basket/JNK

Proteins acting upstream of Basket/JNK

The small GTPases Rac and Cdc42 have been implicated together with JNKK and JNK in the JNK pathway in vertebrates (Coso, 1995 and Minden, 1995). In Drosophila the homologs have been cloned (Dcdc42 and Drac), but no mutations have been isolated (Eaton, 1995). Expression of a dominant negative form of DracA results in dorsal closure phenotypes (Harden, 1995). A dominant negative form of Dcdc42 also shows a dorsal open phenotype. This result suggests that Dcdc42 like DracA acts in dorsal closure regulation (Riesgo-Escovar, 1996).

Recombinant Hemipterous was tested for its ability to phosphorylate and activate DJNK. DJNK acts as a substrate for HEP, providing evidence for a signal transduction pathway between HEP and DJNK. DJUN is also phosphorylated in the presence of HEP and DJNK, but not in the presence of HEP alone. Lipopolysaccharide also serves to stimulate DJUN phosphorylation, suggesting that LPS is able to stimulated the HEP-DJNK-DJUN pathway (Sluss, 1996).

Cdc42 and Rac1 are required for a diverse set of cytoskeleton-membrane interactions in different cell types. These two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. In contrast, Dcdc42 is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types (Eaton, 1995).

DPAK is a Drosophila homolog of the serine/threonine kinase PAK, a protein that is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK binds to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK expression is elevated in the leading edge of epidermal cells, the same cells whose morphological changes drive the dorsal closure of the embryo. The accumulation in these cells of cytoskeletal elements initiating cell shape changes can be inhibited by expression of a dominant-negative DRacA transgene. Leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and/or may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure (Harden, 1996).

Two Drosophila genes, DRacA and DRacB, encode proteins with homology to mammalian Rac1 and Rac2. Transgenic flies were created bearing dominant inhibitory (N17DRacA), and wild-type versions of the DRacA cDNA under control of an Hsp70 promoter. Expression of the N17DRacA transgene during embryonic development causes a high frequency of defects in dorsal closure, due to disruption of cell shape changes in the lateral epidermis. Embryonic expression of N17DRacA also affects germband retraction and head involution. The epidermal cell shape defects caused by expression of N17DRacA are accompanied by disruption of a localized accumulation of actin and myosin, two proteins thought to drive epidermal cell shape change (Harden, 1995).

Rac activation is thought to be important for stimulating dorsal closure because expression of dominant negative forms of Rac (DN Rac) or Cdc42 inhibits dorsal closure in the Drosophila embryo. Since activated Jra/Djun rescues the defect in dorsal closure induced by expression of DN Rac, Rac probably functions upstream of JNK activation to stimulate dorsal closure. To begin to address the mechanism whereby Rac and Misshapen cooperate to activate JNK, cultured cells were transfected with either Msn or NIK, together with DN Rac and an epitope-tagged JNK, and kinase activity assays were performed on JNK precipitates. Although overexpression of either NIK or msn leads to a four- to five-fold increase in JNK activation, coexpression of DN Rac markedly decreases JNK activation. Because PAK family Ste20 kinases are activated by GTP-bound Cdc42 and Rac, it had been assumed that this family of Ste20 kinases rather than an SPS1 Ste20 kinase family member would cooperate with Rac to activate JNK. Thus, discovery of the role of Misshapen in conveying Rac signals to JNK has stimulated consideration of new paradigms for how Rac functions to activate JNK. It is not thought that Rac activates Msn directly. Unlike PAK family members, Msn does not contain a consensus Rac-binding motif and no binding of Msn to activated Rac in vitro can be detected. Rather, it is hypothesized that Rac cooperates with Msn to activate a downstream MKKK. MKKKs have been shown to bind GTP-bound Cdc42 or Rac. Thus, Rac may cooperate with Msn to regulate a downstream MKKK in a manner similar to the way Ras cooperates with a yet to be defined kinase to activate RAF. In this model, binding of an MKKK to activated Rac would facilitate interaction of this MKKK with Msn, thereby enabling its activation by Msn. However, the possibility cannot be excluded that Rac and Msn activate parallel pathways converging on JNK activation (Su, 1998).

The role of DJNK in dorsal closure is to phosphorylate and activate Djun, resulting in transcriptional activation of the dpp gene at the leading edge of the dorsal epidermis. In turn, the binding of Dpp to its receptors Thick veins (Tkv) and Punt (Put) on the ventrally adjacent epithelial cells induces reorganization of the cytoskeleton, leading to epithelial cell elongation and subsequent closure over the amnioserosa. This is supported by the findings that dpp expression is decreased in the dorsal-most epithelial cells in embryos lacking bsk and hep, and that expression of activated forms of Djun or Tkv rescues embryos zygotically mutant for bsk or Jra. Therefore, if msn functions to initiate dorsal closure by activating Basket/JNK, dpp expression in the leading edge epithelial cells should be decreased in msn mutant embryos. In agreement with the idea that msn functions upstream of bsk, dpp expression in leading edge cells surrounding the amnioserosa is observed to be decreased in embryos lacking msn to a degree comparable to bsk¹ embryos. About 20% of embryos derived from the msn¹⁰²/+; msn¹⁰²/+ cross display a decrease in dpp expression in the dorsal leading edge. The decrease in dpp staining in msn mutant embryos is limited to the dorsal rim cells (Su, 1998 and references)

Two families of protein kinases that are closely related to Ste20 in their kinase domain have been identified: the p21-activated protein kinase (Pak) and SPS1 families. In contrast to Pak family members, SPS1 family members do not bind and are not activated by GTP-bound p21Rac and Cdc42. A member of the SPS1 family, called Misshapen (Msn), has been placed genetically upstream of the c-Jun amino-terminal (JNK) mitogen-activated protein (MAP) kinase module in Drosophila. The failure to activate JNK in Drosophila leads to embryonic lethality due to the failure of these embryos to stimulate dorsal closure. Msn probably functions as a MAP kinase kinase kinase kinase in Drosophila, activating the JNK pathway via an as yet undefined MAP kinase kinase kinase. A Drosophila TNF-receptor-associated factor, DTRAF1, has been identified by screening for Msn-interacting proteins using the yeast two-hybrid system. In contrast to the mammalian TRAFs that have been shown to activate JNK, DTRAF1 lacks an amino-terminal 'Ring-finger' domain, and overexpression of a truncated DTRAF1, consisting of only its TRAF domain, activates JNK. Another DTRAF, DTRAF2, has been identified that contains an amino-terminal Ring-finger domain. Msn specifically binds the TRAF domain of DTRAF1 but not that of DTRAF2. The region between the kinase and C-terminal regulatory domains of Msn is sufficient to bind DTRAF1, whereas neither the kinase domain nor the C-terminal domain alone can bind the protein. A stretch of about 250 amono acids that lies in the N-terminal portion of the interdomain is sufficient for Msn to bind DTRAF1. The C-terminal portion of this region does not interact with DTRAF1 but does interact with the SH3 domains of Dreadlocks, supporting the idea that the central region couples Msn and related Ste20 kinases to multiple upstream targets. DTRAF1 and DTRAF2 can dimerize with themselves but cannot form heterodimers. In Drosophila, DTRAF1 is thus a good candidate for an upstream molecule that regulates the JNK pathway by interacting with, and activating, Msn. Consistent with this idea, expression of a dominant-negative Msn mutant protein blocks the activation of JNK by DTRAF1. Furthermore, coexpression of Msn with DTRAF1 leads to the synergistic activation of JNK. A truncated Msn lacking the kinase domain functions as a dominant-negative inhibitor by blocking activation of JNK by DTRAF1. Some of these observations are extended to the mammalian homolog of Msn, Nck-interacting kinase (NIK), suggesting that TRAFs also play a critical role in regulating Ste20 kinases in mammals (Liu, 1999).

The TAK kinases belong to the MAPKKK group and have been implicated in a variety of signaling events. Originally described as a TGFß activated kinase (TAK), the mammalian protein has, however, been demonstrated to signal through p38, Jun N-terminal kinase (JNK) and Nemo types of MAP kinases, and the NFkappaB inducing kinase. Despite these multiple proposed functions, the in vivo role of TAK family kinases remains unclear. The isolation and genetic characterization of the Drosophila TAK homolog (TGF-ß activated kinase 1: Tak1) is reported in this study. Sequence analysis reveals a 678 amino acid long open reading frame (ORF), which shows the highest similarity to vertebrate TAK proteins. Subsequent conceptual translation displays an N-terminal kinase domain of about 280 amino acids, showing 54% identity and 69% similarity to mTAK1, and a long C-terminal domain. The C-terminal domain is less well conserved. However, a 60 amino acid stretch shows a significant level of conservation as compared to the vertebrate and C. elegans orthologs (36% identity and 60% similarity to mTAK1), constituting a conserved protein-protein interaction interface for putative modulators of TAK activity, such as TAB-2 (Mihaly, 2001).

The use of overexpression and double-stranded RNA interference (RNAi) techniques has allowed analysis of Tak1 function during embryogenesis and larval development. Overexpression of Tak1 in the embryonic epidermis is sufficient to induce the transcription of the JNK target genes decapentaplegic and puckered. Furthermore, overexpression of dominant negative (DN) or wild-type forms of Tak1 in wing and eye imaginal discs, respectively, results in defects in thorax closure and ommatidial planar polarity, two well described phenotypes associated with JNK signaling activity. Surprisingly, RNAi and DN-Tak1 expression studies in the embryo argue for a differential requirement of Tak1 during developmental processes controlled by JNK signaling, and a redundant or minor role of Tak1 in dorsal closure. In addition, Tak1-mediated activation of JNK in the Drosophila eye imaginal disc leads to an eye ablation phenotype due to ectopically induced apoptotic cell death. Genetic analyses in the eye indicate that Tak1 can also act through the p38 and Nemo kinases in imaginal discs. These results suggest that dTAK can act as a JNKKK upstream of JNK in multiple contexts and also other MAPKs in the eye. However, the loss-of-function RNAi studies indicate that it is not strictly required and thus either redundant or playing only a minor role in the context of embryonic dorsal closure (Mihaly, 2001).

