EvoprintHD of dock

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).

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: 27 April 99

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