InteractiveFly: GeneBrief

APP-like protein interacting protein 1: Biological Overview | References

Gene name - APP-like protein interacting protein 1

Synonyms -

Cytological map position- 61F3-61F3

Function - scaffold protein

Keywords - Axon transport, JNK pathway, vesicular transport

Symbol - Aplip1

FlyBase ID: FBgn0040281

Genetic map position - 3L: 1,196,321..1,199,341 [+]

Classification - JNK-interacting protein (JIP) Phosphotyrosine-binding (PTB) domain

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene

In a genetic screen for Kinesin heavy chain (Khc)-interacting proteins, APLIP1, a neuronally expressed Drosophila homolog of JIP-1, a JNK scaffolding protein (Taru, 2002), was discovered. JIP-1 and its homologs have been proposed to act as physical linkers between kinesin-1, which is a plus-end-directed microtubule motor, and certain anterograde vesicles in the axons of cultured neurons (Verhey, 2001). Mutation of Aplip1 causes larval paralysis, axonal swellings, and reduced levels of both anterograde and retrograde vesicle transport, similar to the effects of kinesin-1 inhibition. In contrast, Aplip1 mutation causes a decrease only in retrograde transport of mitochondria, suggesting inhibition of the minus-end microtubule motor cytoplasmic dynein (Pilling, 2005). Consistent with dynein defects, combining heterozygous mutations in Aplip1 and Dynein heavy chain (Dhc64C) generate synthetic axonal transport phenotypes. Thus, APLIP1 may be an important part of motor-cargo linkage complexes for both kinesin-1 and dynein. However, it is also worth considering that APLIP1 and its associated JNK signaling proteins could serve as an important signaling module for regulating transport by the two opposing motors (Horiuchi, 2005).

To identify proteins that influence kinesin-1-based axonal transport, genetic interaction tests were done to search for mutations that act as dominant enhancers of Kinesin heavy chain. A number of such E(Khc) mutations were found that caused synthetic axonal transport phenotypes (i.e., larval paralytic 'tail flipping' and organelle-filled 'axon swellings') when combined with a Khc null (Khc27/+; E(Khc)/+). Tail flipping was not seen and swellings were rare in Khc27/+ or E(Khc)/+ single heterozygotes. A subset of E(Khc) loci cause tail flipping and swellings when homozygous mutant in a wild-type Khc background, suggesting that the products of those loci have direct roles in axonal transport. That subset includes Kinesin light chain (Klc), Dynein heavy chain 64C (Dhc64C), Glued and an unknown locus on chromosome 3 initially designated E(Khc)ek4 (abbreviated as ek4) (Horiuchi, 2005).

To gain more insight into the functions of ek4 products, a number of phenotypic tests were done. Homozygous ek4 mutant larvae showed classic posterior paralysis and axonal swelling phenotypes with severities similar to those caused by strong hypomorphic Khc genotypes. However, in contrast to such Khc mutants, which die during larval and pupal stages of development, ek4 mutants survive to become active, fertile adults. Severity comparisons with a null [Df(3L)Fpa2] indicate that the ek4 mutation is a strong hypomorphic allele, causing nearly a complete loss of function. These observations suggest that wild-type products of the ek4 locus have important axonal transport functions in larvae and that they have a positive functional relationship with kinesin-1. However, ek4 is not itself essential, suggesting that its products contribute to only a subset of kinesin-1 functions (Horiuchi, 2005).

To test the effects of ek4 mutations on kinesin-1-dependent fast axonal transport, time-lapse confocal microscopy was used. GFP-neuronal synaptobrevin (GFP-nSyb) was used to image transport vesicles, while cytochrome c oxidase-GFP (mito-GFP) (Pilling, 2005) was used to image mitochondria. These constructs were expressed in motoneurons of larvae by virtue of Gal4-UAS promoters that were activated by P[GawB]D42-Gal4 (abbreviated D42), a motor neuron Gal4 driver. With this system, it has been shown that hypomorphic Khc mutations cause anterograde and retrograde flux reductions for GFP-nSyb (60%-70%) and for mito-GFP (75% and 90%), supporting the hypothesis that normal dynein function in some processes depends on kinesin-1 (Pilling, 2005). Both anterograde and retrograde GFP-nSyb flux were reduced ~35% in ek4 mutant axons, supporting the idea that wild-type ek4 products facilitate some kinesin-1 functions. Surprisingly, ek4 mutant axons showed no change in anterograde mito-GFP flux and a 60% reduction in retrograde flux. Currently, the only mutations known to cause a similar unidirectional inhibition of retrograde mitochondrial flux are in Dhc64C (~80%) (Pilling, 2005), which encodes the motor subunit of cytoplasmic dynein (Horiuchi, 2005).

To further test the possibility that ek4 influences dynein, additional genetic interaction tests were done. Consistent with the original genetic screen for dominant enhancers of Khc, ek4 acts as a dominant enhancer of Kinesin light chain (Klc), causing synthetic tail flipping and axonal swelling phenotypes. No such interaction was seen when ek4 was combined with a mutant allele of Klp64D, which encodes an anterograde axonal motor of the kinesin-2 family. However, when ek4 was combined with a mutant allele of Dhc64C, synthetic tail flipping and axonal swelling phenotypes were seen. In summary, these results support the hypothesis that wild-type ek4 gene products facilitate vesicle transport by kinesin-1 and mitochondrial transport by cytoplasmic dynein (Horiuchi, 2005).