Dorsal closure, taking place in mid-embryogenesis, describes the morphogenetic movements of the epidermis in order to replace the amnioserosa on the dorsal side of the embryo. This event is driven by the concerted spreading of epidermal cells towards the dorsal midline, where the two contralateral epidermal cell layers meet and remain connected. The JNK signaling module and nuclear targets of JNK, the AP-1 transcription factors dJun and dFos, control the process of dorsal closure. Uncompleted or failed dorsal closure is indicative of disrupted JNK signaling. Mutations in all known components of the JNK signaling pathway result in dorsal open embryos. During dorsal closure, the expression of the dpp and puc genes in cells of the leading edge is controlled by the JNK kinase module and the AP-1 transcription factors Jun and Fos. Leading edge cells show loss of puc and dpp expression when deficient for JNK signaling. Conversely, constitutive activation of JNK signaling in the embryonic epidermis by overexpressing activated Rac or Cdc42 induces the upregulation of dpp and puc. To address the question of whether dTAK can activate and act through the JNK MAPK module, Tak1 was expressed under the control of the en-GAL4 and pnr-GAL4 drivers and the induction of dpp and puc was monitered in the epidermis of stage 12-15 embryos (pnr is strongly expressed in leading edge cells and cells neighboring the leading edge). In wild-type, dpp is expressed in two lateral stripes along the Drosophila embryo, and the dorsal most stripe corresponds to the leading edge of the epidermis. Overexpression of Tak1 with either GAL4 driver causes ectopic upregulation of dpp, as monitored by RNA in situ hybridization. Similarly, the analysis of embryos carrying one copy of a puc lacZ enhancer trap by ß-galactosidase activity staining shows a clear and robust ectopic puc expression when Tak1 is overexpressed. These patterns of dpp and puc activation by Tak1 are identical to those observed with activated Jun, suggesting that the effect is direct and mediated by the JNK signaling pathway (Mihaly, 2001). In summary, these data indicate that overexpression of Tak1 in the embryonic ectoderm is sufficient to induce high-level expression of both known JNK target genes. Since the same upregulation of puc and dpp is observed with activated JNKK/Hep and Jun (a JNK activated transcription factor), they strongly suggest that Tak1 acts through the JNK/ Jun(AP-1) module in the context of dorsal closure (Mihaly, 2001).

During Drosophila embryogenesis, Jun kinase (JNK) signaling has been shown to play a key role in regulating the morphogenetic process of dorsal closure, which also serves as a model for epithelial sheet fusion during wound repair. During dorsal closure the JNK signaling cascade in the dorsal-most (leading edge) cells of the epidermis activates the AP-1 transcription factor comprised of Jun and Fos that, in turn, upregulates the expression of the dpp gene. Dpp is a secreted morphogen that signals lateral epidermal cells to elongate along the dorsoventral axis. The leading edge cells contact the peripheral cells of a monolayer extraembryonic epithelium, the amnioserosa, which lies on the dorsal side of the embryo. Focal complexes are present at the dorsal-most membrane of the leading edge cells, where they contact the amnioserosa. The JNK signaling cascade is initially active in both the amnioserosa and the leading edge of the epidermis. JNK signaling is downregulated in the amnioserosa, but not in the leading edge, prior to dorsal closure. The subcellular localization of Fos and Jun is responsive to JNK signaling in the amnioserosa: JNK activation results in nuclear localization of Fos and Jun; the downregulation of JNK signaling results in the relocalization of Fos and Jun to the cytoplasm. The Hindsight (Hng) Zn-finger protein and the Puckered (Puc) JNK phosphatase are essential for downregulation of the JNK cascade in the amnioserosa. Persistent JNK activity in the amnioserosa leads to defective focal complexes in the adjacent leading edge cells and to the failure of dorsal closure. Thus focal complexes are assembled at the boundary between high and low JNK activity. In the absence of focal complexes, miscommunication between the amnioserosa and the leading edge may lead to a premature 'stop' signal that halts dorsalward migration of the leading edge. Spatial and temporal regulation of the JNK signaling cascade may be a general mechanism that controls tissue remodeling during morphogenesis and wound healing (Reed, 2001).

Expression of the Hnt Zn-finger transcription factor in the amnioserosa, particularly in those cells that abut the leading edge of the epidermis, is essential for this morphogenetic process. Hnt function has been shown in this study to be necessary for dorsal closure. A subset of hnt mutant embryos carrying the embryonic lethal alleles hnt^704a and hnt^XO01 successfully complete germ band retraction but do not hatch. Analyses of cuticle preparations have revealed that 60% of hnt^704a and 79% of hnt^XO01 embryos that complete retraction exhibit an anterior-open or dorsal-hole phenotype characteristic of the failure of dorsal closure (Reed, 2001).

hnt and JNK signaling pathway mutants interact genetically. hnt³⁰⁸ single mutants exhibited 41% embryonic lethality. When the dose of the JNK-encoding gene, basket (bsk), is reduced in a hnt³⁰⁸ mutant background (embryonic lethality was suppressed approximately 2-fold). These results suggest that, in hnt mutants, JNK signaling is upregulated (i.e., that the function of Hnt in dorsal closure is to downregulate JNK signaling). Thus, the reduction of JNK function is able to partially suppress the dorsal closure defect in hnt³⁰⁸ mutants (Reed, 2001).

To further test the role of Hnt in regulating JNK signaling, genetic interactions between hnt³⁰⁸ and dpp mutants were examined. A 50% reduction of dpp gene dose led to an 8-fold reduction in hnt³⁰⁸ embryonic lethality (5% versus 41%). Conversely, increasing the dose of the wild-type dpp gene from two to three copies led to a 2-fold increase in embryonic lethality (80% versus 41%). Examination of the hnt³⁰⁸ embryos carrying three doses of dpp revealed that the frequency of embryos with germ band retraction defects had doubled (41% as compared to 20%). These results provide the first evidence that Hnt may regulate both germ band retraction and dorsal closure through the JNK/DPP signaling pathways. The direction of the genetic interaction between hnt³⁰⁸ and dpp is consistent with the hypothesized role of Hnt to downregulate JNK signaling (Reed, 2001).

During normal development, JNK activity is downregulated in the amnioserosa prior to dorsal closure. Hnt is expressed in the amnioserosa, but not in the epidermis of the embryo. Given the genetic interactions between hnt and JNK pathway mutants, it was therefore asked whether JNK signaling occurs in the amnioserosa during normal embryogenesis. JNK signaling is shown to initially be present in the amnioserosa but it is is downregulated prior to dorsal closure (Reed, 2001).

The transcriptional activation of the genes dpp and puc provides a readout of JNK signaling activity in the leading edge. Enhancer trap lines dpp^lacZ and puc^lacZ were used as reporters for the activation state of the JNK pathway in the amnioserosa. These enhancer trap lines are expressed in the amnioserosa prior to germ band retraction. Toward the end of germ band retraction, JNK activity, as assayed by puc^lacZ and dpp^lacZ, decreases in the interior of the amnioserosa but persists in the amnioserosa perimeter cells that abut the leading edge. By the onset of dorsal closure, when JNK activity becomes elevated in the leading edge, the amnioserosa perimeter cells lose JNK activity, and there is reduced dpp^lacZ or puc^lacZ expression throughout the amnioserosa. It should be noted that perdurance of ß-galactosidase protein in the amnioserosa means that these analyses of the timing of loss of puc^lacZ and dpp^lacZ expression define the latest point in development at which JNK signaling is downregulated, not when such downregulation initiates. It is concluded that JNK signaling occurs in the amnioserosa prior to and during germ band retraction but is downregulated at or before the initiation of dorsal closure (Reed, 2001).

DJUN is activated through phosphorylation by JNK, and although it is capable of forming transcriptional activation complexes through homodimerization, it also forms heterodimers with Fos. Jun/Fos heterodimers belong to the AP-1 class of transcription factor complexes, are more stable than Jun homodimers, and are thought to be the biologically relevant protein complex (Reed, 2001).

To further investigate JNK signaling in the amnioserosa during dorsal closure, the expression of Jun and Fos were examined. In wild-type embryos, Jun and Fos accumulate at high levels in the amnioserosa prior to dorsal closure. During dorsal closure, Jun and Fos levels are highest in the leading edge but persist in the amnioserosa. In the amnioserosa, Jun and Fos are strictly nuclear prior to germ band retraction. Strikingly, both proteins begin to accumulate in the cytoplasm as germ band retraction is completed. While Fos becomes nearly exclusively cytoplasmic, Jun can be detected in both the cytoplasm and the nuclei during dorsal closure (Jun is present in a punctate pattern in the cytoplasm) (Reed, 2001).

To determine whether nuclear restriction of Jun and Fos is dependent on JNK signaling, Jun and Fos expression and subcellular localization were examined in genetic backgrounds that are either reduced or elevated with respect to JNK signaling. In bsk² embryos, which are deficient in JNK activity, the amnioserosal cells show strong cytoplasmic localization of Jun and Fos. The cytoplasmic localization is clearly enhanced in bsk²/+ embryos, relative to wild-type, suggesting that nuclear versus cytoplasmic localization of Jun and Fos is particularly sensitive to reduction in JNK signaling levels. To test the effect of increasing JNK activity in the amnioserosa, puc mutant embryos were immunostained. In this background, JNK activity is upregulated, and both Jun and Fos were restricted to the nuclei of the amnioserosal cells throughout embryogenesis. This is the first report of nucleo-cytoplasmic regulation of Jun and Fos localization in Drosophila in response to JNK signaling. Jun and Fos nuclear localization as well as dpp^lacZ and puc^lacZ expression support the conclusion that JNK signaling occurs in the amnioserosa prior to dorsal closure. Reciprocally, the reduction of dpp^lacZ and puc^lacZ expression and the movement of Jun and Fos from the nucleus into the cytoplasm of amnioserosal cells are indicative of downregulation of JNK signaling in this tissue prior to and during dorsal closure (Reed, 2001).

Given the phenotypic similarities between hnt and JNK signaling mutants, the genetic interactions between hnt and the JNK pathway mutants and observations that JNK signaling is normally downregulated in the amnioserosa prior to dorsal closure, it was asked whether molecular confirmation for Hnt as a negative regulator of JNK signaling could be found (Reed, 2001).

Hnt is not required for Jun and Fos expression, since these proteins are present in the amnioserosa of hnt mutant embryos at levels roughly comparable to wild-type. Strikingly, in contrast to wild-type embryos, hnt mutant embryos (hnt³⁰⁸ and hnt^XO01) show persistent nuclear localization of Jun and Fos. These results are consistent with the postulated role of Hnt as a negative regulator of JNK signaling in the amnioserosa (Reed, 2001).

Persistent nuclear localization of Jun and Fos is seen, not only in the amnioserosa of hnt mutants, but also in the amnioserosa of puc mutants in which dorsal closure also fails. Thus hnt and puc mutants provide independent lines of evidence that downregulation of JNK signaling in the amnioserosa is essential for dorsal closure (Reed, 2001).

Formation or maintenance of focal complexes in the leading edge of the epidermis is disrupted by persistent JNK signaling in the amnioserosa. In wild-type embryos, phosphotyrosine and F-actin accumulate conspicuously along the dorsal-most leading edge cell membranes that abut the amnioserosa, representing focal complexes. Focal complexes fail to accumulate in leading edge cells of puc mutants. Similarly, in hnt mutants, phosphotyrosine and F-actin fail to accumulate at the dorsal-most membrane of the leading edge cells. Thus, Hnt function in the amnioserosa is necessary for the adjacent leading edge cells to assemble or maintain focal complexes at their dorsal-most membranes (Reed, 2001).

The failure of focal complex assembly in the leading edge cells of hnt and puc mutants is not a secondary consequence of the failure of JNK signaling in these cells. This conclusion derives from the fact that dpp^lacZ and puc^lacZ are expressed in the leading edge of wild-type, puc, and hnt mutants. Consistent with this result, the cells of the lateral epidermis undergo dorsal-ventral elongation in puc and hnt mutants, a process dependent on JNK-induced signals from the leading edge (Reed, 2001).

