Targets of Activity

hep is required for expression in the dorsal epithelium edges of another dorsal closure gene, puckered (Glise, 1995).

Little is known about the exact role that hemipterous and basket/DJNK play in the process of dorsal closure. Specifically, it is not clear what the target of JNK phosphorylation is in dorsal closure. JNK could directly phosphorylate and modify cytoskeletal components involved in dorsal closure such as Zipper (Nonmuscle myosin), Coracle (A Drosophila homolog of the vertebrate band 4.1 cytoskeletal protein), Inflated or Myospheroid (Integrins involved in cell adhesion). Alternatively JNK could modify the activity of transcription factors that are known to be involved in dorsal closure (See Jun-related antigen and Anterior open/Yan). Mutations in genes coding for several transcripiton factors have dorsal open phenotypes, like pannier and serpent (two GATA transcript factors), and anterior open (an ETS domain protein). The fact that both hep and bsk mutants affect the expression of puckered (a gene with a dorsal closure phenotype), suggests that JNK and HEP act by regulating transcription factors rather than by directly modifying cytoskeletal components involved in the actual process of cell shape change (Rieso-Escovar, 1996).

The expression of most members of the VH-1 family of PTPs is subject to tight transcriptional regulation. The same is likely to be true for puckered because it displays dynamic patterns of expression in the embryo and the adult. During and after germ band shortening, puc is expressed in the dorsal-most epidermal cells that play a leading role in the process of dorsal closure. In embryos mutant for the JNKK encoded by hemipterous or for the JNK encoded by basket, there is no puc expression in these cells, and dorsal closure fails in a manner similar to that produced by the overexpression of puc (Glise, 1995 and Riesgo-Escovar, 1996). These results suggest a model in which signaling through Hep and Bsk leads to the expression of effectors of dorsal closure and a regulator encoded by puc. The function of the latter is to exert a negative feedback on the signaling cascade of hep and bsk. Interestingly, in mutants for Djun (a likely target of JNK activity), puc expression is absent at the leading edge of the epidermis (N. Perrimon, pers. comm. to Martin-Blanco, 1998), suggesting a transcriptional link between the activity of the JNK encoded by bsk and the expression of puc. Thus, the activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities during dorsal closure. In this system, Puckered seems to act in a feedback loop. Puckered expression is upregulated by DJun and in turn, Puckered inactivates MAPK, whose function is the activation of DJun downstream of Rac signaling (Martin-Blanco, 1998).

Protein Interactions

HEP undergoes autophosphorylation, as reported for vertebrate MEKs (Kosako, 1993). The kinase activity is abolished in a deletion mutant removing the entire HEP kinase domain (Glise, 1995)

Many bacterial toxins act on conserved components of essential host-signaling pathways. One consequence of this conservation is that genetic model organisms such as Drosophila can be used for analyzing the mechanism of toxin action. In this study, the activities of two anthrax virulence factors, lethal factor (LF) and edema factor, were characterized in transgenic Drosophila. LF is a zinc metalloprotease that cleaves and inactivates most human mitogen-activated protein kinase (MAPK) kinases (MAPKKs). LF similarly cleaves the Drosophila MAPK kinases Hemipterous (Hep) and Licorne in vitro. Consistent with these observations, expression of LF in Drosophila inhibited the Hep/c-Jun N-terminal kinase pathway during embryonic dorsal closure and the related process of adult thoracic closure. Epistasis experiments confirmed that LF acts at the level of Hep. It was also found that LF inhibits Ras/MAPK signaling during wing development and that LF acts upstream of MAPK and downstream of Raf, consistent with LF acting at the level of Dsor. In addition, edema factor, a potent adenylate cyclase, inhibits the hh pathway during wing development, consistent with the known role of cAMP-dependent PKA in suppressing the Hedgehog response. These results demonstrate that anthrax toxins function in Drosophila as they do in mammalian cells and open the way to using Drosophila as a multicellular host system for studying the in vivo function of diverse toxins and virulence factors (Guichard, 2006).