To identify the ek4 locus, meiotic recombination and deletion mapping approaches were initially used. The results indicated a position near the tip of the left arm of chromosome 3 within the 61F3-4 cytological region. That interval included APP-like interacting protein 1 (Aplip1), a gene that encodes a neuronally expressed Drosophila homolog of c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1), a scaffolding protein that has been shown to bind Kinesin light chain (KLC), a reelin receptor (ApoER2), and Alzheimer's amyloid precursor protein (APP), as well as JNK pathway kinases (Taru, 2000; Verhey; 2001; Yasuda, 1999). It has been proposed that JIP-1 and its close relative JIP-2 link kinesin-1 with axon vesicles to facilitate anterograde vesicle transport. Similar kinesin-1 linker functions have been proposed for an unrelated JNK scaffolding protein, sunday driver (syd, JSAP, JIP-3), and for APP, although the APP-kinesin relationship may be mediated by APLIP1/JIP-1. A P element transgene that included Aplip1 and flanking sequences fully rescued the tail flipping and partially rescued the axonal swelling phenotypes of larvae that were doubly heterozygous for Khc27 and ek4. Finally, sequencing of the Aplip1 locus from ek4 mutant animals revealed a single base change that converts a conserved proline at position 483 to leucine. This proline is within a conserved 11 amino acid C-terminal region (KBD) that has been shown to be important for binding of mammalian JIP-1 to KLC (Verhey, 2001). The transgenic rescue and sequencing results confirm that ek4 is a mutant allele of the Aplip1 gene, and hence it will be referred to as Aplip1ek4 (Horiuchi, 2005).

To determine whether the P483L mutation affects KLC-APLIP1 binding, epitope-tagged versions of KLC and APLIP1 were used for immunoprecipitation studies. After coexpression of Myc-KLC and wild-type Flag-APLIP1 in S2 cultured cells, anti-Myc antibody precipitated both proteins. Removal of the 11 amino acid KBD from Flag-APLIP1 eliminated detectable binding to Myc-KLC. Furthermore, changing proline 483 to either leucine or alanine substantially reduced KLC binding. This shows that P483 is indeed important for KLC binding, which suggests that at least some of the Aplip1ek4 mutant phenotypes are due to poor association of APLIP1 and kinesin-1 (Horiuchi, 2005).

If APLIP1 links kinesin-1 to anterograde transport vesicles in Drosophila axons, as has been proposed for JIP-1 in vertebrates (Verhey, 2001), APLIP1 should localize in axons and such localization should depend on its ability to bind KLC. To test those predictions, flies were transformed with P elements that carried either full-length UAS-Flag-Aplip1 or UAS-Flag-Aplip1ΔKBD. When driven by D42-Gal4, the two constructs produced equivalent levels of mRNA, which were many times in excess relative to the endogenous gene in larvae. Western blots of larvae with anti-Flag were not successful, but both the full-length and the ΔKBD Flag-tagged proteins were seen at equivalent levels in Westerns of transfected S2 cells, suggesting that both were stable. Interestingly, D42-Gal4-driven expression of one copy of full-length UAS-Flag-Aplip1 in motoneurons causes dramatic tail flipping and nearly 100% lethality during late larval and pupal stages. In larval nerves, it causes axon swellings that stain intensely for vesicles (anti-Syt) and APLIP1 (anti-Flag). D42-Gal4-driven expression of the deletion construct caused no tail flipping or lethality. It did cause some axon swellings in larval nerves, and Flag-APLIP1ΔKBD staining was visible in those swellings. However, the overall amount of staining in nerves was substantially reduced relative to the amount seen after expression of the full-length protein (Horiuchi, 2005).

The presence of residual Flag-APLIP1ΔKBD in larval nerves indicates that some is transported into axons despite the fact that its binding to kinesin-1 is compromised. JIP-1 as well as APLIP1 is known to form multimers (Taru, 2002; Yasuda, 1999). Indeed, immunoprecipitation tests indicate that tagged APLIP1 and APLIP1ΔKBD can form stable multimers with one another. Thus, it is possible that in larval neurons, endogenous wild-type APLIP1 mediates linkage of some transgenic Flag-APLIP1ΔKBD to kinesin-1. Overall, these results suggest that binding between APLIP1 and KLC is an important factor in the presence of APLIP1 in axons, providing in vivo support for the hypothesis that APLIP1 is transported anterograde by kinesin-1 (Horiuchi, 2005).

To test the possibility that APLIP1 is associated with dynein-driven retrograde transport as well as with kinesin-1-driven anterograde transport, transgenic flies were developed carrying a UAS-GFP-Aplip1 transgene that expressed a stable fusion protein. When combined with the D42-Gal4 driver, some transformant lines showed paralysis and GFP-filled swellings, similar to the Flag-APLIP1 lines. Time-lapse imaging did not reveal obvious transport, suggesting that the GFP-APLIP1 was transported in a form too dispersed for imaging of discrete punctate signals. Turning to a classic axonal transport approach, a method was developed for nerve ligation in Drosophila larvae. A homozygous UAS-GFP-Aplip1 D42-Gal4 transformant line was used in which there were few axonal swellings and little visible axonal GFP fluorescence, presumably because of low expression. Intact live larvae were constricted with a fine synthetic fiber midway between head and tail to compress their segmental nerves. After 4 hr, they were partially dissected, the ligation threads were cut, dissection was completed, and the nerves were imaged. Distinct compressed regions were flanked by bright accumulations of GFP-APLIP1 on both the proximal and distal sides. This provides a strong indication that APLIP1 is carried not only by anterograde, but also by retrograde axonal transport (Horiuchi, 2005).

By using an in vivo genetic approach to identify proteins that contribute to the mechanism of kinesin-1-driven anterograde axonal transport, this study has identified APLIP1, a Drosophila homolog of the JNK-interacting protein JIP-1. In vivo axonal transport analysis with intact nervous systems suggests roles for APLIP1 in anterograde and retrograde transport of nSyb-tagged vesicles and in retrograde transport of mitochondria. Similar neuronal phenotypes were seen with either Aplip1 inhibition or overexpression, suggesting that correct stoichiometry of APLIP1 and its interacting proteins is critical for normal organelle transport. The influence of APLIP1 on nSyb vesicle transport in both directions could be explained simply by its importance for kinesin-1 function. Khc is required for normal retrograde dynein activity as well as for anterograde kinesin-1 activity, probably because of a physical or regulatory relationship between the two motors. Alternatively, APLIP1 might make separate contributions to kinesin-1-driven anterograde and dynein-driven retrograde vesicle transport (Horiuchi, 2005).

