The synaptic function of the receptor protein tyrosine phosphatase (RPTP), Dlar, and an associated intracellular protein, Liprin-α, has been examined at the Drosophila larval neuromuscular junction. Liprin-α and Dlar are required for normal synaptic morphology. Synapse complexity is proportional to the amount of Dlar gene product, suggesting that Dlar activity determines synapse size. Ultrastructural analysis reveals that Dliprin-α and Dlar are required to define the size and shape of the presynaptic active zone. Accordingly, there is a concomitant decrease in synaptic transmission in both mutants. Finally, epistasis analysis indicates that Dliprin-α is required for Dlar's action at the synapse. These data suggest a model where Dliprin-α and Dlar cooperate to regulate the formation and/or maintenance of a network of presynaptic proteins (Kaufmann, 2002; full text of article).
The yeast two hybrid system was used to searched for Drosophila proteins that would bind directly to Dlar in the yeast interaction trap assay. Using the entire cytoplasmic domain of Dlar as bait, approximately ten million Drosophila cDNA clones were examined. Sequence of the first strong interacting clone revealed it to be the Drosophila homolog of Liprin-α. Mammalian Liprin-α belongs to a family of related mammalian proteins; however, scans of the Drosophila genome identify only one Liprin-α gene (Kaufmann, 2002).
The structural motifs in Liprin-α family members consist of an N-terminal coiled-coil domain, shown to multimerize the protein and a C-terminal LAR binding region. While Drosophila Liprin-α is 47% identical to mammalian Liprin-α overall, much higher levels of conservation are seen in individual domains. For example, a region of 70 amino acids near the N terminus of Drosophila Liprin-α is over 90% identical to mLiprin-α. In addition, the LAR binding domain of Drosophila Liprin-α recovered in the protein interaction screen is 75% identical to mLiprin-α and 68% identical to its C. elegans homolog (Kaufmann, 2002).
As a first step toward a functional analysis, the expression of Liprin-α was examined. Using in situ hybridization, Liprin-α mRNA was observed to be abundantly expressed in the embryo, from cellular blastoderm through hatching. A strong signal at early stages (stages 4 and 5) indicates that there is a substantial maternal contribution of Liprin-α. Liprin-α mRNA accumulates at highest levels in the developing central and peripheral nervous systems (CNS and PNS, respectively) and is not detected outside of the CNS and PNS late in embryonic development. In situs at the third instar stage also reveal persistent expression of liprin-α in the larval CNS and bodywall muscle (Kaufmann, 2002).
To determine Liprin-α protein localization, polyclonal antibodies were raised and affinity purified. In late stage embryos (stages 16 and 17), the highest levels of Liprin-α are restricted to the neuropil of the ventral nerve cord. This precisely matches the pattern of Dlar protein localization at these stages, consistent with direct biochemical association between Liprin and LAR family members. While Liprin-α protein localizes to both longitudinal and commissural neuropil in stage 16, it was noticed that the protein becomes restricted to longitudinal neuropil at stage 17 and first instar. Since developing synapses are found primarily in the longitudinal regions, this raised the question of whether Liprin-α is primarily synaptic in its localization. To address this, peripheral motor pathways were examined at larval stages when the NMJ is well formed and distinct from the axon. At this stage, Liprin-α accumulates specifically at the synapse, showing little or no staining along the axon. Higher resolution confocal images show that Liprin-α is both presynaptic and postsynaptic (compared to a neuronal membrane marker), often forming puncta at the membrane reminiscent of active zones. This pattern is nearly identical to the localization of the synaptic protein Discs large (Dlg) and strongly suggests a function for Liprin-α at the NMJ. Interestingly, Liprin-α localization appears grossly normal at Dlar null mutant NMJs. Although in situ hybridizations confirm that Dlar is expressed throughout the larval CNS, it is not detected in muscle during the same larval stages (Kaufmann, 2002).