The simplest interpretation of these data is that assembly or maintenance of focal complexes in the leading edge occurs only if there is a boundary between high and low JNK signaling at the junction of the leading edge (high) and the amnioserosa (low). In hnt and puc mutants, since JNK signaling persists in the amnioserosa, such a high/low JNK activity boundary never forms, and therefore focal complexes are either not assembled or are not maintained at the dorsal membrane of the leading edge. In the absence of focal complexes, the leading edge is unable to move over the amnioserosa (Reed, 2001).

The hypothesis that focal complexes form only when there is a high/low JNK signaling boundary at the juxtaposition of the leading edge and the amnioserosa predicts that conversion of a high/high back to a high/low condition will lead to the restoration of focal complexes. Therefore JNK activity was downregulated in the amnioserosa of hnt mutants during the stages at which JNK signaling would abnormally persist. This was accomplished by expressing PUC or dominant-negative JNK using an amnioserosa-specific GAL4 driver. In hnt mutants (the high/high situation), focal complexes are absent from the leading edge, and the morphology of the leading edge cells is highly abnormal. Consistent with the hypothesis, when either Puk or dominant-negative JNK is expressed in the amnioserosa of hnt mutants, focal complexes are restored to the dorsal-most membrane of the leading edge, and the morphology of the leading edge is shifted toward wild-type (Reed, 2001).

In summary, these analyses show that focal complexes fail to accumulate in the leading edge when there is no JNK signaling boundary between the leading edge and the amnioserosa. The restoration of a high/low situation by the expression of either PUC or dominant-negative JNK in a hnt mutant results in the restoration of focal complexes in the leading edge (Reed, 2001).

Wnt signaling regulates ß-catenin-dependent developmental processes through the Dishevelled protein (Dsh). Dsh regulates two distinct pathways, one mediated by ß-catenin and the other by Jun kinase (JNK). A Dsh-associated kinase has been purified from Drosophila that encodes a homologue of Caenorhabditis elegans PAR-1, a known determinant of polarity during asymmetric cell divisions. Treating cells with Wnt increases endogenous PAR-1 activity coincident with Dsh phosphorylation. PAR-1 potentiates Wnt activation of the ß-catenin pathway but blocks the JNK pathway. Suppressing endogenous PAR-1 function inhibits Wnt signaling through ß-catenin in mammalian cells, and Xenopus and Drosophila embryos. PAR-1 seems to be a positive regulator of the ß-catenin pathway and an inhibitor of the JNK pathway. These findings show that PAR-1, a regulator of polarity, is also a modulator of Wnt-ß-catenin signaling, indicating a link between two important developmental pathways (Sun, 2001).

Dsh also activates an alternative pathway that culminates in the stimulation of JNK in mammalian cells and controls of cell polarity in Drosophila and in vertebrates. Expression of Dvl in NIH3T3 cells induces JNK activation. Interestingly, hPAR-1 strongly suppresses the ability of Dvl3 to activate JNK in NIH3T3 cells. This inhibitory effect depends on the kinase activity of hPAR-1, because a mutation in hPAR-1, which is expected to inactivate its kinase activity (hPAR-1 KN), leads to a loss of its inhibitory effect on JNK activation. Together, these data indicate that PAR-1 promotes the participation of Dsh in the Wnt-ß-catenin pathway but suppresses its function in the JNK pathway, thereby acting as a switch for the downstream responses mediated by Dsh protein (Sun, 2001).

The small GTPase Rho is a molecular switch that is best known for its role in regulating the actomyosin cytoskeleton. Its role in the developing Drosophila embryonic epidermis during the process of dorsal closure has been investigated. By expressing the dominant negative DRhoA^N19 construct in stripes of epidermal cells, it has been confirmed that Rho function is required for dorsal closure and it is necessary to maintain the integrity of the ventral epidermis. Defects in actin organization, nonmuscle myosin II localization, the regulation of gene transcription, DE-cadherin-based cell-cell adhesion and cell polarity underlie the effects of DRhoA^N19 expression. Furthermore, these changes in cell physiology have a differential effect on the epidermis that is dependent upon position in the dorsoventral axis. In the ventral epidermis, cells either lose their adhesiveness and fall out of the epidermis or undergo apoptosis. At the leading edge, cells show altered adhesive properties such that they form ectopic contacts with other DRhoA^N19-expressing cells (Bloor, 2002).

RhoA^N19 expression causes ectopic activation of the JNK pathway in the lateral epidermis, suggesting that RhoA normally functions to inhibit JNK signaling. JNK activation is antagonized by the protein phosphatase encoded by puc, and in puc mutants JNK signaling is increased at the leading edge and is activated in the lateral epidermis. Thus, JNK signaling in wild-type embryos is not maximal and basal JNK activity in the lateral epidermis is revealed in the absence of either puc or RhoA mediated repression. Interestingly, RhoA^N19 expression does not increase JNK signaling in the leading edge. This difference between the effect of puc mutations and RhoA^N19 expression could be due to ectopic RhoA^N19 suppressing an upstream JNK activator that is itself maximally activated in the leading edge (Bloor, 2002).

Tensin is an actin-binding protein that is localized in focal adhesions. At focal adhesion sites, tensin participates in the protein complex that establishes transmembrane linkage between the extracellular matrix and cytoskeletal actin filaments. Even though there have been many studies of tensin as an adaptor protein, the role of tensin during development has not yet been clearly elucidated. Thus, this study was designed to dissect the developmental role of tensin by isolating Drosophila tensin mutants and characterizing its role in wing development. The Drosophila tensin loss-of-function mutations results in the formation of blisters in the wings, that are due to a defective wing unfolding process. Interestingly, by¹ -- the mutant allele of the gene blistery (by) -- also shows a blistered wing phenotype, but fails to complement the wing blister phenotype of the Drosophila tensin mutants, and contains Y62N/T163R point mutations in Drosophila tensin coding sequences. These results demonstrate that by encodes Drosophila Tensin protein and that the Drosophila tensin mutants are alleles of by. Using a genetic approach, it has been demonstrated that Tensin interacts with integrin and also with the components of the JNK signaling pathway during wing development; overexpression of by in wing imaginal discs significantly increases JNK activity and induces apoptotic cell death. Besides the defects in wing cell adhesion process, another distinct mutant phenotype was observed in the by mutants; they lay rounded eggs due to defective oocyte elongation during oogenesis. Collectively, these data suggest that Tensin relays signals from the extracellular matrix to the cytoskeleton through interaction with integrin, and through the modulation of the JNK signal transduction pathway during Drosophila wing development (Lee, 2003).

Drosophila wing development after pupariation (AP) consists of two distinct stages: prepupal and pupal wing morphogenesis. Pupal wing morphogenesis is further divided into three stages: separation (11-12 hours AP) of the ventral cell layer from the dorsal layer, re-apposition of the inter-vein cells (21-36 hours AP) and re-separation (60 hours AP) of the two cell layers. Shortly after eclosion, wings expand and unfold by an influx of hemolymph. PS integrins are required for the attachment of the two wing surfaces during pupal wing re-apposition and for the maintenance of the wing bilayer (Lee, 2003).

To determine the detailed roles of tensin during wing morphogenesis, the pupal wings of the by² flies were examined. No differences were observed in the attachment of two wing surfaces and in the integrin localization between wild type and by² wings during both prepupal apposition (4-6 hours AP) and pupal reapposition stages (30-36 hours AP) (Lee, 2003).

Because the pupal wing development was not disturbed in the by² mutants, the expansion and unfolding processes of adult wings in the by² mutants was investigated. After eclosion, the by² flies display folded wings similar to the controls. Then, a sudden and rapid influx of hemolymph induces the unfolding of folded wings in the by² mutants. However, as soon as the wings of by² flies unfold, fluid-filled blisters began to appear at the distal part of the wings, and the boundary of the blisters expand to a certain extent. After the fluid dries, the wing blisters are fixed in place. Taken together, although the dorsal and ventral layers of a wing can be brought into close association during apposition and re-apposition processes in the by² flies, the link between them may not be strong enough to resist the hydrostatic pressures during the wing unfolding process (Lee, 2003).

The functional significance of each domain of tensin in normal wing development was examined. UAS lines overexpressing either full-length tensin protein or various deletion mutant forms of tensin were generated. Unlike DeltaN and DeltaC, overexpression of DeltaPTB by MS1096-GAL4 driver completely rescues the blistered wing phenotype of by². These data suggest that both the N-terminal region and the SH2 domain of tensin are required for proper attachment of two wing surfaces (Lee, 2003).

Since mammalian tensin is known to participate in the integrin signaling, whether tensin genetically interacts with integrin was examined. As expected, the blistered wing phenotype become more severe in the if³; by²/+ mutants and extremely severe in the double homozygotic mutants for if³ and by², compared with if³ homozygotes or by² heterozygotes. In addition, the rate of flies showing blistered wings in the total population greatly increases in the double mutants (Lee, 2003).

In mammalian cells, tensin has been implicated in signal transduction related to cell adhesion such as Src, JNK and PI3K. To examine the role of tensin in the signaling processes related to wing development, the in vivo interaction between tensin and signaling molecules including rl/Erk, Src, JNK and PI3K was investigated. Interestingly, it was found that the JNK signaling pathway is tightly correlated with tensin in the wing development, while other signaling molecules including rl/Erk do not show any interactions with tensin. Homozygous by² mutants with heterozygotic mutations of the JNK signaling components bsk¹ or hep¹ (the loss-of-function mutants for Drosophila JNK and MKK7, respectively) display a highly severe blistered wing phenotype, compared with either homozygous by², heterozygous bsk¹ or heterozygous hep¹ mutants. Notably, the rate of flies, which show Class II blistered wings, increases from 46.5% to 70% for these double mutants compared with homozygous by² mutants, and about 15% of these flies had multiple blisters in their wings. Furthermore, the double homozygotic mutants for by² and hep¹ die at pharate adult stage. The lethality of these double mutants may be due to an impairment of essential in vivo interactions between tensin and the JNK signaling pathway in Drosophila (Lee, 2003).

Next, whether overexpressed by also interacts with the components of the JNK signaling pathway was tested. Overexpression of by using MS1096-GAL4 driver turns the adult wings into a convex shape with a smaller overall size, and this phenotype becomes more severe when two copies of the by gene are overexpressed. Simultaneous overexpression of bsk or hep with by results in a severely curled wing phenotype, which is fully penetrant, whereas overexpression of bsk or hep alone by MS1096-GAL4 driver did not induce any detectable phenotypes in the wing. Collectively, these data suggest that tensin activity is highly related to the JNK signaling pathway during wing development in Drosophila (Lee, 2003).

To further confirm the genetic interaction between tensin and the JNK pathway, the effect of tensin on JNK activity in vivo was tested. The extent of JNK phosphorylation was tested using anti-phosphospecific JNK antibody in the by overexpression line and the by² mutants. As expected, JNK phosphorylation is dramatically increased in the wing imaginal discs overexpressing by; this directly demonstrates increased JNK activity by by. On the contrary, JNK phosphorylation in the imaginal discs of the by² mutants is reduced compared with the control (Lee, 2003).

Moreover, the reduced size and the convex wing phenotype observed in the wings overexpressing by can be most easily explained by apoptosis in the wings. Since the induction of apoptosis by the JNK signaling is well established, it was expected that the wing phenotype induced by by overexpression might be due to apoptosis. To confirm the by-induced apoptosis in vivo, Acridine Orange staining of the relevant wing imaginal discs was carried out. As expected, the overexpression of by dramatically increases apoptotic cell death compared with the control (Lee, 2003).