The selective influence of APLIP1 on retrograde, but not anterograde, transport of mitochondria, as well as Aplip1-Dhc64C genetic interactions, suggests that APLIP1 does have distinct, kinesin-independent functions in dynein-driven transport, at least for mitochondria. Considering how APLIP1 and other JIP-1-related proteins contribute to axonal transport mechanisms, binding studies suggest they may be structural components of kinesin-1-cargo linkage complexes. However, the APLIP1 influence on retrograde mitochondria, the well-known scaffolding role of APLIP1/JIP-1 in the JNK signaling pathway, and indications that JNK may influence motor linkage must also be kept in mind. Mitochondrial transport and distribution in axons responds dramatically to extracellular signaling and may also respond to intracellular signaling stimulated by changes in mitochondrial membrane potential. APLIP1 might be important in these or in other pathways that regulate dynein-cargo linkage and/or mechanochemistry. Future tests for a physical APLIP1-dynein association and for influences of JNK signaling on axonal transport may provide important insights into the microtubule-based transport mechanisms required to sustain neurons and other large asymmetric cells (Horiuchi, 2005).

Control of a Kinesin-Cargo linkage mechanism by JNK pathway kinases

Long-distance organelle transport toward axon terminals, critical for neuron development and function, is driven along microtubules by kinesins. The biophysics of force production by various kinesins is known in detail. However, the mechanisms of in vivo transport processes are poorly understood because little is known about how motor-cargo linkages are controlled. A c-Jun N-terminal kinase (JNK)-interacting protein (JIP1) has been identified previously as a linker between kinesin-1 and certain vesicle membrane proteins, such as Alzheimer's APP protein and a reelin receptor ApoER2 (Taru, 2002, Verhey, 2001). JIPs are also known to be scaffolding proteins for JNK pathway kinases (Kelkar, 2005; Yasuda, 1999). Evidence is presented that a Drosophila ubiquitin-specific hydrolase (Fat facets) and a JNK signaling pathway that it modulates can regulate a JIP1-kinesin linkage. The JNK pathway includes a MAPKKK (Wallenda/DLK), a MAPKK (Hemipterous/MKK7), and the Drosophila JNK homolog Basket. Genetic tests indicate that those kinases are required for normal axonal transport. Biochemical tests show that activation of Wallenda (DLK) and Hemipterous (MKK7) disrupts binding between kinesin-1 and APLIP1, which is the Drosophila JIP1 homolog. This suggests a control mechanism in which an activated JNK pathway influences axonal transport by functioning as a kinesin-cargo dissociation factor (Horiuchi, 2007).

Maintaining proper distributions of protein complexes, RNAs, vesicles, and other organelles in axons is critical for the development, function, and survival of neurons. The primary distribution mechanism relies on long-distance transport driven by microtubule motor proteins. Components newly synthesized in the cell body, but needed in the axon, bind kinesin motors that carry them toward microtubule plus ends and the axon terminal (anterograde transport). Neurotrophic signals and endosomes, examples of axonal components that require transport to the cell body, bind dynein motors that carry them toward minus ends (retrograde transport). The importance of these processes is highlighted by the observation that mutation of motors and other transport machinery components can cause neurodegenerative diseases in humans and analogous phenotypes in model organisms (Horiuchi, 2007).

Two key questions are (1) how do cargoes link to particular motors, and (2) how are such linkages regulated to ensure appropriate pickup and dropoff dynamics? For kinesin-vesicle linkages, scaffolding proteins have emerged as key connectors. For example, the cargo-binding kinesin light chain (Klc) subunit of kinesin-1 binds not only the kinesin-1 heavy chain (Khc) but also JNK-interacting proteins (JIPs) (Verhey, 2001; Kelkar, 2005; Bowman, 2000). Vertebrate JIPs can bind multiple components of the JNK signaling pathway, e.g., JNK itself, upstream activating kinases (MAPKKs), and regulatory kinases (MAPKKKs) (Kelkar, 2005; Yasuda, 1999). JIPs can also bind vesicle-associated membrane proteins, such as ApoER2, which is a reelin receptor, and APP, a key factor in Alzheimer's disease (Verhey, 2001; Inomata, 2003; Matsuda, 2003). Therefore, JIP scaffolding proteins are likely to link JNK pathway kinases and kinesin-1 to vesicles carrying these membrane proteins. This raises an interesting question: Are the JNK pathway kinases simply passive hitchhikers on the kinesin-1/JIP/vesicle complex, or can they actively regulate its transport (Horiuchi, 2007)?

A genetic screen was conducted for factors that control kinesin-JIP linkage during axonal transport. The screen was based on the previous observation that neuron-specific overexpression of Aplip1, which encodes the Drosophila JIP1, causes synaptic protein accumulation in axons, larval paralysis, and larval-pupal lethality (Horiuchi, 2005), the classic axonal-transport-disruption phenotypes caused by Khc and Klc mutations. Why might overexpression of the JIP1 cargo linker for kinesin-1 disrupt axonal transport? The disruptive effect requires APLIP1 (JIP1)-Klc binding. It may be that excess APLIP1 (JIP1) competes with other Klc-binding proteins, for example, different linkers that may attach kinesin-1 to other cargoes. In search of factors that can disrupt or antagonize APLIP1 (JIP1)-Klc binding, a screen was performed for genes that can suppress the axonal-transport phenotypes when co-overexpressed with Aplip1. An 'EP' collection of fly strains capable of the targeted overexpression of endogenous Drosophila genes was screened and P{EP}fafEP381, a line that overexpresses fat facets (faf), was identified as a strong suppressor of the APLIP1 (JIP1)-induced lethality and other neuronal overexpression phenotypes (Horiuchi, 2007).

Faf protein antagonizes ubiquitination and proteasome-mediated degradation of its target proteins. Interestingly, Faf was recently reported to stimulate a Drosophila neuronal JNK signaling pathway that is regulated by the MAPKKK Wallenda (Wnd; Collins, 2006), a homolog of dual leucine zipper-bearing kinase (DLK) that is known to bind JIP1 (Yasuda, 1999; Nihalani, 2000). Overexpression of faf leads to increased levels of Wnd (MAPKKK) protein and thereby causes excessive synaptic sprouting through a pathway that requires the Drosophila JNK homolog Basket. It was found that mutating just one copy of wnd blocked the suppression of Aplip1 overexpression by P{EP}fafEP381. This suggests that faf overexpression suppresses APLIP1 (JIP1)-Klc interaction by elevating the level of Wnd (MAPKKK). Consistent with this, direct overexpression of wnd in neurons with a wild-type transgene (UAS-wnd) was as effective as P{EP}fafEP381 in suppressing UAS-Aplip1-induced axonal accumulation of synaptic proteins. Equivalent expression of a 'kinase-dead' mutant transgene (UAS-wndKD) did not suppress the defects. Thus, Wnd (MAPKKK) and its downstream phosphorylation targets may actively regulate APLIP1 (JIP1)-Klc binding in neurons (Horiuchi, 2007).