The liprin-α gene maps to the cytological location 27B1. The closest P element was about 40 kb away from liprin-α at that time, though there is now an EP insertion (EP2141) approximately 600 base pairs upstream of liprin-α. To generate mutations in liprin-α, P elements from two separate lines, l(2)k00605 and l(2)k13315, were remobilized. Two independent strains were recovered from the l(2)k13315 remobilization with insertions in the liprin-α transcription unit. Both insertions fall in an intron in the 5'UTR of liprin-α within a few nucleotides of each other but are oriented in opposite directions. Staining for Liprin-α clearly shows lower levels of the protein in homozygous embryos from one of the insertion lines (J1), while protein levels seemed approximately normal in the other line (F3). Though dramatically lighter than wild-type controls, traces of residual Liprin-α are still seen in the CNS of the J1 line. This suggests that J1 is hypomorphic; however, the pattern is also consistent with the perdurance of maternally expressed Liprin-α; abundant staining in early embryos (stage 4) supports this theory (Kaufmann, 2002).
To generate stronger mutant alleles, the P element in the F3 line was remobilized. A number of liprin-α deficiency lines were generated by this method. One line, liprin-αF3ex15, was selected for detailed study. DNA sequence analysis shows that liprin-αF3ex15 retains some of the P element and leaves a small piece of the 5'UTR of liprin-α while the rest of the gene is deleted. Genomic mapping of this excision shows that the liprin-α coding region is completely removed in this line; however, in embryos, traces of protein staining are still observed in the embryonic neuropil, similar to that seen in the liprin-αJ1 insertion line; the localization of this antigen appeared normal. It is believed this is due to maternal contribution to the embryo. To generate additional alleles, a P element from the l(2)k00605 mobilization screen, which had inserted 600 base pairs upstream of liprin-α, was excised and excisions were analyzed. This latter screen generated deficiencies extending in both directions, some of which removed liprin-α, including liprin-α77ex17. While generating these alleles, two lines derived from excision of EP2141, liprin-αR60 and liprin-αR117 became available. Mapping of these excisions shows they are both small deficiencies, removing most of the upstream region between liprin-α and an adjacent transcription unit. Liprin-α protein levels are reduced in both lines. Dlar expression and localization is normal in all of these liprin-α mutant backgrounds (Kaufmann, 2002).
To determine the function of Liprin-α, a series of allelic combinations that disrupt liprin-α was created with removal of only one copy of flanking genes. Since a percentage of motor axons (~20%) from the intersegmental nerves b and d (ISNb and ISNd, respectively) require Dlar for guidance to their target muscles in embryonic stages, embryonic motor pathways were examined in liprin-α mutants. No defects in axon guidance were seen. Thus, no evidence is found that zygotic liprin-α is necessary for motor axons to reach their targets, although maternal expression may mask an early function. However, the absence of axon guidance defects in zygotic liprin-α mutants afforded the opportunity to examine the role of liprin-α in synapse development at the larval NMJ (Kaufmann, 2002).
The most thoroughly characterized NMJ in the Drosophila larva is the glutamatergic synapse at muscles 6 and 7. This synapse (6/7NMJ) is innervated by two neurons: RP3, which elaborates a terminal decorated with large boutons (type Ib), and MN6/7b, which generates smaller boutons (type Is). The number of boutons and the branch complexity in the 6/7NMJ at abdominal segments A2 and A3 is highly stereotyped during the wandering third larval instar stage. When liprin-α mutants are compared to the genetically matched parental control strains using immunohistochemistry, a 30%-50% reduction is found in the size of the 6/7NMJ, as assessed by the number of boutons per NMJ. Both type Ib and type Is boutons are reduced in number. Although more variable than bouton number, branch complexity of the terminal arbor is also decreased compared to wild-type. In addition, the same phenotype is seen in three liprin-α loss-of-function combinations derived from two independent parental strains, ruling out genetic background effects. Bouton number is normal in liprin-α/+ heterozygotes, demonstrating that liprin-α is not haploinsufficient (Kaufmann, 2002).
In order to prove that the reduction in synapse size was due solely to loss of liprin-α function, the J1 P element insertion was mobilized and revertants were sought that completely restored the liprin-α gene. Two precise excision lines were isolated, and anatomical analysis shows that they have normal bouton numbers and branching complexity at the 6/7NMJ. liprin-α is thus required for normal NMJ development (Kaufmann, 2002).