Thus, tensin genetically interacts with the components of the JNK signaling pathway, and regulates JNK activity during wing development. The supporting evidence for the engagement of tensin in the JNK signaling pathway comes from a recent report that transfected mammalian tensin activates JNK signaling in HEK 293T cells (Katz, 2000). Interestingly, in mammalian cells, JNK is also activated via adaptor proteins p130 CAS and Crk which receive a signal from the FAK/Src tyrosine kinase complex in the cell adhesion sites when cells attach to the ECM. Since tensin is a possible substrate for FAK, and p130 CAS is able to interact with the C terminus of tensin, it is highly possible that tensin is involved in this signaling cascade and mediates signals from integrin and FAK to the JNK signaling pathway (Lee, 2003).

Immune activation of NF-kappaB and JNK requires Drosophila TAK1

Stimulation of the Drosophila immune response activates NF-kappaB and JNK signaling pathways. For example, infection by Gram-negative bacteria induces the Imd signaling pathway, leading to the activation of the NF-kappaB-like transcription factor Relish and the expression of a battery of genes encoding antimicrobial peptides. Bacterial infection also activates the JNK pathway, but the role of this pathway in the immune response has not yet been established. Genetic experiments suggest that the Drosophila homolog of the mammalian MAPK kinase kinase, TAK1 (transforming growth factor ß-activated kinase 1), activates both the JNK and NF-kappaB pathways following immune stimulation. Drosophila TAK1 functions as both the Drosophila IkappaB kinase-activating kinase and the JNK kinase-activating kinase. However, JNK signaling is not required for antimicrobial peptide gene expression but is required for the activation of other immune inducible genes, including Punch, Sulfated, and Malvolio (Mlv). Thus, JNK signaling appears to play an important role in the cellular immune response and the stress response (Silverman, 2003).

JNK signaling is also activated during the immune response in both flies and humans. However, the exact mechanism by which LPS leads to JNK activation in Drosophila is unclear, as is the role of JNK signaling during the immune response. Gene expression profiling has been used to infer that TAK1 is required for the activation of JNK signaling and that JNK signaling is important for wound healing. This study directly demonstrates that JNK activation requires TAK1. Thus, TAK1 appears to function both as a JNKK activating kinase and an IKK activating kinase, as proposed for mammalian TAK1. Furthermore, microarray results suggest that JNK signaling may have important functions in cellular immunity and the stress response (Silverman, 2003).

The gene expression profiling data presented in this study identifies a relatively small number of genes that specifically require the JNK signaling pathway for their LPS-induced expression. The expression of two genes (Punch and sulfated) identified in these experiments has been validated by real time RT-PCR. Punch is an immune inducible gene in cells in culture and in adult flies. However, although it has been shown that in adult flies the immune induction of Punch requires Relish (De Gregorio, 2001), the data presented in this study demonstrate that Punch induction in S2 cells requires JNK pathway components (hep, bsk, and TAK1) but not the Relish-activating kinase IKK. The experiments presented here were performed in an embryonic Drosophila cell line (that has macrophage-like qualities), whereas the data from De Gregorio (2001) was generated from entire adult flies. Thus, it is possible that the signaling pathways required for Punch induction vary depending on the developmental stage and cell type examined. In fact, Punch has at least two promoters that direct developmentally specific expression (Silverman, 2003).

Punch encodes the enzyme for GTP cyclohydrolase I, which is the first enzyme (and rate-determining step) in the formation of the cofactor tetrahydrobiopterin (BH4). This cofactor is required for the conversion of tyrosine to dopamine, which has at least two possible roles in immunity: (1) dopamine is one of two main Drosophila catecholamines, which are important for the stress response in both insects and mammals; (2) dopamine is the precursor of melanin, which is produced during wound healing and encapsulation processes in the fly. In fact, it has been proposed that increased Punch activity could lead to increased melanization (Silverman, 2003 and references therein).

The cofactor BH4 is also an essential cofactor for nitric oxide synthase (NOS). NO itself has at least two possible roles in the immune response: (1) NO is known to be a major microbicidal compound in mammalian phagocytic cells and is likely to function similarly in Drosophila macrophages; (2) NO has also been implicated in immune signaling in Drosophila. NO is required for transmitting a signal from the site of infection to the fat body, the major organ of immune responsive gene expression. Thus, Punch may contribute to the insect immune response in several ways, including protection against stress, melanization of wound sites, and activation of cellular and humoral immunity (Silverman, 2003 and references therein).

The potential role of sulfated in the immune response is less obvious. sulfated encodes an extracellular sulfatase that removes sulfate groups from heparin sulfate proteoglycans (HSPGs). In avian and Drosophila systems, it is thought that sulfated activity is crucial for the regulation of Wnt signaling, possibly by controlling the extracellular milieu in which the Wnt ligand travels (Silverman, 2003).

One of the most intriguing targets of both the JNK and IKK pathways is Mvl, the Drosophila NRAMP-1 homolog. Mvl mutants were first identified in the fly because they display gustatory behavioral defects caused by the inability to properly process sensory neuronal input. Mvl is expressed in both the nervous system and circulating hemocytes. In the mouse, NRAMP-1 is expressed in macrophages, and mutations in the NRAMP-1 gene are responsible for the sensitivity of some inbred mice strains to the Mycobacterium bovis bacille Calmette-Guérin (BCG) and other intracellular bacterial pathogens. NRAMP-1 is thought to control the levels of cations, possibly Fe²⁺ or Mn²⁺, in lysosomal compartments of mouse macrophages. A current model suggests that NRAMP-1 pumps cations out of the phagolysosome, thereby starving microbes of cations required by the enzymes (superoxide dismutase and catalase), which protect the bacteria from reactive oxygen intermediate (ROI)- and reactive nitrogen intermediate (RNI)-induced damage. In the fly, the role of Mvl in immunity is not yet characterized, but its induction during an immune response coupled with the activity of this protein in vertebrate macrophages suggests that it may play an important role in the cellular immune response (Silverman, 2003).

Microarray analysis has provided evidence that LPS-induced JNK activation is important for the stimulation of a gene expression program similar to that seen during dorsal closure. Thus, JNK may be important for wound healing. Expression of only a few of the JNK target genes (for example, Filamin) is reported in this study. The data argue that JNK signaling is required for the activation of cellular immunity and stress protection, whereas a connection to wound healing cannot be excluded by these data (Silverman, 2003).

Certain antimicrobial genes (e.g., diptericin) require a combination of transcription factors for their proper induction. It has been suggested, based on DNA footprinting and DNA sequence analysis, that Diptericin activation requires a kappaB binding site (now believed to be the site of Relish binding) as well as putative NF-IL6-like, and interferon regulatory factor (IRF)-like binding sites. However, of these only Relish is required for the immune inducible expression of diptericin. The data presented here show that the JNK signaling pathway and the AP-1-like factors activated by Drosophila JNK signaling are not involved in antimicrobial peptide gene induction in phagocytes. This would be quite different from immune activation of many mammalian cytokine genes, which require the coordination of several signaling pathways and the activity of several transcription factors for full immune induction. For example, IFN-ß induction requires the activation of three independent signaling cascades and the cooperative binding of three transcription factors, NF-kappaB, c-Jun/activating transcription factor 2 (ATF-2), and the interferon regulatory factor, to the enhancer region. Together, these transcription factors form a higher order complex known as the enhanceosome. Control of the insect antimicrobial genes may not require this complex enhancer architecture (Silverman, 2003).

These studies clearly demonstrate that activation of the innate immune response in Drosophila leads to the activation of JNK and NF-kappaB signaling pathways through a branched signal transduction cascade. The MAP3K TAK1 lies at the branch point of this cascade and likely functions as the JNKK activating kinase and the IKK activating kinase. These signaling pathways are highly conserved. TAK1 also serves similar functions in mammalian innate immune signaling. Furthermore, novel immune-induced targets of the JNK pathway have been identified, that may function in cellular immunity and stress protection (Silverman, 2003).

The TAK1 plays a pivotal role in the innate immune response of Drosophila by controlling the activation of JNK and NF-kappaB. Activation of TAK1 in mammals is mediated by two TAK1-binding proteins, TAB1 and TAB2, but the role of the TAB proteins in the immune response of Drosophila has not yet been established. A TAB2-like protein has been identified in Drosophila called dTAB2. Similar to mammalian TAB2/3, dTAB2 contains a ubiquitin-binding domain (N-CUE) at its N-terminal, and a highly conserved Zinc Finger (ZnF) domain at its C-terminal, respectively. dTAB2 can interact with TAK1, and stimulate the activation of the JNK and NF-kappaB signaling pathway. Furthermore, silencing of dTAB2 expression by dsRNAi inhibits JNK activation by peptidoglycans (PGN), but not by NaCl or sorbitol. In addition, suppression of dTAB2 blocks PGN-induced expression of antibacterial peptide genes, a function normally mediated by the activation of NF-kappaB signaling pathway. No significant effect on p38 activation by dTAB2 was found. These results suggest that dTAB2 is specifically required for PGN-induced activation of JNK and NF-kappaB signaling pathways (Zhuang, 2006).

Studies in mammalian system have shown that activation of TAK1 is regulated by two TAK1 binding proteins, TAB1 and TAB2/3. The dTAB2 appears to associate with dTAK1, thus dTAB2 also acts as a dTAK1-associating protein in Drosophila. In mammalian cells, TAB2 not only binds with TAK1, but also associates with an upstream regulator TRAF6 to form a TAK1-TAB2-TRAF6 complex. TRAF6 contains an N-terminal RING domain, and functions as a ubiquitin ligase, which, in conjunction with a dimeric Ub-conjugating enzyme complex consisting of Ubc13 and Uev1A.Mms2, to catalyze the K63-linked ubiquitination of TRAF6. TAB2 binds to K63-linked polyubiquitinated TRAF6 though a highly conserved C-terminal zinc finger (ZnF) domain, thus facilitating the activation of TAK1. In Drosophila, TAB2 also contains a conserved ZnF domain at its C-terminal, and deletion of this region eliminated its ability to activate the NF-kappaB signaling pathway. In addition, two TRAF homologues, dTRAF1 and dTRAF2, have been identified in Drosophila. Only dTRAF2 contains a RING domain, and dTRAF2 mutant has a reduced level of Diptericin and Drosomycin induction after E. coli infection. However, whether dTRAF2 can be polyubiquitinated and whether dTAB2 binds with polyubiquitinated dTRAF2 to facilitate the activation of dTAK1 still requires further biochemical study (Zhuang, 2006).