If Wnd (MAPKKK) signaling plays a role in normal axonal transport, disrupting its function should cause axonal-transport phenotypes. Consistent with this, wnd loss-of-function mutations (wnd1/wnd2) in an otherwise wild-type background caused accumulation of synaptic proteins in axons. The accumulation phenotype was rescued by motoneuron expression of the wild-type wnd transgene but not by equivalent expression of the kinase-dead mutant transgene. The likely target of Wnd (MAPKKK) kinase activity is the Drosophila homolog of MKK7, Hemipterous (Hep), a MAPKK that activates Bsk (JNK). Mutation of hep also causes axonal accumulations, as does neuronal expression of a dominant-negative mutant bsk transgene. The results of these genetic-inhibition tests combined with those of the Aplip1-overexpression-suppression tests suggest that a Wnd (MAPKKK)-activated JNK pathway influences fast axonal transport by regulating APLIP1 (JIP1)-Klc binding (Horiuchi, 2007).

Is a Wnd (MAPKKK)-Hep (MAPKK)-Bsk (JNK) signaling module bound by APLIP1 (JIP1)? Although all three components of the homologous vertebrate module (DLK-MKK7-JNK) bind JIP1 (Kelkar, 2005; Yasuda, 1999), APLIP1 (JIP1) lacks a conserved JNK-binding domain, and it does not bind directly to Bsk (JNK) (Taru, 2002). However, APLIP1 (JIP1) does bind Hep (MAPKK), Klc, and the Drosophila APP homolog APPL (Taru, 2002). To determine whether Wnd (MAPKKK) associates with Hep (MAPKK) and APLIP1 (JIP1), coexpression and immunoprecipitation tests were performed in Drosophila S2 cultured cells. Wnd (MAPKKK) did not coprecipitate with APLIP1 (JIP1). However, Hep (MAPKK) did coprecipitate with APLIP1 (JIP1), and Wnd (MAPKKK) coprecipitated with Hep (MAPKK). Thus, Wnd (MAPKKK) may bind and influence the APLIP1 (JIP1)-kinesin complex via Hep (MAPKK) (Horiuchi, 2007).

Can Wnd (MAPKKK) and Hep (MAPKK) control the binding of APLIP1 (JIP1) to Klc? When expressed in S2 cells, APLIP1 (JIP1) and Klc exhibit strong binding, as assessed by coimmunoprecipitation. Coexpression of wild-type Wnd (MAPKKK) partially inhibited APLIP1 (JIP1) binding to Klc, but coexpression of a kinase-dead mutant Wnd (MAPKKK) did not. Wild-type Hep (MAPKK) also caused a partial inhibition of APLIP1 (JIP1)-Klc binding, and a constitutively active mutant Hep (MAPKK) caused nearly complete inhibition. Finally, coexpression of wild-type Wnd (MAPKKK) and Hep (MAPKK) together caused an almost complete inhibition of APLIP1 (JIP1)-Klc binding. In addition to inhibiting APLIP1 (JIP1)-Klc binding, Wnd-Hep activation in S2 cells increased the level of Bsk (JNK) activation. Hence, there is a correlation between decreased levels of APLIP1 (JIP1)-Klc binding and elevated levels of Bsk (JNK) activation. This suggests that, despite the lack of a known JNK-binding site on APLIP1 (JIP1), Bsk (JNK) may be the kinase that disrupts the APLIP1 (JIP1)-Klc complex. These results suggest that Wnd (MAPKKK) activation of Hep (MAPKK), and perhaps also Hep (MAPKK) activation of Bsk (JNK), can regulate the linkage between kinesin-1 and a cargo complex via the JIP1-like scaffolding protein, APLIP1 (Horiuchi, 2007).

Hep (MAPKK) may regulate the APLIP1 (JIP1) complex either by activating JNK or by a mechanism independent of JNK. Observations that motoneuron-specific inhibition of Bsk (JNK) caused transport defects similar to those caused by mutations in wnd and hep and that decreased APLIP1 (JIP1)-Klc binding in S2-cell lysates coincided with increased phosphorylated Bsk (JNK) support pathway 1, i.e., Hep (MAPKK) activation of Bsk (JNK), which then directly or indirectly inhibits APLIP1 (JIP1)-Klc binding. A second pathway, Pathway 2, employs an alternative mechanism in which activated Hep (MAPKK) does not need Bsk (JNK) to inhibit APLIP1 (JIP1)-Klc binding. There is little current evidence that Hep or its vertebrate MAPKK homolog MKK7 have phosphorylation targets other than Bsk (JNK). However, that does not exclude the possibility that activated Hep induces in APLIP1 (JIP1) a direct conformational change that causes Klc dissociation. Regardless of how Hep (MAPKK) disrupts binding, when kinesin-1 is not attached to cargo via JIP1, it can fold into a compact form that does not interact with microtubules (Verhey, 2001). Hence, the activated Wnd (MAPKKK) pathway could both inhibit APLIP1 (JIP1)-Klc binding and cause dissociation of kinesin-1 from microtubules. Consistent with this, recent studies report that stimulation of JNK pathways in cultured cells or axoplasm can disrupt the association of kinesin-1 with microtubules (Horiuchi, 2007 and references therein).

From a broader perspective on axonal-transport regulation, it is interesting to consider that there are multiple types of kinesin-1 cargoes, that there are various JIPs that could be specific for different cargoes, and that different MAPKKKs can associate with different JIPs. By sitting at the top of a classic signaling cascade, MAPKKKs such as Wnd are in a good position to differentially control the transport of specific subsets of anterograde kinesin-1 cargoes in response to specific cellular signals. It is known in mammals that other MAPKKKs such as MLK, ASK1, and MEKK1 can bind JIP scaffolding proteins (Whitmarsh, 2006). It will be interesting to determine whether they too influence kinesin-cargo interactions (Horiuchi, 2007).

