment of Rac to Robo. Based on the known physical interactions between Dock and Pak and between Pak and Rac, it is likely that the recruitment of Rac is dependent on Dock. Alternatively, another protein interacting through CC2 and/or CC3 could function to recruit Rac in a Dock-independent fashion (Fan, 2003).

Regardless of whether the recruitment of Rac to Robo is dependent on Dock and Pak or is an independent event, the data cannot explain how Slit stimulation of Robo results in increased Rac activity. Two obvious types of molecules that are missing from the model and the protein complex are the upstream regulators of Rac, the GEF and GAP proteins. Intriguingly, in the course of a genome-wide analysis of all RhoGEFs and RhoGAPs in Drosophila, one Rac-specific GAP has been identified that when overexpressed results in phenotypes reminiscent of robo loss of function (H. Hu et al., submitted, reported in Fan, 2003). There are a number of candidate GEFs that could explain how Rac activity is upregulated by Slit activation of Robo, most notably Sos, rtGEF (pix), and Trio. It will be interesting to determine which if any of these molecules could play such a role in Robo signaling (Fan, 2003).

GENE STRUCTURE

Exons - 5

PROTEIN STRUCTURE

Amino Acids - 410

Structural Domains

Database comparisons indicate that Dock is similar to the mammalian Nck oncoprotein. Both molecules consist of three SH3 domains and one C-terminal SH2 domain and are similar in length and domain order. The most highly conserved sequences shared by these molecules are within the SH3 and SH2 domains (55%-72% identity). Comparison of the SH3 domains from Dock with SH3 domains from proteins other than Nck yield percent identities averaging 38%, suggesting that Dock and Nck must be either homologs or two members of a family of Nck-like molecules. Dock and Nck contain the two key arginine residues within the SH2 domains that contact phosphotyrosine (Clemens, 1996).

date revised: 5 August 2021

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