In order to better understand the defects in synaptogenesis, NMJ morphology was examined in liprin-α mutants using confocal microscopy. At the tips of wild-type synaptic arbors, a large end bouton is frequently surrounded by smaller boutons, presumably formed by budding of the parent bouton in the process of synapse growth. In contrast, liprin-α mutant terminals frequently lack these nascent bouton structures. Quantification of these structures in wild-type and heterozygous controls, compared to two different strong liprin-α mutant combinations, reveals a substantial reduction in the mutants. This suggests that Dliprin-α is important for the growth of the synapse through formation of new boutons at the ends of terminal branches. The reduction in branch complexity in liprin-α mutants is consistent with this hypothesis. Despite the reduction of nascent boutons, the expression and localization of several synaptic markers are unaffected by the liprin-α mutations; the markers include the scaffolding protein Discs large (Dlg), the cytoskeletal protein Futsch, and the glutamate receptor GluRII (Kaufmann, 2002).
The function of liprin-α at the synapse and the late larval expression of Dlar raised the question of whether Dlar might also be required for NMJ morphogenesis. In this regard, the low penetrance (~20%) of motor axon guidance defects in Dlar null alleles (e.g., Dlar13.2 and Dlar5.5) provided an opportunity to examine NMJ morphology at synapses innervated by the correct number of motor neurons. To identify the ~80% of larval segments where axon guidance was unaffected in Dlar mutants, the analysis was limited to 6/7NMJs that contained both type Ib and type Is boutons from axons that entered the target domain along the correct trajectory (indicating that both RP3 and MN6/7b were present). Antibodies that recognize the synaptic protein Dlg were used in order to distinguish between the two classes of boutons. Examination of bouton number and terminal branch complexity revealed that complete loss of Dlar results in a reduction in synapse size very similar to that observed in liprin-α loss-of-function mutants. Both type Ib and type Is boutons were reduced, suggesting that both neurons at the 6/7NMJ require Dlar activity, as observed in liprin-α mutants (Kaufmann, 2002).
The synapse is very sensitive to the amount of Dlar expressed. Although a null allele of Dlar (Dlar5.5) would be expected to show more penetrant phenotypes than a weaker hypomorph (Dlarbyp), reduction of Dlar gene dose by 50% (Dlar5.5/+) decreases NMJ size relative to wild-type. This is in contrast to all other known functions of Dlar and suggests that synapse growth is directly proportional to the amount of Dlar activity. If this model is correct, it is predicted that raising Dlar activity above wild-type levels should increase synapse size. Indeed, expression of a full-length Dlar transgene under control of two different postmitotic neural-specific GAL4 drivers increases bouton number in comparison to a wild-type strain. These experiments also suggest that Dlar acts presynaptically, as initially suggested by the neural-specific expression of endogenous Dlar (Kaufmann, 2002).
To determine if the effect of increased Dlar expression was manifest specifically during larval stages when the NMJ undergoes massive growth and to ask if the activity could be supplied by the catalytic domains alone, the cytoplasmic region of Dlar was expressed under a neural-specific GAL4 fused to a hormone receptor (GS-GAL4). This expression system provides both tissue and temporal specificity. Induction of Dlar expression during midlarval development with 50 µg/ml mifepristone increases bouton number (Kaufmann, 2002).
Previous studies have suggested a model where Liprin-α acts upstream of Dlar to localize the protein on the cell surface (Serra-Pagès, 1998). The Dlar gain-of-function NMJ phenotype presented an opportunity to perform an epistasis test by simultaneously removing liprin-α function while overexpressing Dlar in postmitotic neurons. While overexpression of Dlar does not rescue the liprin-α phenotype, the absence of Liprin-α protein prevents the increase in NMJ bouton number observed when Dlar is overexpressed in a wild-type background. This puts Liprin-α genetically downstream of Dlar, providing evidence that Liprin-α is required for the normal output of the LAR pathway (Kaufmann, 2002).