In mammals, the physiological importance of TAB2 has been clarified by studies on TAB2-deficient mice and RNAi studies. The phenotype generated from the TAB2 deficiency is very similar to that of NF-kappaB-, IKK-beta-, and NEMO/IKK-deficient mice. However, IL-1 or TNF-induced activation of NF-kappaB and JNK signaling pathways appears to be normal in TAB2-deficient embryonic fibroblasts. Recently, another mammalian TAB2 homologue, TAB3 has been identified, suppression of both TAB2 and TAB3 by RNAi inhibit the activation of IKK and JNK by IL-1 and TNF. In Drosophila, the generation of dTAB2-deficient fly has not been reported so far. However, silencing of dTAB2 by using dsRNAi inhibits PGN-induced activation of JNK and the expression of antibacterial peptide diptericin and attacin gene, two typical peptides produced when the IMD signaling pathway is activated by Gram-negative bacterial pathogens. Since the dTAB2 is associated with dTAK1 and TAB2 is believed to be a key regulator of TAK1, these results are consistent with previous studies that have shown that the Drosophila TAK1 is essential for the activation of both NF-kappaB and JNK MAP kinase signaling after PGN stimulation. Considering the association of dTAB2 with dTAK1 and the functional role of its mammalian homologue TAB2 in the activation of TAK1, the physiological function of dTAB2 in the stimulation of PGN is most likely mediated through TAK1 (Zhuang, 2006).

Compared to the studies of TAB2 and TAK1 in the activation of IKK and JNK, the functional role of TAB2 and TAK1 in the activation of p38 MAP kinase is less clear. TAK1 has been shown to directly phosphorylate and activate the MKK6, a MAP2K of p38 in vitro. However, no significant defect of PGN-induced p38 activation in TAB2-silenced Drosophila cells was observed. In addition, PGN-induced p38 activation is normal in TAK1-silenced cells. These results suggest that TAK1 and its associating protein TAB2 may not be involved in p38 activation in PGN stimulation. The activation of p38 by TAK1 in previous reports may have been caused by TAB1 protein, another component of TAK1 activation complex (Zhuang, 2006).

The Drosophila homolog of the putative phosphatidylserine receptor functions to inhibit apoptosis, perhaps acting through the JNK pathway

Exposure of phosphatidylserine is a conserved feature of apoptotic cells and is thought to act as a signal for engulfment of the cell corpse. A putative receptor for phosphatidylserine (PSR) was previously identified in mammalian systems. This receptor is proposed to function in engulfment of apoptotic cells, although gene ablation of PSR has resulted in a variety of phenotypes. The role of the predicted Drosophila homolog of PSR (dPSR) in apoptotic cell engulfment was examined and no obvious role for dPSR in apoptotic cell engulfment by phagocytes was found in the embryo. In addition, dPSR is localized to the nucleus, inconsistent with a role in apoptotic cell recognition. However, it was surprisind to find that overexpression of dPSR protects from apoptosis, while loss of dPSR enhances apoptosis in the developing eye. The increased apoptosis is mediated by the head involution defective (Wrinkled) gene product. In addition, the data suggest that dPSR acts through the c-Jun-NH2 terminal kinase pathway to alter the sensitivity to cell death (Krieser, 2007).

Evidence against a role for PSR in engulfment also comes from two other knockout models and from data on the localization of the protein. One of the reported mouse knockouts showed no difference in engulfment of apoptotic cells by macrophages in the mutant, although PSR^-/- macrophages were generally inhibited in their release of pro- and anti-inflammatory cytokines. In addition, fibroblast lines established from PSR^-/- embryos showed no defect in apoptotic cell engulfment or in their response to apoptotic cells. Zebrafish lacking PSR accumulated dead cells, but were not definitively shown to have defects in apoptotic cell engulfment. Finally, localization data from the current work and from a number of labs strongly supports a nuclear localization for the protein. This is not consistent with a role for PSR as a surface receptor for the recognition for apoptotic cells, although PS could theoretically modulate the activity of this protein within the cell (Krieser, 2007).

The observations support a role for dPSR in cell survival. In zebrafish, reduction of PSR resulted in an increase in the number of apoptotic cells present during development. In particular, the brains of these fish were shrunken and had an increase in apoptotic cells. In two of the mouse knockout models an increase in apoptotic cells was detected. However, all three knockouts resulted in perinatal lethality, with defects in differentiation in a variety of tissues. It is speculated that defects in engulfment detected in some of the gene ablation models could reflect a role for PSR in the proper differentiation of macrophages. Increased apoptosis seen in the current studies and by others might also be due to defects in proper differentiation in the absence of PSR (Krieser, 2007).

What insight can be gained from these studies into the function of dPSR in differentiation and cell survival? Increased dPSR results in a cell survival phenotype that is suppressed by activation of the JNK pathway, while loss of dPSR results in apoptosis, activated by the cell death regulator Hid, a known target of JNK activation in apoptosis. Taken together, these data suggest that dPSR may normally act to suppress JNK activation of Hid-induced apoptosis (Krieser, 2007).

JNK activation is important for many processes in cells, including cell death, proliferation and differentiation. A role for JNK in apoptosis was found in many mammalian cell types. Data from mouse knockouts of JNK also suggest a role for JNK in proliferation and differentiation. In addition, JNK activation in dying cells is required for proliferative signals originating from apoptotic cells in Drosophila. Interestingly, defects in proliferation and differentiation of many tissues were observed in mice that lack PSR. Taken together with observations of increased cell death in dPSR mutant flies, these observations suggest that some of the phenotypes seen in mouse and fish models of PSR gene ablation might be due to inappropriate activation of the JNK pathway (Krieser, 2007).

Based on genetic assays, it is proposed that one function of dPSR is to suppress Hid activation. Flies that lack dPSR show increased apoptosis in the developing pupal eye, which is suppressed in the absence of hid, while overexpression of dPSR results in ectopic cell survival. Hid function is required for the death of the interommatidial cells in the pupal retina. The results also showed that expression of dPSR can inhibit death induced by the expression of Hid- or Grim in the eye, and that loss of dPSR enhances Rpr-, Hid- or Grim-induced death in the eye. Interestingly, loss of one copy of hid can also suppress cell death induced by Rpr or Hid expression in the eye. Therefore alterations of dPSR levels in the eye may be altering Hid activity to modify the Grim- and Rprinduced eye phenotypes (Krieser, 2007).

JNK activation has been shown to increase hid activity. However, Hid activity is also modulated by activation of the Ras/Erk pathway. Ras activation results in the survival of ectopic interommatidial cells, through the downregulation of Hid activity. Ectopic Ras activation also results in genital rotation defects, similar to those seen with dPSR overexpression. This suggests that PSR overexpression might activate the Ras/Erk pathway. Based on the current data, it is not clear whether dPSR might activate Ras and thus suppress JNK activity, whether dPSR could suppress JNK and thus activate Ras, or whether dPSR might act independently in an opposing manner on the JNK and Ras pathways (Krieser, 2007).

By examining the function of dPSR in the Drosophila system, new insight has been provided into the controversy regarding this protein. Although no evidence was found that this protein plays a role in engulfment, it is important in cell survival. This is consistent with phenotypes seen in gene ablation models in other organisms. Furthermore, dPSR affects the JNK pathway, and this may provide a clue as to its diverse functions in mammals (Krieser, 2007).

The nonmuscle myosin phosphatase PP1β (flapwing) negatively regulates Jun N-terminal kinase in wing imaginal discs of Drosophila

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1ß (PP1ß). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1α genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. This study shows genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw¹, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. puc expression is upregulated in wing imaginal discs mutant for flw¹ and puc^A251, and this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw¹ enhanced by puc. Together, this study shows that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs (Kirchner, 2007; full text of article).

This study shows that the nonmuscle myosin phosphatase flw interacts genetically with components of the JNK signaling pathway. The proteins JNK and PP1ß (Flw), as well as the mechanisms of JNK signal transduction and nonmuscle myosin activation, are highly conserved between Drosophila and humans. This suggests that findings of Drosophila PP1ß regulating JNK through nonmuscle myosin may be relevant for similar processes in human cells and tissues (Kirchner, 2007).

bsk (JNK) mutants suppressed the semilethality of the strong allele flw⁶, while puc and constitutively active hep^CA enhanced the weak viable allele flw¹, resulting in severe wing defects. The level of diphosphorylated (active) JNK was elevated in a flw¹/Y ; puc^A251 mutant background. This, together with the finding that puc expression was upregulated in wing imaginal discs of flw¹/Y ; puc^A251/+, implies that flw can act as a negative regulator of JNK. This was further supported by the fact that puc expression was not upregulated in flw¹/Y ; bsk¹/+ ; puc^A251/+ wing imaginal discs, where reduced amounts of overall JNK (Bsk) protein probably compensate for elevated activity of JNK in flw¹/Y ; puc^A251/+. A possible difficulty with using puc^A251 (or puc^E69, another frequently used puc enhancer trap) as a reporter of puc expression is that both lines are also puc mutants, and puc probably regulates its own expression through a negative feedback loop involving JNK. However, it was confirmed in an independent assay with an anti-P-JNK antibody that JNK was indeed ectopically activated in flw¹/Y ; puc^A251/+. Wing imaginal disc extracts from both flw¹ and puc^A251/+ showed somewhat elevated levels of monophosphorylated, but not diphosphorylated, JNK. In flw¹/Y ; puc^A251/+, it is suggested that dephosphorylation of JNK fails to such an extent that JNK is substantially diphosphorylated and thereby activated. Since it has been shown that activation of JNK in wing imaginal discs induces apoptosis, it is likely that the flw¹/Y ; puc/+ wing phenotype is due to increased death of cells with aberrantly activated JNK (Kirchner, 2007).

A zip mutant suppresses the upregulation of puc in wing imaginal discs as well as the adult wing phenotype of flw¹/Y ; puc^A251/+. This shows that nonmuscle myosin acts upstream of JNK and mediates an activating signal on JNK in a flw¹/Y ; puc/+ mutant background; this is also consistent with the finding that a mutant in the myosin phosphatase-targeting subunit Mbs enhances flw¹/Y ; puc^A251/+ to lethality. Drosophila encodes two myosin phosphatase-targeting subunits, Mbs and MYPT-75D. Mbs binds both Flw and Pp1-87B, while MYPT-75D binds Flw specifically Vereshchagina, 2004). Unfortunately, it was not possible to test for genetic interaction between flw¹/Y ; puc^A251/+ and MYPT-75D because no MYPT-75D mutants have been described. It was also found that a Rho1 mutant abolishes ectopic lacZ staining in flw¹/Y ; puc^A251 wing imaginal discs. Rho1 is an activator of Rho-dependent kinase, which phosphorylates and activates myosin light chain, as well as phosphorylating and inhibiting Mbs. Furthermore, both zip and Rho1, in combination with other genetic interactors, have been reported to show a malformed third-leg phenotype that resembles that of flw¹/Y ; puc/+ (Kirchner, 2007).

Dorsal closure and wound healing depend on both nonmuscle myosin and JNK activity, but a clear genetic or molecular interaction between these pathways has not been previously demonstrated. This is the first study that shows that disruption of nonmuscle myosin can induce activation of JNK, although the mechanism of signal transduction is not clear. The single essential and nonredundant function of flw is the inhibition of nonmuscle myosin activity, presumably by dephosphorylating Sqh at T21 and S22. However, the results suggest that hyperphosphorylation of Sqh at these residues may not explain the elevated expression of puc in flw¹/Y ; puc^A251/+ flies. Additional mechanisms may exist to regulate nonmuscle myosin assembly and activity through phosphorylation, and the complex Flw/Mbs may have other targets in the actomyosin network in addition to Sqh. For example, moesin and focal adhesion kinase are potential targets for mammalian myosin phosphatase. Interestingly, overexpression of the focal adhesion protein tensin (blistery) in Drosophila wing imaginal discs activates JNK and induces apoptosis (Kirchner, 2007).