This work provides the first demonstration that a kinesin and its transport functions can be influenced by a MAPKKK. More specifically, the MAPKKK Wnd and its downstream MAPKK Hep can regulate attachment of a JIP1 cargo linker to kinesin-1. These results also provide the first indication that ubiquitination pathways, by way of MAPKKKs, could be important for proper regulation of axonal transport. Finally, these results suggest that JNK pathway kinases are not just hitchhikers on the axonal kinesin-1/JIP/cargo complex; rather, they can actively regulate its transport dynamics (Horiuchi, 2007).

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). This study demonstrates axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. RBP C-terminal SH3 domains were shown in atomic detail to bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Point mutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes (Siebert, 2015).

Large multi-domain scaffold proteins such as BRP/RBP are ultimately destined to form stable scaffolds, characterized by remarkable tenacity and a low turnover, likely due to stabilization by multiple homo- and heterotypic interactions simultaneously. How these large and 'sticky'; AZ scaffold components engage into axonal transport processes to ensure their 'safe'; arrival at the synaptic terminal remains to be addressed. This study found that the AZ scaffold protein RBP binds the transport adaptor Aplip1 using a 'classic'; PxxP/SH3 interaction. Notably, the same RBP SH3 domain (II and III) interaction surfaces are used for binding the synaptic AZ ligands of RBP, that is, RIM and the voltage gated Ca2+ channel, though with clearly lower affinity than for Aplip1. A point mutation which disrupts the Aplip1-RBP interaction provoked a 'premature'; capture of RBP and the co-transported BRP at the axonal membrane, thus forming ectopic but, concerning T-bar shape and BRP/RBP arrangement, WT-like AZ scaffolds. The Aplip1 orthologue Jip1 has been shown to homo-dimerize via interaction of its SH3 domain. Thus, the multiplicity of interactions, with Aplip1 dimers binding to two SH3 domains of RBP as well as to KLC, might form transport complexes of sufficient avidity to ensure tight adaptor–cargo interaction and prevent premature capture of the scaffold components (Siebert, 2015).

Intravital imaging experiments showed that within axons RBP and BRP are co-transport in shared complexes together with Aplip1, whereas, despite efforts, no any co-transport of other AZ scaffold components, that is, Syd-1 or Liprin-α with BRP/RBP, were detected. In addition, STED analysis of axonal aggregates in srpk79D mutants showed BRP/RBP in stoichiometric amounts, but also failed to detect other AZ scaffold components. Moreover, BRP and RBP co-aggregated in the axoplasm of several other transport mutants tested (acsl, unc-51, appl, unc-76), consistent with both proteins entering synaptic AZ assembly from a common transport complex. Of note, during AZ assembly at the NMJ, BRP incorporation is invariably delayed compared to the 'early assembly'; phase which is driven by the accumulation of Syd-1/Liprin-α scaffolds. As the early assembly phase is, per se, still reversible, the transport of 'stoichiometric RBP/BRP complexes'; delivering building blocks for the 'mature scaffold'; might drive AZ assembly into a mature, irreversible state, and seems mechanistically distinct from early scaffold assembly mechanisms (Siebert, 2015).

Previous work suggested that AZ scaffold components (Piccolo, Bassoon, Munc-13 and ELKS) in rodent neurons are transported to assembling synapses as 'preformed complexes';, so-called Piccolo-Bassoon-Transport Vesicles (PTVs). The PTVs are thought to be co-transported with SV precursors anterogradely mediated via a KHC(KIF5B)/Syntabuli/Syntaxin-1 complex and retrogradely via a direct interaction between Dynein light chain and Bassoon. Since their initial description, however, further investigations of PTVs have been hampered by the apparent relative scarcity of PTVs, and by the lack of genetic or biochemical options for specifically interfering with their transport or final incorporation into AZs (Siebert, 2015).

A direct interaction of Aplip1 and BRP was not detected although their common transport can be uncoupled from the presence of RBP. One possible explanation could be a direct interaction of Aplip1 to other AZ proteins that are co-transported together with BRP and RBP. It is interesting that the very C-terminus of BRP is essential for SV clustering around the BRP-based AZ cytomatrix. Thus, it is tempting to speculate that adaptor/transport complex binding might block premature AZ protein/SV interactions before AZ assembly, but further analysis will have to await more atomic details than were obtained for the RBP::Aplip1 interaction (Siebert, 2015).

The down-regulation of the motor protein KHC also provoked severe axonal co-accumulations of BRP and RBP but per se should leave the adaptor protein-AZ cargo interaction intact. In contrast to aplip1, the axonal aggregations in khc mutants adapted irregular shapes most of the time, likely not representing T-bar-like structures. Thus, the data suggest a mechanistic difference when comparing the consequences between eliminating adaptor cargo interactions with a direct impairment of motor functions. Still, it cannot be excluded that trafficking of AZ complexes naturally antagonizes their ability to assemble into T-bars (Siebert, 2015).

The idea that proteins/molecules are held in an inactive state till they reach their final target has been observed in many other cell types. For example, in the context of local translation control, mRNAs are shielded or hidden in messenger ribonucleoprotein particles during transport so that they are withheld from cellular processing events such as translation and degradation. Shielding is thought to operate through proteins that bind to the mRNA and alter its conformation while at the correct time or place the masking protein is influenced by a signal that alleviates its shielding effect. As another example, hydrolytic enzymes, for example, lysosomes, are transported as proteolytically inactive precursors that become matured by proteolytic processing only within late endosomes or lysosomes. Particularly relevant in the context of AZ proteins involved in exocytosis, the Habc domain of Syntaxin-1 folds back on the central helix of the SNARE motif to generate a closed and inactive conformation which might prevent the interaction of Syntaxin-1 with other AZ proteins during diffusion (Siebert, 2015).