Genetic analysis in C. elegans shows that the Liprin-α homolog syd-2 is required to constrain the size of cholinergic active zones (AZs). To ask whether this Liprin-α function is conserved in Drosophila and whether it generalizes to a glutamatergic synapse, an analysis of liprin-α mutant NMJ ultrastructure was undertaken. liprin-α active zones (AZs) fall across a range of sizes from smaller than normal to far greater in size. Mutant AZs were always abnormal in either total size (area) or shape; the mean maximum dimension of mutant AZs (1319 nm) is nearly double that of wild-type (684 nm). Moreover, the mean total area of mutant AZs is 2.4-fold higher than wild-type (Kaufmann, 2002).
Since Liprin-α and Dlar associate directly and display nearly identical gross NMJ phenotypes, it was asked if Dlar is also required to define the dimensions of the presynaptic active zone. Like the liprin-α mutants, overall ultrastructure is normal in the Dlar mutants on both presynaptic and postsynaptic sides of the NMJ. However, 61% of individual AZs are larger than the largest AZ found in wild-type NMJs (Kaufmann, 2002).
To elucidate a potential synaptic function of Liprin-α and Dlar, the synaptic physiology of mutant larval NMJs were examined by whole-cell recordings of evoked excitatory junctional potentials (EJPs) and spontaneous excitatory junctional potentials (mEJPs). EJPs were reduced 36% in liprin-α and 34% in Dlar mutants, compared to precise excision controls; this was highly significant. The resting potential of the mutant muscle was normal (Kaufmann, 2002).
The decrease of synaptic transmission in liprin-α and Dlar mutants was not caused by a postsynaptic defect since quantal events (mEJPs) were normal. The mean amplitude and the frequency of unitary mEJPs in liprin-α and Dlar larvae also showed no significant differences from control. This indicates that the postsynaptic sensitivity to neurotransmitter and the neurotransmitter content of synaptic vesicles are not altered in mutants. However, the normal postsynaptic neurotransmitter reception pointed to a defect intrinsic to the evoked release process (Kaufmann, 2002).
To better quantify mutant synaptic transmission, the number of quanta released per stimulus was determined. Quantal content is defined by the ratio of EJP/mEJP amplitudes after correcting for nonlinear summation using a reversal potential of 0 mV. The quantal content of evoked release for both liprin-α and Dlar was reduced by over 50%. This highly significant reduction in presynaptic vesicle release is consistent with a fundamental defect in active zone structure and function (Kaufmann, 2002).
Analysis of the C. elegans Liprin-α gene syd-2 provided the first support for the model that Liprins are integral to the presynaptic protein complex that organizes neurotransmitter release (Zhen, 1999). This function is conserved. Data from C. elegans, Drosophila, and mammals indicate that the protein localization of Liprin-α family members to the synapse is also conserved (Zhen, 1999; Wyszynski, 2002). Interestingly, in all organisms, Liprin-α is found on both sides of the synapse. Analyses in C. elegans and mammalian neurons support both presynaptic and postsynaptic roles. In C. elegans, loss of syd-2 increases the dimensions of the AZ by approximately 2-fold, with a concomitant decrease in the efficiency of synaptic transmission (Zhen and Jin, 1999). Analysis in Drosophila not only confirms an increase in AZ size, but also reveals that liprin-α mutations have a dramatic effect on AZ shape. This latter phenotype is fully penetrant, suggesting that liprin-α is a requisite structural component of the AZ (Kaufmann, 2002).
Exactly what Liprin-α is doing on the postsynaptic side is unknown. Since Dlar expression is not detected in muscle, this also raises the question of what proteins act in partnership with Liprin-α in the subsynaptic reticulum. Studies in mammalian synapses suggest that Liprins interact with several pre- and post-synaptic proteins (Kaufmann, 2002).
But how do Liprins act to support LAR-family receptor function? Several models are possible. Liprin-α may act in the signaling mechanism downstream of the receptor. Although Liprins have not been shown to be phosphorylated PTP substrates, they could serve to recruit other necessary signaling proteins. The fact that mLiprin-α associates with synaptic scaffolding proteins is consistent with this hypothesis (e.g., GRIP; Wyszynski, 2002). Alternatively, Liprin-α may function to deliver LAR-family receptors to their sites of action in the cell, as suggested by experiments in nonneuronal cells. Without a functional delivery system, Dlar overexpression may fail to increase RPTP activity in the appropriate location (Kaufmann, 2002).