Two important questions remain unanswered regarding the results on the activation of JNK through nonmuscle myosin. (1) What is the molecular pathway from nonmuscle myosin to JNK? Several mechanisms have been identified that activate JNK and induce apoptosis in wing imaginal discs. For example, mutations in the caspase inhibitor DTraf1 (Drosophila tumor necrosis factor receptor-associated factor 1), as well as inhibition of DTraf1 by overexpression of Hid (head involution defective), Rpr (reaper), or Grim, induces JNK-mediated apoptosis, possibly through Msn (misshapen) or Ask1 (apoptosis signal-regulating kinase 1). Other factors involved in inducing apoptosis and activating JNK are Eiger (Drosophila tumor-necrosis factor superfamily ligand), the serine C-palmitoyltransferase Lace, Blistery (Drosophila Tensin), and Decapentaplegic and Wingless. It is not clear how these factors signal to JNK or whether they act in a single pathway, let alone whether any of them interact with nonmuscle myosin. It is likely that there are several independent ways of activating JNK, and it has been suggested that induction of apoptosis through JNK activation is a regulatory mechanism to eliminate abnormally developing cells in wing imaginal discs. (2)This leads directly to the second question: are the results of nonmuscle myosin signaling to JNK significant in a developmental context? The fact that bsk¹ suppresses the semilethality of flw⁶ indicates that the interactions that uncovered are not confined to the main experimental model of ectopic JNK activation in wing imaginal discs. The obvious system for studying possible interactions between nonmuscle myosin and JNK would be dorsal closure, which depends on both the coordinated assembly and the contraction of the actomyosin cytoskeleton and on activation of JNK. So far, there is no evidence that dorsal closure is affected in flw mutant embryos; however, there is a maternal contribution of flw that may conceal embryonic phenotypes. Actomyosin and JNK do not promote dorsal closure completely independently from each other; for example, the expression of many components of the actomyosin cytoskeleton is upregulated in response to JNK. Also, actin and myosin failed to accumulate along the leading edge of the epidermis in the puc mutant background. Both findings would place the actomyosin cytoskeleton downstream of JNK, whereas genetic data place flw and zip upstream of JNK and puc. It is possible, however, that actomyosin acts both upstream and downstream of JNK during dorsal closure. Because of the conserved nature of the components involved, it is likely that the finding that nonmuscle myosin can signal to and activate JNK is relevant to furthering the understanding of processes like dorsal closure and wound healing in Drosophila and humans (Kirchner, 2007).

Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila

Oncogenic alterations in epithelial tissues often trigger apoptosis, suggesting an evolutionary mechanism by which organisms eliminate aberrant cells from epithelia. In Drosophila imaginal epithelia, clones of cells mutant for tumor suppressors, such as scrib or dlg, lose their polarity and are eliminated by cell death. This study shows that Eiger, the Drosophila tumor necrosis factor (TNF), behaves like a tumor suppressor that eliminates oncogenic cells from epithelia through a local endocytic JNK-activation mechanism. In the absence of Eiger, these polarity-deficient clones are no longer eliminated; instead, they grow aggressively into tumors. In scrib clones endocytosis is elevated, which translocates Eiger to endocytic vesicles and leads to activation of apoptotic JNK signaling. Furthermore, blocking endocytosis prevents both JNK activation and cell elimination. These data indicate that TNF signaling and the endocytic machinery could be components of an evolutionarily conserved fail-safe mechanism by which animals protect against neoplastic development (Igaki, 2009).

Clones of cells mutant for Drosophila tumor suppressor genes, such as scrib or dlg, are eliminated from imaginal discs, suggesting an evolutionarily conserved fail-safe mechanism that eliminates oncogenic cells from epithelia. This study reports that this elimination of mutant cells is accomplished by endocytic activation of Eiger/TNF signaling. Eiger is a conserved member of the TNF superfamily in Drosophila, but its physiological function has been elusive. Although ectopic overexpression of Eiger can trigger apoptosis, flies deficient for eiger develop normally and exhibit no morphological or cell death defect. This study shows that Eiger is required for the elimination of oncogenic mutant cells from imaginal epithelia. This not only provides an explanation for previous unexplained observations, but also argues that Eiger is a putative intrinsic tumor suppressor in a fashion similar to mammalian p53 or ATM, which causes no phenotype when mutated, but protects animals as tumor suppressors when their somatic cells are damaged (Igaki, 2009).

The intrinsic tumor suppression found in scrib mutant clones was also observed in dlg mutant clones, suggesting that this is a mechanism triggered by loss of epithelial basolateral determinants. Intriguingly, it was found that mutant clones of salvador, the hippo pathway tumor suppressor, are not susceptible to similar effect of Eiger. These data suggest that the Eiger-JNK pathway behaves as an intrinsic tumor suppressor that eliminates cells with disrupted cell polarity (Igaki, 2009).

It is intriguing that Eiger's tumor suppressor-like function is dependent on endocytosis. The data show that Eiger is translocated to endosomes through endocytosis and activates JNK signaling in these vesicles. Moreover, blocking endocytosis abolishes both JNK activation and Eiger-dependent cell elimination. Endocytic activation of signal transduction has been observed for EGF and β₂-adrenergic receptor signaling in mammalian cells. After endocytosis, these ligand/receptor complexes localize to endosomes, where they meet adaptor or scaffold proteins that recruit downstream signaling components. Therefore, the endocytic activation of Eiger/TNF-JNK signaling might also be achieved by the recruitment of its downstream signaling complex to the endosomes, possibly through a scaffold protein that resides in endosomes. Recent studies in Drosophila have shown that components of the endocytic pathway -- vps25, erupted, and avalanche -- function as tumor suppressors (Lu, 2005; Moberg, 2005; Thompson, 2005; Vaccari, 2005). Furthermore, mutations in endocytosis proteins have been reported in human cancers. Thus, deregulation of endocytosis may contribute to tumorigenesis. This study provides new mechanistic insights into the role of endocytosis in tumorigenesis (Igaki, 2009).

Mammalian TNF superfamily consists of at least 19 members. While many have been shown to play important roles in immune responses, hematopoiesis, and morphogenesis, the physiological functions for other members have yet to be determined. Mechanisms that eliminate damaged or oncogenic cells from epithelial tissues are essential for multicellular organisms, especially for long-lived mammals like humans. The tumor suppressor role of Eiger might have evolved for host defense or elimination of dying/damaged cells, such as cancerous cells, very early in animal evolution. Given that components of the Eiger tumor suppressor-like machinery (such as Eiger, endocytic pathway components, and JNK pathway components) are conserved from flies to humans, it is also possible that Eiger and its mammalian counterparts are components of an evolutionarily conserved fail-safe by which animals maintain their epithelial integrity to protect against neoplastic development (Igaki, 2009).

MAP4K3 is a component of the TORC1 signalling complex that modulates cell growth and viability in Drosophila melanogaster

MAP4K3 is a conserved Ser/Thr kinase that has being found in connection with several signalling pathways, including the Imd, EGFR, TORC1 and JNK modules, in different organisms and experimental assays. This study analyzed the consequences of changing the levels of MAP4K3 expression in the development of the Drosophila wing, a convenient model system to characterize gene function during epithelial development. Using loss-of-function mutants and over-expression conditions it was found that MAP4K3 activity affects cell growth and viability in the Drosophila wing. These requirements are related to the modulation of the TORC1 and JNK signalling pathways, and are best detected when the larvae grow in a medium with low protein concentration (TORC1) or are exposed to irradiation (JNK). MAP4K3 was also shown to display strong genetic interactions with different components of the InR/Tor signalling pathway, and can interact directly with the GTPases RagA and RagC and with the multi-domain kinase Tor. It is suggested that MAP4K3 has two independent functions during wing development, one related to the activation of the JNK pathway in response to stress and other in the assembling or activation of the TORC1 complex, being critical to modulate cellular responses to changes in nutrient availability (Resnik-Docampo, 2011).

This study has characterised the consequences of changing the amount of MAP4K3, encoded by hppy, in the development of the wing disc, focussing on its relationships with the TORC1 signalling pathway. Previous data suggested that MAP4K3 might be related with a variety of signalling pathways, including EGFR, ImD, JNK and TOR. For these reasons, the advantages of the wing model was used to analyse hppy, as in this system changes in the level of signalling by a variety of pathways lead to pathway-specific phenotypes. A reduction of hppy expression in the wing, using interference RNA or loss-of-function alleles, did not uncover a critical requirement of the gene for embryonic or larval viability. In hppy mutant wings, only a weak reduction was found in wing size and cell size, compatible with a moderate reduction of TORC1 activity. It has been recently reported (Bryk, 2010) that the developmental delay caused by protein starvation is similar in wild type and hppy mutant larvae, suggesting that MAP4K3 is required in vivo to activate TOR and promote growth mostly when amino acid conditions are rich. In contract, this study found a significant requirement for the gene when hppy mutant larvae grow under starvation conditions. Thus, these flies still develop smaller wings than controls, indicating a functional requirement of hppy when the availability of proteins is reduced. This difference could be due to the parameters measured (developmental delay vs. cell and wing size) or to the remnants of hppy function in the alleles used in each experiment (Resnik-Docampo, 2011).

It was also found that, loss of hppy does not affect cell viability or JNK signalling, but that in a hppy loss-of-function genetic background the activation of JNK signalling in response to irradiation is reduced. Thus, the function of hppy might become significant mostly when the organism is challenged by stress signals induced for example by irradiation, indicating a role for the gene in the modulation of JNK signalling in vivo (Resnik-Docampo, 2011).

The increase in happyhour expression does have more dramatic consequences than its loss, causing a severe reduction in the size of the wing independently of environmental conditions. Wing size reduction is associated with both apoptosis and a smaller than normal cell size. The overall morphology and pattern of these wings is normal, with only a weak phenotype of extra-veins in the strongest combinations. Cell death induction and reduced cell size are the diagnostic phenotypes of increased JNK and reduced InR/Tor signalling, respectively. The same processes are affected by loss of MAP4K3 expression in the wing, and therefore, from this analysis it is concluded that MAP4K3 has the potential to activate cell death through the JNK signalling pathway, and also that it can interfere with some component/s of the InR/Tor cascade. The effects of loss- and gain of MAP4K3 on JNK activity are opposite, which is expected from a protein with kinase activity. In contrast both loss and gain of MAP4K3 seem to reduce the function of TORC1. It is likely that in this case MAP4K3 acts as part of a protein complex that can be made non-functional by changes in the stochiometry of its components. What seems clear is that the effects of MAP4K3 on JNK and TORC1 are exerted through independent mechanisms, because the contribution of cell death to the wing phenotype of MAP4K3 over-expression is very modest, and Tor reductions only lead to cell death when cells with different levels of Tor activity are confronted (Resnik-Docampo, 2011).