Previous genetic analysis of C. elegans axons forming en passant synapses suggested a tight balance between capture and dissociation of protein transport complexes to ensure proper positioning of presynaptic AZs. In this study, overexpression of the kinesin motor Unc-104/KIF1A reduced the capture rate and could suppress the premature axonal accumulations of AZ/SV proteins in mutants of the small, ARF-family G-protein Arl-8. Interestingly, large axonal accumulations in arl-8 mutants displayed a particularly high capture rate. Similarly, both aplip1 alleles exhibited enlarged axonal BRP/RBP accumulations. Thus, the capture/dissociation balance for AZ components might be shifted towards 'capture'; in these mutants, consistent with the ectopic axonal T-bar formation. It is tempting to speculate that loss of Aplip1-dependent scaffolding and/or kinesin binding provokes the exposure of critical 'sticky'; patches of scaffold components such as RBP and BRP. Such opening of interaction surfaces might increase 'premature'; interactions of cargo proteins actually destined for AZ assembly, thus increase overall size of the cargo complexes by oligomerization between AZ proteins and, finally, promote premature capture and ultimately ectopic AZ-like assembly. On the other hand, the need for the system to unload the AZ cargo at places of physiological assembly (i.e., presynaptic AZ) might pose a limit to the 'wrapping'; of AZ components and ask for a fine-tuned capture/dissociation balance (Siebert, 2015).

Several mechanisms for motor/cargo separation such as (1) conformational changes induced by guanosine-5′-triphosphate hydrolysis, (2) posttranslational modification as de/phosphorylation, or (3) acetylation affecting motor-tubulin affinity, have been suggested for cargo unloading. Notably, Aplip1 also functions as a scaffold for JNK pathway kinases, whose activity causes motor-cargo dissociation. JNK probably converges with a mitogen-activated protein kinase (MAPK) cascade (MAPK kinase kinase Wallenda phosphorylating MAPK kinase Hemipterous) in the phosphorylation of Aplip1, thereby dissociating Aplip1 from KLC. Thus, JNK signaling, co-ordinated by the Aplip1 scaffold, provides an attractive candidate mechanism for local unloading of SVs and, as shown in this study, AZ cargo at synaptic boutons. This study further emphasizes the role of the Aplip1 adaptor, whose direct scaffolding role through binding AZ proteins might well be integrated with upstream controls via JNK and MAP kinases. Intravital imaging in combination with genetics of newly assembling NMJ synapses should be ideally suited to further dissect the obviously delicate interplay between local cues mediating capturing and axonal transport with motor-cargo dissociation (Siebert, 2015).

Interaction of APP family proteins with APLIP1 and its mammalian homologs, APLIP1 is a scaffold protein of the JNK signaling cascade

A novel protein has been isolated based on its association with Drosophila APP-like protein (APPL), a homolog of the beta-amyloid precursor protein (APP) that is implicated in Alzheimer's disease. This novel APPL-interacting protein 1 (APLIP1) contains a Src homology 3 domain and a phosphotyrosine interaction domain and is expressed abundantly in neural tissues. The phosphotyrosine interaction domain of APLIP1 interacts with a sequence containing GYENPTY in the cytoplasmic domain of APPL. APLIP1 is highly homologous to the carboxyl-terminal halves of mammalian c-Jun NH(2)-terminal kinase (JNK)-interacting protein 1b (JIP1b) and 2 (JIP2), which also contain Src homology 3 and phosphotyrosine interaction domains. The similarity of APLIP1 to JIP1b and JIP2 includes interaction with component(s) of the JNK signaling pathway and with the motor protein kinesin and the formation of homo-oligomers. JIP1b interacts strongly with the cytoplasmic domain of APP (APPcyt), as APLIP1 does with APPL, but the interaction of JIP2 with APPcyt is weak. Overexpression of JIP1b slightly enhances the JNK-dependent threonine phosphorylation of APP in cultured cells, but that of JIP2 suppresses it. These observations suggest that the interactions of APP family proteins with APLIP1, JIP1b, and JIP2 are conserved and play important roles in the metabolism and/or the function of APPs including the regulation of APP phosphorylation by JNK. Analysis of APP family proteins and their associated proteins is expected to contribute to understanding the molecular process of neural degeneration in Alzheimer's disease (Taru, 2002; full text of article).

APPs possess a membrane-associated receptor-like structure, and the amino acid sequence of their short cytoplasmic region is highly conserved among a wide variety of species. Protein interactions between the cytoplasmic domains of APPs and cytoplasmic proteins are thought to be important for regulating the metabolism of APPs and/or for putative physiological function of APP. Drosophila APLIP1 and its putative mammalian homologs JIP1b and JIP2 can interact with the cytoplasmic domain of the APPs. These proteins, APLIP1, JIP1b, and JIP2, resemble each other in their structure, especially in their carboxyl-terminal regions that contain SH3 and PI domains. They also share properties such as interactions with APPs, MAP kinase kinase, and kinesin; an abundant expression in the nervous system; and the formation of homo-oligomers. These similarities suggest that APLIP1, JIP1b, and JIP2 belong to same protein family functionally conserved in various species. In mammals, JIP3/JSAP was reported as another member of the JIP family of proteins, which display scaffold function in the JNK signaling pathway as do JIP1 and JIP2. In Drosophila, a putative homolog of JIP3 has been designed Sunday Driver protein (SYD). However, they differ from APLIP1, JIP1b, and JIP2 in their domain structure; they do not possess the SH3 and PI domains that are important regions for binding several proteins including APPs, and they may have some different roles from APLIP1, JIP1, and JIP2 (Taru, 2002).