Axonal transport is required for the elaboration and maintenance of synaptic morphology and function; this study demonstrates that Liprin-α is required for trafficking of synaptic vesicles. Liprin-αs are scaffolding proteins important for synapse structure and electrophysiology. A reported interaction with Kinesin-3 (Kif1a) suggested Liprin-α may also be involved in axonal transport. Aberrant accumulations have been discovered, at the light and ultrastructural levels, of synaptic vesicle markers (Synaptotagmin and Synaptobrevin-GFP) and clear-core vesicles along Drosophila Liprin-α mutant axons. Analysis of presynaptic markers reveals reduced levels at Liprin-α synapses. Direct visualization of Synaptobrevin-GFP transport in living animals demonstrates a decrease in anterograde processivity in Liprin-α mutants but also an increase in retrograde transport initiation. Pull-down assays reveal that Liprin-α interacts with Drosophila Kinesin-1 (Khc) but not dynein. Together, these findings suggest that Liprin-α promotes the delivery of synaptic material by a direct increase in kinesin processivity and an indirect suppression of dynein activation. This work is the first to use live observation in Drosophila mutants to demonstrate the role of a scaffolding protein in the regulation of bidirectional transport. It suggests the synaptic strength and morphology defects linked to Liprin-α may in part be due to a failure in the delivery of synaptic-vesicle precursors (Miller, 2005; full text of article).
Three prominent models have been proposed to explain the regulation of bidirectional transport: (1) a substitution model in which only one set of motors is present on the cargo at a given time, (2) a tug-of-war model in which both anterograde and retrograde motors are bound and always active but differ in their number on the cargo, and (3) a coordinate-regulation model in which both sets of motors are bound but one group is inactive. The observations that dynein is associated with anterograde transported cargos and kinesin is associated with retrogradely transported vesicles containing synaptic components argue against the substitution model of transport for SVPs. If the tug-of-war model were correct, then the shift in flux that was observe in Liprin-α mutants would correspond to a change in the number of bound active motors and a skewing of the velocity profile. However, because no such shift is observed, the current results are most consistent with a model in which coordinate regulation mediated through Liprin-α modulates transport. Because Liprin-α interacts with kinesins but not dynein, the data suggest that Liprin-α directly promotes kinesin activity or cargo-association, which then leads to dynein inhibition through some additional component(s) (Miller, 2005).
In light of the observations that disruption of kinesin alters the morphology and electrophysiological properties of synapses, these observations suggest that the synaptic defects seen in mutants of LAR and the Anaphase Promoting Complex may be mediated in part by Liprin-α’s role in axonal transport. As a scaffolding protein with multiple known partners and motors, Liprin-α is in an ideal position for integrating and transducing information to regulate the delivery of cargoes to and from the synapse (Miller, 2005).
The multiprotein complexes that receive and transmit axon pathfinding cues during development are essential to circuit generation. Identified and characterized the Drosophila sterile α-motif (SAM) domain-containing protein Caskin, which shares homology with vertebrate Caskin, a CASK [calcium/calmodulin-(CaM)-activated serine-threonine kinase]-interacting protein. Drosophila caskin (ckn) is necessary for embryonic motor axon pathfinding and interacts genetically and physically with the leukocyte common antigen-related (Lar) receptor protein tyrosine phosphatase. In vivo and in vitro analyses of a panel of ckn loss-of-function alleles indicate that the N-terminal SAM domain of Ckn mediates its interaction with Lar. Like Caskin, Liprin-α is a neuronal adaptor protein that interacts with Lar via a SAM domain-mediated interaction. Evidence is presented that Lar does not bind Caskin and Liprin-α concurrently, suggesting they may assemble functionally distinct signaling complexes on Lar. Furthermore, a vertebrate Caskin homolog interacts with LAR family members, arguing that the role of ckn in Lar signal transduction is evolutionarily conserved. Last, several ckn mutants were characterized that retain Lar binding yet display guidance defects, implying the existence of additional Ckn binding partners. Indeed, the SH2/SH3 adaptor protein Dock was identified as a second Caskin-binding protein and it was found that Caskin binds Lar and Dock through distinct domains. Furthermore, whereas ckn has a nonredundant function in Lar-dependent signaling during motor axon targeting, ckn and dock have overlapping roles in axon outgrowth in the CNS. Together, these studies identify caskin as a neuronal adaptor protein required for axon growth and guidance (Weng, 2011).