The phenotype of MAP4K3 over-expression is very sensitive to changes in the levels or activity of several members of the InR/Tor pathway. Thus, strong synergistic interactions were observed when Akt, raptor and Tor are reduced in the background of MAP4K3 over-expression, and the presence of the dominant-negative form Tor^TED in this background entirely eliminates the wing. Conversely, loss of hppy expression rescues the effects of Tor^TED expression. These results suggest that MAP4K3 could act at the level of TORC1. This possibility is compatible with the suppression by MAP4K3 over-expression of phenotypes caused by increased levels of InR/Tor signalling generated by lower than normal levels of PTEN and TSC1/2. In addition to genetic data in the wing, experiments in cell culture with both the fly and human MAP4K3 homologue proteins indicated that MAP4K3 is required to generate maximal activity of TORC1 in response to aminoacids. Therefore, it is suggested that although MAP4K3 is normally required to promote TORC1 signalling, when the protein is over-expressed the balance between TORC1 components required for its normal function in vivo is modified. This effect appears to depend exclusively on the kinase domain of MAP4K3, because the over-expression of this domain causes a strong reduction in wing and cell size. This sstudy has shown that MAP4K3 can interact with RagA, RagC and Tor in pull-down experiments in vitro, and therefore it is speculated that the excess of MAP4K3 alters the phosphorylation levels of TORC1 components and this leads to the assembly of inactive complexes. A similar mechanism might explain the dominant-negative effect of Tor, as it was suggested that Tor over-expression leads to the sequestering of TORC1 components in non-functional complexes. In summary, it is suggested that MAP4K3 normally potentiate TORC1 and JNK functions in response to environmental challenges, without being strictly required to generate some levels of TORC1 or JNK activity, and that MAP4K3 hyper-activity leads to high levels of JNK signalling and to reduced TORC1 function, in this case due to the formation of inactive TORC1 complexes (Resnik-Docampo, 2011).

A dual function of Drosophila capping protein on DE-cadherin maintains epithelial integrity and prevents JNK-mediated apoptosis

E-cadherin plays a pivotal role in epithelial cell polarity, cell signalling and tumour suppression. However, how E-cadherin dysfunction promotes tumour progression is poorly understood. This study shows that the actin-capping protein heterodimer, which regulates actin filament polymerization, has a dual function on DE-cadherin in restricted Drosophila epithelia. Knocking down Capping Protein in the distal wing disc epithelium disrupts DE-cadherin and Armadillo localization at adherens junctions and upregulates DE-cadherin transcription. In turn, DE-cadherin provides an active signal, which prevents Wingless signalling and promotes JNK-mediated apoptosis. However, when cells are kept alive with the Caspase inhibitor P35, the activity of the JNK pathway and of the Yorkie oncogene trigger massive proliferation of cells that fail to stably retain associations with their neighbours. Moreover, loss of capping protein cooperates with the Ras oncogene to induce massive tissue overgrowth. Taken together, these findings argue that in some epithelia, the dual effect of capping protein loss on DE-cadherin triggers the elimination of mutant cells, preventing them from proliferating. However, the appearance of a second mutation that blocks cell death may allow for the development of some epithelial tumours (Jezowska, 2011).

Actin filament (F-actin) turnover and organization is a critical regulator of AJs assembly, maintenance, and remodelling. F-actin growth, stability, disassembly and also their organization into functional higher-order networks are controlled by a plethora of actin-binding proteins (ABPs), strongly conserved between species. Capping protein (CP), composed of an α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer by restricting accessibility of the filament barbed end, inhibiting addition or loss of actin monomers. In Drosophila, removing either cpa or cpb, promotes accumulation of F-actin within the cell and gives rise to identical developmental phenotypes. In the whole larval wing disc epithelium, loss of CP activity reduces Hpo pathway activity and leads to ectopic expression of several Yki target genes that promote cell survival and proliferation. However, inappropriate growth can only be observed in the proximal wing domain. In the distal wing primordium, cpa or cpb mutant cells mislocalize the AJs components DE-Cad and Arm, upregulate puc expression, extrude and die. This indicates that while loss of CP can under certain conditions result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue (Jezowska, 2011).

This study investigated the role of the actin-CP heterodimer in survival of cells in the distal wing disc epithelium. CP has a dual function in regulating DE-Cad: it stabilizes DE-Cad at cell-cell junctions, thereby preventing loss of epithelial integrity and inhibits upregulation of the DE-cad gene. DE-Cad would otherwise provide an active signal, which affects Wg signalling and promotes JNK-mediated apoptosis. However, when cells lacking CP are kept alive, JNK is converted into a potent inducer of proliferation (Jezowska, 2011).

This study demonstrates that in the distal wing disc epithelium, JNK signalling triggers apoptosis of cells with reduced CP expression but induces massive proliferation when apoptosis is blocked with P35. Yki activity is also required to allow overgrowth of 'undead' Cpa-depleted tissues. Induction of apoptosis has been shown to activate Yki through the JNK pathway and triggers compensatory cell proliferation. Thus, in CP-depleted cells kept alive with P35, Yki may act downstream of JNK signalling. Consistent with this, targeting Yki degradation in these tissues fully suppresses ectopic N-Cad expression but not MMP1 upregulation. Because CP also prevents Yki activity in the whole wing disc epithelium, independently of its effect on JNK signalling, (Fernandez, 2011; Sansores-Garcia, 2011), in the distal wing domain, excess Yki activity of 'undead' CP-depleted tissues may result from a dual effect, which involves a JNK-dependent and independent mechanisms (Jezowska, 2011).

JNK signalling has been reported to propagate from cell to cell in the wing disc, where it could trigger apoptosis or Yki-dependent compensatory proliferation. Neither non-autonomous apoptosis nor activation of JNK signalling was observed when patches of CP mutant cells were induced or dsRNA for CP was expressed with or without P35. Therefore, the propagation of JNK activation might be impaired in tissues knocked down for CP. However, increase proliferation was observed of wild-type cells apposed to 'undead' Cpa-depleted tissues. This suggests that JNK propagation is not required to trigger compensatory cell proliferation (Jezowska, 2011).

Several observations argue that in cells lacking CP, a DE-Cad-dependent signal promotes JNK-mediated apoptosis by inhibiting Wg signalling. First, knocking down Cpa affects Wg signalling, which has been shown to prevent JNK-dependent cell death in this region. Second, removing one copy of DE-cad in Cpa-depleted cells partially suppresses apoptosis and ectopic MMP1 expression and restores Wg target genes expression. Third, loss of CP is associated with upregulation of the DE-cad gene and increased levels of the DE-Cad protein. One way by which DE-Cad may block Wg signalling is by tethering Arm. In agreement with this possibility, in the distal wing disc epithelium, overexpression of DE-cad compromises Wg signalling, while co-expression of Arm rescues the DE-cad overexpression phenotype. Moreover, in mouse, overexpression of E-Cad induces apoptosis and sequesters the transcriptionally competent pool of β-cat, effectively shutting off expression of Lef/TCF/β-cat-responsive genes. Interestingly, in Cpa-depleted tissues, the faster mobility form of Arm is enriched. Because this form was proposed to correspond to the cytoplasmic pool of Arm, following CP loss, increase DE-Cad levels might tether and stabilize Arm in the cytoplasm, preventing it to transduce Wg signalling. How a defect in Wg signalling triggers JNK-mediated cell death is not known. In cells lacking CP, JNK activation may occur in response to loss of DIAP1 since overexpressing DIAP1 strongly reduces ectopic MMP1 expression. However, it cannot be excluded that JNK signalling reduces DIAP1 levels since JNK signalling can also function upstream of DIAP1 (Jezowska, 2011).

In the distal wing domain, cells lacking CP mislocalize DE-Cad and Arm at AJs, upregulate expression of DE-cad and extrude from the epithelium (Janody, 2006). DE-cad appears to be a direct transcriptional target of the Hpo signalling pathway. CP inhibits Yki activity (Fernandez, 2011; Sansores-Garcia, 2011) and prevents shg-LacZ upregulation, even in mutant clones that maintain a polarized epithelial architecture in the proximal wing domain. Thus, increased DE-cad expression likely results from inhibition of Hpo pathway activity. However, while mutant clones for Hpo pathway components accumulate DE-Cad, mutant cells do not extrude from the wing disc epithelium. Therefore, the polarity defect of cells lacking CP is unlikely to result from increased DE-Cad levels. Different observations also argue that altered cell-cell adhesion does not result from a defect in Wg signalling or from ectopic activation of JNK signalling, as previously reported. First, reducing DE-cad levels do not restore Arm localization at AJs. Second, in Cpa-depleted tissues in which JNK signalling is blocked, dividing nuclei surrounded by dense F-actin patches are recovered on the basal surface of the distal wing disc epithelium. Third, unlike cells lacking CP, tissues expressing P35 and defective for Wg signalling or overexpressing DE-cad or in which high apoptotic levels were induced maintain a polarized epithelial architecture). Therefore, following loss of CP, the mislocalization of DE-Cad and Arm and the loss of cell-cell contacts are likely upstream or parallel events to DE-cad upregulation and JNK-mediated cell death. Because disruption of apical-basal polarity can trigger JNK activation, a model is favored by which CP prevents JNK-mediated cell death though a dual function on DE-Cad: it promotes DE-Cad-mediated cell adhesion and restricts DE-cad expression (Jezowska, 2011).

While the effect of CP loss on DE-cad transcription is not context dependent, the polarity defect is mainly observed in the distal wing domain. Different regions of the wing disc may have specific requirements in terms of AJs stability and remodelling. Because the distal wing disc is under higher mechanical stress, this epithelium may require higher dynamics of DE-Cad remobilization. CP might be critical to control this kinetic, making distal wing cells lacking CP more prone to lose cell-cell adhesion and extrude from the epithelium (Jezowska, 2011).

Interestingly, the proto-oncogene of the Src family kinases Src42A antagonizes DE-Cad-mediated cell adhesion and stimulates the transcription of DE-cad. Moreover, in the distal wing disc epithelium, the major inhibitor of Src family kinases C-terminal Src kinase (Csk), maintains AJs stability, prevents JNK-mediated apoptosis, whereas halving the genetic dose of DE-cad suppresses the apoptotic phenotype of dCsk-depleted cells. CP and mammalian c-Src both regulate F-actin. Conversely, the control of F-actin impacts on the kinase activity of c-Src. Thus, whether the main role of CP is to regulate Src activity in the distal wing disc is an exciting possibility to be tested in the future (Jezowska, 2011).

