InteractiveFly: GeneBrief

Caskin: Biological Overview | References

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 (Fawcett, 2007; Balázs, 2009). Caskin was first identified as a novel protein binding the CaM kinase domain of CASK (Tabuchi, 2002). 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 (Tabuchi, 2002). 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 (Fawcett, 2007; Balázs, 2009). 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, ckn^C 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 ckn^C mutant phenotype since ISNb bypass is not associated with dock LOF. The pathfinding phenotype observed in ckn^C 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).

Search PubMed for articles about Drosophila Caskin

Balázs A, et al. (2009). High levels of structural disorder in scaffold proteins as exemplified by a novel neuronal protein, CASK-interactive protein1. FEBS J. 276: 3744-3756. PubMed ID: 19523119

Fawcett JP, et al. (2007) Nck adaptor proteins control the organization of neuronal circuits important for walking. Proc. Natl. Acad. Sci. 104: 20973-20978. PubMed ID: 18093944

Tabuchi K, Biederer T, Butz S, and Sudhof TC (2002). CASK participates in alternative tripartite complexes in which Mint 1 competes for binding with caskin 1, a novel CASK-binding protein. J. Neurosci. 22: 4264-4273. PubMed ID: 12040031

Weng, Y. L., Liu, N., Diantonio, A. and Broihier, H. T. (2011). The cytoplasmic adaptor protein Caskin mediates Lar signal transduction during Drosophila motor axon guidance. J. Neurosci. 31(12): 4421-33. PubMed ID: 21430143

Wills, Z., et al. (1999). The tyrosine kinase Abl and its substrate enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance. Neuron 22(2): 301-12. PubMed ID: 10069336

date revised: 7 June 2011

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