The Drosophila neuromuscular system is an excellent paradigm to decipher the molecular signals orchestrating the precise matching between individual motorneurons and their muscle partners. A number of guidance cues and receptors coordinately regulate motor axon pathfinding assuring the high fidelity of this process. The axon must integrate these disparate signals as it navigates through its environment. Multidomain adaptor proteins promote such integration since they serve as platforms to facilitate communication between signal transduction cascades (Weng, 2011).
The leukocyte common antigen-related (LAR)-related subfamily of receptor protein tyrosine phosphatases (RPTPs type IIA) are conserved regulators of axon pathfinding and synaptogenesis. This subfamily includes the Drosophila receptors Lar and PTP69D, and the vertebrate receptors LAR, protein tyrosine phosphatase ς (PTPς), and PTPδ. Family members contain a variable number of Ig and fibronectin (FN) III domains extracellularly, and two intracellular phosphatase domains. The membrane-proximal D1 phosphatase domain (D1) confers most if not all of the catalytic activity of the receptor, whereas the membrane-distal D2 domain is catalytically inactive and may contribute to LAR family function via interaction with downstream signaling components. These receptors exhibit neuronal expression patterns, and loss-of-function (LOF) mutants display defects in axon targeting and synapse formation. Heparan sulfate proteoglycans (HSPGs) are binding partners of LAR family members in axon pathfinding and synaptogenesis. In vertebrates, PTPδ is a neuronal receptor for chondroitin sulfate proteoglycan (CSPG) and inhibits axon regeneration after CNS injury. On the intracellular side, Lar activity in some contexts requires phosphatase activity, whereas in other contexts its function is independent of catalytic activity, suggesting a diversity of downstream signaling pathways. Indeed, a number of Lar-interacting proteins have been identified. Lar function in synaptic maturation requires Liprin-α, a sterile α-motif (SAM) domain-containing adaptor protein that interacts with Lar in vertebrates and invertebrates (Weng, 2011).
Drosophila Dock and vertebrate Nck are neuronal SH2/SH3-containing adaptor proteins that link guidance receptors to cytoskeletal remodeling. Given the widespread expression of Dock in the embryonic CNS and its central position linking guidance receptors to the actin cytoskeleton, it is notable that motor axons in dock LOF mutant embryos display only subtle defects, raising the possibility of compensation or redundancy. Dock/Nck interact directly with a number of receptors, including Robo, DSCAM (Down syndrome cell adhesion molecule), and the insulin receptor and bind directly to cytoskeletal effectors such as p21-activated protein kinase (Pak) and WASP (Wiskott-Aldrich syndrome protein), thereby presumably linking receptor activation to cytoskeletal rearrangement. Of particular relevance, vertebrate Caskin has been identified as a potential Nck interactor. Caskin was first identified as a novel protein binding the CaM kinase domain of CASK. Vertebrate Caskins are predicted scaffolding proteins with multiple ankyrin repeats, an SH3 domain, and two SAM domains, suggesting that Caskin is a component of a multiprotein complex. This study presents genetic, cell-biological, and biochemical evidence arguing that Drosophila Caskin is a Lar-binding partner and is required for Lar signal transduction in motor axon guidance (Weng, 2011).
This study has demonstrate that Caskin mediates a novel Lar RPTP signaling cascade during axonogenesis. Analysis of a panel of ckn LOF alleles indicates that ckn is necessary for motor axon pathfinding, since homozygous mutants display classic bypass defects in the ISNb motor nerve. This phenotype is identical with that displayed by Lar mutants, and genetic and biochemical interaction data demonstrate that Ckn is a Lar-interacting protein. These studies position Caskin to be a core member of a Lar-associated signaling complex that mediates its function during axonogenesis (Weng, 2011).