In mammal several proteins bind the cytoplasmic domain of APP (APPcyt), whereas the physiological role(s) of these interaction have not been sufficiently revealed. JIP1b and JIP2, mammalian counterparts of APLIP1 interact with APPcyt. Regarding JIP1b, the binding to APPcyt is relatively lower than the binding of other APP-binding proteins, such as mDab1, X11, and Fe65. It was also observed that the binding of JIP1b was slightly lower than that of the other binding protein X11L in vitro but not in the cell. However, the faint differences in the binding activities do not necessarily deny the physiological importance of JIP1b for APP. In fact, a novel function of JIP1 and JIP2 was found to be the modulation of the phosphorylation of APP at Thr-668 residue induced by the activation of JNK. Expression of JIP1b slightly enhances the phosphorylation of APP, whereas the expression of JIP2 or JIP1a suppresses the phosphorylation. From the previous reports that JIP1b, JIP1a, or JIP2 equally facilitate the activation of JNK signaling, it was expected that these proteins similarly regulate the phosphorylation of APP when JNK is activated. Nevertheless, only JIP1b facilitates the phosphorylation, and others decrease the level of the phosphorylation of APP. The interaction of JIP2 and JIP1a with APP is remarkably weaker than that of JIP1b in the cell. Therefore, it is conceivable that the effect of JIP1a or JIP2 to decrease the level of the phosphorylation of APP reflects their weaker binding properties to APP rather than their properties of regulating the JNK signaling cascade. It is assumed that formation of the complex between JIP1a or JIP2 and JNK may suppress the approach of JNK to the phosphorylation site of APP, whereas the complex of JIP1b and JNK can easily approach APPcyt. Indeed ut has been reported that formation of the tripartite complex composed of JIP1b, JNK, and APP can be observed in cultured cells. Phosphorylation of APP at Thr-668 has been implicated in the metabolism and/or putative function(s) of APP, and modulation of the phosphorylation level of APP by JIPs in mammal possibly has physiological importance (Taru, 2002).

In invertebrates only Drosophila APLIP1 and dX11L have been reported to interact with APPs except kinesin interacts genetically with APPL. They may all implicated in evolutionarily conserved roles relative to metabolism and/or function of APPs, besides the role of mammalian JIP in the phosphorylation of APP. In Drosophila, APPL does not have a phosphorylation site corresponding to the Thr-668 residue of mammalian APP695. In addition, there are some differences in the function of APLIP1 on the JNK signaling pathway from that of mammalian JIP1 and JIP2 because APLIP1 could not interact with DJNK, whereas it can interact with Drosophila JNK kinase Hep. Thus APLIP1 cannot form a complex with DJNK and facilitate JNK activation in Drosophila in the same manner as JIPs do in mammals, whereas a possibility of regulating JNK signaling through an interaction with Hep still remains. Therefore, the effect modulating the phosphorylation of APP by JIP1 and JIP2 may be acquired in the evolutionary process. Questions of what the evolutionarily conserved role(s) of the interaction of APLIP1, JIP1b, and JIP2 with APPs are remain to be elucidated. Several physiological roles for the mammalian JIP family proteins have been proposed other than acting as scaffold molecules of JNK cascades: as a transactivator of the GLUT2 gene and as cargo for kinesin to mediate the transportation of several transmembrane proteins. In Drosophila APLIP1 interacts with the kinesin light chain as well as mammalian JIP1 and JIP2 do, but interaction with the molecules of the JNK cascades is only partly conserved. The metabolic scheme of APPs is basically conserved between Drosophila and mammals. Kinesin is involved in intracellular transport and metabolism of APP in mammals and is associated with APPL in Drosophila. Accordingly, it is assumed that Drosophila APLIP1 and mammalian JIP share a role in the intracellular metabolism of APPs (Taru, 2002).

In conclusion, Drosophila APLIP1 and mammalian JIP1b and JIP2 are binding proteins of APPs. APLIP1, JIP1b, and JIP2 comprise an evolutionary conserved protein family and share properties in their domain structure, expression pattern, and interaction profiles with proteins such as APPs, kinesin, and JNK kinase, although a few exceptions are observed. It is proposed that a novel function of mammalian JIP1 and JIP2 is to modulate the phosphorylation of APP. Further analysis of conserved or different roles of APLIP1, JIP1b, and JIP2 may contribute to an understanding of the mechanisms of APPs metabolism and the pathogenesis of Alzheimer's disease (Taru, 2002).

Requirement of JIP scaffold proteins for NMDA-mediated signal transduction

JIP scaffold proteins are implicated in the regulation of protein kinase signal transduction pathways. To test the physiological role of these scaffold proteins, the phenotype was examined of compound mutant mice that lack expression of JIP proteins. These mice were found to exhibit severe defects in NMDA receptor function, including decreased NMDA-evoked current amplitude, cytoplasmic Ca++, and gene expression. The decreased NMDA receptor activity in JIP-deficient neurons is associated with reduced tyrosine phosphorylation of NR2 subunits of the NMDA receptor. JIP complexes interact with the SH2 domain of cFyn and may therefore promote tyrosine phosphorylation and activity of the NMDA receptor. It is concluded that JIP scaffold proteins are critically required for normal NMDA receptor function (Kennedy, 2007).

JIP scaffold proteins are important for the normal activity of NMDA receptors. It is established that JIP proteins are localized at post-synaptic densities in neurons, but the mechanism that accounts for the functional interaction of JIP proteins with NMDA receptors is unclear. One possibility is that the PTB domain of JIP1 and JIP2 may contribute to this regulatory process. Indeed, several ligands for this PTB domain have been described, including the low-density lipoprotein receptor-related protein LRP8, the Rac exchange factor Tiam1, and the Ras exchange factor Ras-Grf. Interestingly, all three of these proteins (LRP8, Tiam1, and Ras-Grf) are known to bind NMDA receptors. These proteins may therefore recruit JIP/cFyn complexes to the NMDA receptor to regulate NR2 subunit tyrosine phosphorylation (Kennedy, 2007).

The interaction of LRP8 with JIP1/2 is particularly intriguing because it is established that the site of interaction of the JIP1/2 PTB domain with the cytoplasmic domain of LRP8 is encoded by exon 19 (Stockinger, 2000). This is an alternative spliced exon that is selectively included in Lrp8 mRNA in response to neuronal activity. Thus, only exon 19-positive LRP8 can bind to the JIP1/2 PTB domain. Gene targeting studies have demonstrated that ablation of Lrp8 exon 19 results in mice that exhibit defects in NMDA receptor signaling associated with markedly decreased NR2 subunit tyrosine phosphorylation (Beffert, 2005). Exon 19-positive LRP8 may therefore regulate NMDA receptor activity. Interestingly, the defect in NMDA receptor signaling and NR2 subunit phosphorylation caused by loss of Lrp8 exon 19 (Beffert, 2005) is similar to that caused by compound mutation of JIP1/2. Together, these data suggest that the physical interaction between the JIP1/2 PTB domain and the segment of the LRP8 cytoplasmic tail encoded by Lrp8 exon 19 is functionally significant. Indeed, it is possible that JIP/cFyn complexes may mediate the effects of LRP8 on NMDA receptors. Engagement of cell surface LRP8 by its ligand Reelin may trigger this regulatory pathway to control NMDA receptor function (Kennedy, 2007).