Anthrax is caused by Bacillus anthracis, a Gram-positive bacterium that infects primarily herbivores and occasionally humans. B. anthracis secretes three exotoxins [lethal factor (LF), edema factor (EF), and protective antigen (PA)] that are required for its virulence. Anthrax toxins belong to the A/B subfamily of exotoxins, in which the B subunit (PA) binds to a host membrane component and promotes the entry of catalytic A subunits (LF and EF) into host cells. PA binds to the human cell-surface receptors Tumor endothelial marker 8 or Capillary morphogenesis protein 2, two related, widely expressed transmembrane proteins of unknown function. After cleavage by furin proteases, PA becomes activated and forms a heptameric prepore, which binds three molecules of EF, LF, or a combination of both, after which the complex undergoes endocytosis. A pH drop in endocytic vesicles triggers a conformational change in the PA ring, leading to translocation of EF and LF into the cytosol. LF is a zinc metalloprotease that cleaves six of the seven known human mitogen-activated protein kinase (MAPK) kinases (MAPKKs) in their N-terminal proline-rich regulatory domain, which prevents them from binding to their substrates and thereby inhibits phosphorylation and activation of downstream MAPKs. EF, the second catalytic anthrax toxin, is a Ca2+/calmodulin-dependent adenylate cyclase with a specific activity ~1,000-fold higher than that of endogenous mammalian counterparts. Because bacteria lack calmodulin, EF becomes active only after entering host eukaryotic cells, in which it causes an unregulated rise in cAMP levels (Guichard, 2006).

Both LF and EF play a central role in anthrax pathogenesis, as demonstrated by the greatly reduced infectivity of B. anthracis strains lacking either toxin. In addition, the isolated toxins can cause death (LF) or edema (EF) when coinjected with PA. The best characterized cellular response to LF is in macrophages, which undergo programmed cell death and lysis after LF exposure. There is also evidence that LF induces defects in permeability of the vascular endothelium, which, in combination with cytokines produced by dying macrophages, may contribute to the shock-like death of animals exposed to LF. The cellular basis for EF action is less well characterized than that of LF, but it has been reported that EF blocks phagocytosis in monocytes, impairs the function of dendritic cells, and inhibits antigen presentation to T cells. In addition, this toxin causes severe tissue damage and multiple organ failure followed by rapid death in mice (Guichard, 2006).

It is noted that, while there is strong evidence for LF acting at least in part by cleaving and inactivating MAPKK targets, this protease may also have other targets contributing to its lethal effects. Another important question is how LF and EF toxins cooperate to achieve optimal virulence in the host. Recent reports indicate that EF and LF can act in either opposing or synergistic fashions depending on the cellular context. In preliminary experiments, other phenotypes caused by expression of LF and EF were observed in various cell types in addition to the expected phenotypes reported in this study. This study therefore provides a starting point for analyzing potentially novel effects of LF and EF and may lead to the identification of new targets mediating cooperative effects of these two toxins (Guichard, 2006).

B. anthracis is not known to infect hosts other than mammals. Consistent with this observation, no homolog of anthrax toxin receptors tumor endothelial marker 8 and capillary morphogenesis protein 2 is encoded by the Drosophila genome, suggesting that Drosophila is not a suitable model for infection by anthrax. This is also likely to be true for many human pathogens, which have evolved to infect mammals via multiple sequential events, including host recognition, adherence, induction of virulence genes, virulence factor delivery, or evasion of host defenses. In some cases, however, it has been possible to infect Drosophila with human pathogens, such as Vibrio cholerae, Pseudomonas aeruginosa, or Staphylococcus aureus. In contrast to infection with a pathogenic organism, expression of a single virulence factor, which affects only a limited set of conserved host targets, is more likely to produce a specific and interpretable response. Because many pathogens act on specific protein targets that have been highly conserved in Drosophila, it is anticipated that Drosophila will become a widely used in vivo system for the analysis of bacterial toxins or viral virulence factors with unknown activities or unidentified targets. In addition, toxins such as LF that have multiple host target proteins may be used to simultaneously reduce or eliminate the activities of several related proteins that perform overlapping functions. Thus, pharmacogenetic strategies can complement classic loss-of-function genetics in cases where multiple genes carry out related functions (Guichard, 2006).

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. JIPs are also known to be scaffolding proteins for JNK pathway kinases. 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). Vertebrate JIPs can bind multiple components of the JNK signaling pathway, e.g., JNK itself, upstream activating kinases (MAPKKs), and regulatory kinases (MAPKKKs). 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. 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, 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), a homolog of dual leucine zipper-bearing kinase (DLK) that is known to bind JIP1. 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, APLIP1 (JIP1) lacks a conserved JNK-binding domain, and it does not bind directly to Bsk (JNK). However, APLIP1 (JIP1) does bind Hep (MAPKK), Klc, and the Drosophila APP homolog APPL. 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. 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. 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).

hemipterous: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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