Vertebrate Caskin was identified as a binding partner of the synaptic adaptor protein CASK and competes for binding to the CaM kinase domain of CASK with the PDZ (postsynaptic density-95/Discs large/zona occludens-1) protein Mint1. The CASK-binding site on Caskin maps to an N-terminal region not conserved in Drosophila, suggesting that fly Caskin does not bind CASK. Consistent with this finding, Drosophila Caskin and CASK do not interact in a yeast interaction assay. However, both mouse and fly Caskin homologs bind LAR family members and Nck/Dock, in support of considerable shared functions. Furthermore, overexpression of the Lar-binding domain of mouse Caskin in Drosophila neurons yields a pathfinding phenotype like that of Lar and ckn LOF, suggesting that mouse Caskin competes with fly Ckn for binding to the Lar receptor to function as a dominant negative. These biochemical studies indicate that, whereas Caskins may have species-specific binding partners, Ckn function in Lar signal transduction is conserved. Drosophila Lar also physically interacts with the Abl tyrosine kinase and its substrate the cytoskeletal regulator Ena (Wills, 1999). This study was unable to detect physical interactions between Ckn and Abl or Ena, suggesting they bind Lar independently. This raises the possibility that Caskin and Abl/Ena constitute parallel pathways downstream of the Lar receptor (Weng, 2011).
The allelic series enabled analysis of the in vitro and in vivo activities of four Caskin mutant proteins: Ckn-A, Ckn-C, Ckn-K, and Ckn-Y. Ckn-A and Ckn-K contain alterations in the first SAM domain and block the interaction of Ckn with Lar, pointing to the importance of this domain for Lar/Ckn complex formation. The in vivo analysis of Ckn-A and Ckn-K is in strong agreement with the in vitro data as motor axon phenotypes are not associated with their overexpression, suggesting they do not interfere with Lar signaling in vivo. The behavior of Ckn-A and Ckn-K contrasts that of Ckn-C, which contains a C-terminal deletion. Ckn-C interacts with Lar, and its neuronal overexpression yields dominant-negative-like effects. In fact, the penetrance of ISNb bypass associated with Ckn-C overexpression is comparable with that observed in embryos lacking both maternal and zygotic Lar, suggesting that it effectively interferes with Lar activity. Although Ckn-C binds Lar, cknC homozygous LOF mutants display a 'Lar-like' ISNb phenotype, indicating that Lar signaling is blocked downstream of receptor binding. Ckn-C does not interact with Dock, but this interaction is insufficient to explain the cknC mutant phenotype since ISNb bypass is not associated with dock LOF. The pathfinding phenotype observed in cknC embryos argues that the allele also disrupts the interaction between Caskin and another downstream protein(s) essential for Lar signaling (Weng, 2011).
Dock/Nck are SH2/SH3-containing adaptor proteins that couple phosphotyrosines on activated receptors to downstream signaling molecules via SH2 and SH3 domain interactions, respectively. Dock also engages in a ligand-regulated SH3 domain interaction with the Robo receptor, demonstrating that it is involved in diverse interactions downstream of guidance receptors. This work has demonstrate that Caskin interacts with the second SH3 domain of Dock (SH3-2). This domain has also been shown to interact with the cytoskeletal effector Pak, raising the issue of the relationship between Caskin and Pak. It will be informative to determine whether Dock forms alternative complexes with Caskin and Pak, or whether Dock binds Caskin and Pak simultaneously (Weng, 2011).
The contrast between the ckn and dock single- and double-mutant phenotypes demonstrates that the adaptors have mostly redundant functions. Single-mutant analyses indicate that ckn plays a nonredundant role in Lar signaling, whereas dock has a unique role in synaptogenesis of the RP3 motorneuron. However, the outgrowth defects observed in dock ckn double mutants argue that these adaptors have overlapping roles in a number of signaling events. These data caution against drawing conclusions of cellular function based solely on single mutant analysis, as this obviously uncovers only the nonredundant functions of a protein. The issue of genetic redundancy may be particularly acute in signaling systems involving multi-subunit complexes with many opportunities for parallel functions. It will be important to identify additional binding partners of dock and ckn to determine whether they have a common set of interactors, or whether they impinge on the cytoskeleton via distinct, yet redundant, paths (Weng, 2011).