The postnatal role of LRP8 to regulate NMDA receptor activity requires Lrp8 exon 19. However, LRP8 also plays an important developmental role in determining the positioning of neurons in the brain. Mice lacking the ligand Reelin (reeler mice) or mice with targeted ablation of the Lrp8 gene exhibit marked defects in neuronal positioning. However, neither targeted ablation of the alternatively spliced Lrp8 exon 19 nor compound deficiency of JIP1/2 causes a reeler-like neuronal positioning defect during development. Thus, the JIP1/2 PTB domain interaction with the cytoplasmic tail of LRP8 does not play a role in the early developmental function of LRP8 to regulate neuronal positioning (Kennedy, 2007).

A critical role for cFyn in NMDA receptor regulation is established by the finding that cFyn-/- mice exhibit severely reduced tyrosine phosphorylation of NR2A and NR2B and display major defects in long-term potentiation and spatial learning. Nevertheless, other members of the SRC family of tyrosine kinases (including Lck, Lyn, Src, and Yes) have also been implicated in the regulation of NMDA receptor function. cFyn may therefore represent only one member of a larger group of SRC family kinases that are recruited by JIP scaffold proteins. This function of JIP proteins may be coordinated with the actions of other scaffold proteins that can bind SRC family kinases and are present within post-synaptic densities (e.g., PSD-95 or RACK1). Since Jip1-/- and Jip2-/- neurons exhibit different defects in NMDA receptor function, it is possible that these JIP scaffold proteins may cooperate by influencing different steps in the regulatory process. It is also possible that the structurally unrelated scaffold proteins JIP3 and JIP4 may also contribute to NMDA receptor regulation (Kennedy, 2007).

In conclusion, this study has demonstrated that JIP1/2 scaffold proteins represent novel components of the NMDA receptor regulatory network required for normal NMDA-mediated signal transduction (Kennedy, 2007).


Search PubMed for articles about Drosophila Aplip1

Beffert, U., et al. (2005). Modulation of synaptic plasticity and memory by Reelin involves differential splicing of the lipoprotein receptor Apoer2. Neuron 47: 567-579. PubMed ID: 16102539

Bowman, A. B., et al. (2000). Kinesin-dependent axonal transport is mediated by the sunday driver (SYD) protein. Cell 103: 583-594. PubMed ID: 11106729

Collins, C. A., Wairkar, Y. P., Johnson, S. L. and DiAntonio, A. (2006). Highwire restrains synaptic growth by attenuating a MAP kinase signal. Neuron 51: 57-69. PubMed ID: 16815332

Horiuchi, D., Barkus, R. V., Pilling, A. D., Gassman, A. and Saxton, W. M. (2005). APLIP1, a kinesin binding JIP-1/JNK scaffold protein, influences the axonal transport of both vesicles and mitochondria in Drosophila. Curr. Biol. 15(23): 2137-41. PubMed ID: 16332540

Horiuchi, D., Collins, C. A., Bhat, P., Barkus, R. V., Diantonio, A. and Saxton, W. M. (2007). Control of a kinesin-cargo linkage mechanism by JNK pathway kinases. Curr. Biol. 17(15): 1313-7. PubMed ID: 17658258

Inomata, H., et al. (2003). A scaffold protein JIP-1b enhances amyloid precursor protein phosphorylation by JNK and its association with kinesin light chain 1. J. Biol. Chem. 278: 22946-22955. PubMed ID: 12665528

Kelkar, N., Standen, C. L. and Davis, R. J. (2005). Role of the JIP4 scaffold protein in the regulation of mitogen-activated protein kinase signaling pathways. Mol. Cell. Biol. 25: 2733-2743. PubMed ID: 15767678

Kennedy, N. J., et al. (2007). Requirement of JIP scaffold proteins for NMDA-mediated signal transduction. Genes Dev. 21(18): 2336-46. PubMed ID: 17875667

Matsuda, S., Matsuda, Y. and D'Adamio, L. (2003). Amyloid beta protein precursor (AbetaPP), but not AbetaPP-like protein 2, is bridged to the kinesin light chain by the scaffold protein JNK-interacting protein 1. J. Biol. Chem. 278: 38601-38606. PubMed ID: 12893827

Nihalani, D., Merritt, S. and Holzman, L. B. (2000). Identification of structural and functional domains in mixed lineage kinase dual leucine zipper-bearing kinase required for complex formation and stress-activated protein kinase activation. J. Biol. Chem. 275: 7273-7279. PubMed ID: 10702297

Pilling, A. (2005). Analysis of the role of kinesin-1 and cytoplasmic dynein in axonal organelle transport in Drosophila melanogaster. PhD thesis, Indiana University, Bloomington, Indiana.

Siebert, M., et al. (2015). A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones. Elife 4 [Epub ahead of print]. PubMed ID: 26274777

Stockinger, W., Brandes, C., Fasching, D., Hermann, M., Gotthardt, M., Herz, J., Schneider, W.J., and Nimpf, J. (2000). The reelin receptor ApoER2 recruits JNK-interacting proteins-1 and -2. J. Biol. Chem. 275: 25625-25632. PubMed ID: 10827199

Taru, H., Iijima, K., Hase, M., Kirino, Y., Yagi Y. and Suzuki.T. (2002). Interaction of Alzheimer's beta-amyloid precursor family proteins with scaffold proteins of the JNK signaling cascade. J. Biol. Chem. 277: 20070-20078. PubMed ID: 11912189

Verhey, K.J., et al. (2001). Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules. J. Cell Biol. 152: 959-970. PubMed ID: 11238452

Whitmarsh, A. J. (2006). The JIP family of MAPK scaffold proteins. Biochem. Soc. Trans. 34: 828-832. PubMed ID: 17052208

Yasuda, J., Whitmarsh, A.J., Cavanagh, J., Sharma, M. and Davis, R. J. (1999). The JIP group of mitogen-activated protein kinase scaffold proteins. Mol. Cell. Biol. 19(10): 7245-54. PubMed ID: 10490659

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date revised: 21 November 2016

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