The Lar receptor is a member of the type IIA subfamily of RPTPs, comprising Lar and PTP69D in flies. The single-mutant phenotypes of Lar and PTP69D indicate they have nonredundant functions in motor axon guidance, NMJ growth, and photoreceptor axon targeting. Several observations hint that the unique functions implied by the divergent phenotypes of Lar and PTP69D stem in part from distinct ligand-binding activities. Lar and PTP69D alkaline phosphatase fusion proteins have be shown to possess distinct embryonic staining patterns suggesting the presence of unique ligands. Furthermore, overexpression of a chimeric receptor composed of the Lar extracellular domain fused to the PTP69D intracellular domain rescues the LOF photoreceptor defect of Lar, whereas a PTP69D extracellular domain fusion to the Lar intracellular domain does not, arguing that Lar and PTP69D have overlapping intracellular partners and (at least partially) nonoverlapping extracellular ones. However, more recent data open the door for functional differences between the intracellular pathways activated by Lar and PTP69D. R7 photoreceptor axon targeting is independent of Lar phosphatase activity, but dependent on PTP69D phosphatase activity, suggesting that the receptors have distinct binding partners. These findings are consistent with the work presented in this study. Both fly and vertebrate Caskins interact with subsets of LAR family receptors, raising the possibility that the intracellular signaling cascade(s) organized by Ckn contributes to the functional differences between Lar and PTP69D (Weng, 2011).
the physical relationship between Lar, Ckn, and Liprin-α was investigated, and no ternary complex was detected. These binding data support mapping studies indicating that Ckn and Liprin-α both interact with the D2 phosphatase domain of Lar via SAM domain-mediated interactions. They further suggest sequential/competitive binding of Ckn and Liprin-α to the Lar receptor and raise the possibility of distinct neuronal functions. It is conceivable that Ckn and Liprin-α both act downstream of Lar to mediate its activity during axon outgrowth/pathfinding and synaptogenesis, respectively. To determine whether Ckn function is specific for Lar signaling during axonogenesis, it will be informative to test whether ckn LOF mutants exhibit defects in the assembly/localization of presynaptic components similar to that observed in Lar mutants. Alternatively, the function of Liprin-α in Lar signaling may be primarily to localize or maintain Lar at the presynaptic terminal, whereas Ckn functions in downstream signal transduction. This hypothesis is supported by evidence for a conserved function for Liprin-α in synaptic protein targeting or anchoring. A role for Liprin-α in trafficking is further bolstered by conserved physical interactions between Liprin-α and Kinesin, suggesting it is an adaptor protein for anterograde transport of synaptic proteins. In this scenario, it is notable that Liprin-α function is not required for pathfinding, arguing either that another protein serves to localize Lar during guidance or that Lar activity in this process does not require its tight localization to the axon terminal. This model is consistent with the broad axonal localization of Lar during embryogenesis (Weng, 2011).
Extracellularly, LAR family members interact with HSPGs and CSPGs. In Drosophila, mutations in the HSPG syndecan (sdc) interact with Lar in motor axon guidance, but homozygous LOF sdc embryos do not display appreciable bypass phenotypes, arguing that other ligands are involved. Once these ligands are identified, it will be critical to determine whether ligand binding influences the association of intracellular adaptors such as Liprin-α and Caskin with Lar. Recently, vertebrate LAR family members have moved into the spotlight in the field of axon regeneration, as PTPsigma has been shown to be a receptor for CSPGs, which are dramatically upregulated at the lesion site and are strongly inhibitory to axon growth. Strikingly, axons in PTPsigma mutant mice have a greatly enhanced ability for long-distance regeneration relative to wild-type mice. These studies suggest that blocking PTPsigma signaling in injured axons might enhance recovery after spinal cord injury. Hence, the truncated forms of fly and vertebrate Caskins that interfere with Lar signaling are particularly interesting. The identification of such dominant-negative reagents allowing the blockade of Lar signal transduction in vivo may have clinical implications in neuronal regeneration (Weng, 2011).
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