This study and others have previously shown that the CP heterodimer acts as tumour suppressor through its control of Hpo pathway activity. This study now shows that in specific epithelia, loss of CP also affects cell-cell adhesion, which is a fundamental step to an epithelial-to-mesenchymal transition (EMT), triggers MMP1 expression, which degrades the basal extracellular matrix, induces cell invasion and promotes massive proliferation of cells that fail to stably retain associations with their neighbours when cell death is blocked with P35. Moreover 'undead' CP-depleted cells show ectopic N-Cad expression, whose de novo expression promotes the transition from a benign to a malignant tumour phenotype. Finally, like other tumour suppressors, loss of CP cooperates with Ras^V12 in tissue overgrowth. These findings argue that in some epithelia in which CP activity is affected, the appearance of a second mutation that prevents apoptotic cell death may trigger the development of aggressive tumours in humans. However, in contrast to tumour progression, which correlates with loss of overall E-Cad expression and stimulation of canonical Wnt signalling, this study observed increase DE-Cad levels and inhibition of Wg signalling in tissues knocked down for CP. Interestingly, in flies, shg-LacZ expression is also enhanced in response to ectopic expression of the two oncogenes Src42A and Yki. This suggests the interesting hypothesis that transcriptional stimulation of DE-cad is an early mechanism of tumour suppression, which would promote the elimination of deleterious cells, possibly through inhibition of Wg signalling, rather than allowing them to proliferate and form tumours. Malignant cells that become resistant to cell death may compete successfully by losing the overall E-Cad expression and upregulating mesenchymal cadherins such as N-Cad to reinforce their fitness (Jezowska, 2011).

Domain specificity of MAP3K family members, MLK and Tak1, for JNK signaling in Drosophila

A highly diverse set of protein kinases function as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess fourteen MAPK kinase kinases (MAP3Ks) that activate Jun Kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, MAP3K signaling was assessed specificity in several JNK-dependent processes during development and stress response. The approach taken was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slipper and the TGF-beta activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure; then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. It was found that overexpression of the wildtype kinases stimulated JNK signaling in alternate contexts, so cells were capable to respond to both MAP3Ks, but with distinct outcomes. Relative to wildtype, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts including TNF-dependent cell death and innate immune signaling, however depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, these findings suggest that the selective deployment of a particular MAP3K can be attributed in part to their inherent sequence differences, cellular localization, and binding partner availability (Stronach, 2014).

Proteins acting downstream of JNK

The activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities. The latter is mediated by a subset of phosphatases with dual specificity (VH-1 family). This paper describes a new member of this family, encoded by the puckered gene of Drosophila. Mutations in this gene lead to cytoskeletal defects that result in a failure in dorsal closure related to defects associated with mutations in basket, the Drosophila JNK homolog. puckered mutations result in the hyperactivation of DJNK, and overexpression of puc mimics basket mutant phenotypes. puckered expression is itself a consequence of the activity of the JNK pathway. During dorsal closure, JNK signaling has a dual role: to activate an effector, encoded by decapentaplegic, and as an element of negative feedback regulation encoded by puckered. Evidence has been found that Puckered is a JNK specific phosphatase. When extracts from wild-type and puc mutant embryos are assayed for their endogenous JNK activity, puc mutants show a significant increase in JNK activity, when compared with wild type. When similar extracts are tested for their ability to inactivate preactivated JNK, up to 50% inhibition is obtained after 30 min from wild-type embryos, as compared with abolition of activity in extracts from puc mutants (Martin-Blanco, 1998).

A model is proposed for the role of Puckered in dorsal closure 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).

What role is played by hemipterous and basket/DJNK in the process of dorsal closure? Specifically, what are the JNK phosphorylation targets in the process of 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 known to be involved in dorsal closure. Mutations in genes coding for several transcription factors produce dorsal open phenotypes, like pannier and serpent (two GATA transcript factors), and anterior open (an ETS domain protein also known as yan). 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 (Riego-Escovar, 1996).

Dorsal closure depends on the activities of a Jun amino (N)-terminal kinase kinase (JNKK) encoded by the hemipterous gene, and of a JNK encoded by basket. Hep is required for cell determination in the leading edge of migrating epithelia, by controlling specific expression of the puckered gene in these cells. puc encodes a protein related to vertebrate dual-specificity MAPK phosphatases of the CL100 family (E. Martin-Blanco, A. Gampel, and A. Martinez Arias, pers. comm. to Glise, 1997). During dorsal closure, decapentaplegic is expressed in the row of cells making up the leading edge of the epithelia. The small GTPases Dcdc42, Drac1, and the Hep JNKK control dpp expression in this migratory process. Activated Drac1 and Dcdc42 induce distinct, although partly overlapping, responses. Dcdc42 appears to be a good inducer of ectopic puc and dpp expression in ectodermal cells located more ventrally, whereas Drac1 seems more active in cells nearest to the leading edge. Appropriate dpp and puc expression in the leading edge also depends on the inhibitory function of the puc gene. puc acts as a repressor of dpp expression in the ectoderm, likely acting to inhibit Basket, the Jun N-terminal kinase. In puc mutants ectopic expression of puc and dpp is induced in the ectoderm, that is, outside the normal domain of puc expression in the leading edge. In addition, puc (but not dpp) is expressed ectopically in amnioserosa cells. These observations indicate a cell non-autonomous effect of puc mutations. The data suggest that the leading edge is the source of a JNK autocrine signal, and exclude a role of Dpp as such a ligand. Dorsal closure couples JNK and Dpp signaling pathways, a situation that may be conserved in vertebrate development (Glise, 1997).

The p150Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization

A direct link between JNK signaling and actin organization has not been found previously. The Jun N-terminal kinase (JNK, known as Basket in Drosophila) is a downstream effector of Rac and Cdc42 GTPases involved in actin reorganization. Basket plays a role in the regulation of cell shape changes and actin reorganization during the process of dorsal closure. One potential mechanism for induction of cytoskeletal changes by Basket is via transcriptional activation of the decapentaplegic gene, a member of the TGFbeta superfamily. Spire, termed p150-Spire in this study, has a docking site for Basket/JNK, at its carboxy-terminal end. In mouse fibroblasts, p150-Spir colocalizes with F-actin; its overexpression induces clustering of filamentous actin around the nucleus. When coexpressed with p150-Spire in NIH 3T3 cells, JNK translocates to and colocalizes with p150-Spire at discrete spots around the nucleus. Carboxy-termina l sequences of p150-Spir are phosphorylated by JNK both in vitro and in vivo. It is concluded that p150-Spir is a downstream target of JNK function and provides a direct link between JNK and actin organization (Otto, 2000).

p150Spire was identified in a yeast two-hybrid screen to search for novel Basket interaction partners. The cDNA encoding p150Spire contains an open reading frame of 1,020 amino acids. Transient transfection experiments in mouse fibroblasts reveal that the cDNA directs expression of a protein that migrates with an apparent molecular weight of approximately 150 kDa on an SDS-polyacrylamide gel. The spire gene exhibits significant homology to a maternal gene pem-5 isolated from the ascidian Ciona savignyi. Comparison of Drosophila p150Spire sequences to human EST clones reveals a high conservation between the p150Spire sequences of Drosophila and human (Otto, 2000).

The p150Spire protein contains an acidic domain, a cluster of four WH2 domains, a modified FYVE zinc finger domain and a carboxy-terminal DEJL motif (docking site for Erk and JNK containing an LXL motif). WH2 domains bind monomeric actin: WH2 family proteins such as WASP and WAVE are involved in actin reorganization. Sequences encompassing the Spir WH2 domains interact directly with monomeric actin. As has been shown for WASP and WAVE, transient expression of p150Spire in adherent mammalian cells induces clusters of filamentous actin around the nucleus that colocalize with p150Spire in all transfected cells. A modified FYVE zinc-finger motif is located in the central region of the protein. The modified structure lacks a pocket of basic amino acids between cysteines 3 and 4 of the FYVE finger structures that is necessary for the binding of phosphatidylinositol 3-phosphate (Otto, 2000).

In this study, p150Spire was identified in a yeast two-hybrid screen as a DJNK-interacting protein. The smallest fragment that still binds DJNK consists of the carboxy-terminal 53 amino acids. A DEJL motif, characterized by a cluster of basic amino acids amino-terminal to an L/I-X-L/I motif (in the single-letter amino acid code), is located in this carboxy-terminal sequence (amino acids 997-1014 of p150Spire, KQKRSSARNRTIQNLTLD). DEJL domains have been shown to mediate the docking of the Erk and JNK mitogen-activated protein (MAP) kinases to either activating kinases or substrate proteins. To further characterize the interaction between p150Spire and JNK, the localization of the two proteins was determined when transiently expressed in NIH 3T3 cells. p150Spire accumulates in punctate spots located around the nucleus. Expression of a fusion protein between glutathione-S-transferase and JNK (GST-JNK) reveals both a cytoplasmic and nuclear distribution of the kinase. In the presence of p150Spire, GST-JNK is translocated from these areas and accumulates at places where p150Spire is located. The colocalization of the proteins is detected in all cells expressing both proteins but is not found in cells coexpressing GST alone with p150Spire (Otto, 2000).

Erk and JNK MAP kinases are recruited to substrate proteins via docking sites, enabling the kinases to phosphorylate serine or threonine residues adjacent to prolines (S/TP motifs). As p150Spire contains a JNK docking site (the DEJL motif) and several potential S/TP phosphorylation motifs, attempts were made to determine whether p150Spire is a phosphorylation target of JNK. A highly specific, constitutively active Erk can be generated by the fusion of Erk2 to its upstream activator Mek1 (Erk2-Mek1-LA). Similarly, JNK2 (rat) has been fused to its upstream activator MKK7 (mouse) via a linker region. The fusion protein, JNK-MKK7, is a constitutively active Jun N-terminal kinase. JNK-MKK7 phosphorylates amino-terminal c-Jun sequences in vitro and induces an electrophoretic mobility shift of the c-Jun protein when coexpressed in NIH 3T3 cells, indicating an in vivo c-Jun phosphorylation. MKK7 activates JNK by phosphorylating a TPY motif in the central region of JNK. JNK-MKK7 exhibits autophosphorylation and interacts with a phospho-specific antibody that recognizes activated JNK protein. A similar construct (JNKK2-JNK1) has been shown to be a specific, constitutively active c-Jun kinase (Otto, 2000).

An investigation was carried out to see whether p150Spire sequences are phosphorylated by JNK in vitro. Indeed, arsenite-activated JNK1, immunoprecipitated from NIH 3T3 cell lysates, phosphorylates a carboxy-terminal fragment of the p150Spire protein in an in vitro immune-complex kinase assay. Erk and p38 MAP kinases precipitated from the same lysates exhibit a basal phosphorylation activity that could not be increased by arsenite stimulation of the cells. Coexpression of p150Spire with MLK3-activated JNK2 [mixed lineage kinase (MLK3) is an upstream activator of JNKs] or JNK-MKK7 in mouse fibroblasts, induces several slower migrating forms of the p150Spire protein during SDS-PAGE (Otto, 2000).

The electrophoretic mobility shift of p150Spire strongly suggests that it is phosphorylated by JNK on multiple sites. The slower migrating forms are abolished by phosphatase treatment, demonstrating that the mobility shifts are due to phosphorylation of p150Spire protein. Supporting these data, a kinase-inactive mutant of JNK-MKK7 (containing the JNK mutations K55A and K56A and the MKK7 mutation K76E, called JNK-MKK7-KD) does not induce an electrophoretic mobility shift of p150Spire. To analyse the specificity of JNK phosphorylation of p150Spire in vivo, p150Spire was coexpressed with the constitutively active Erk2-Mek1-LA fusion protein. Although expressed to the same levels as JNK-MKK7, Erk2-Mek1-LA induces only a very weak electrophoretic mobility shift of p150Spire, indicating a preferential phosphorylation of p150Spire by JNK (Otto, 2000).

Drosophila Fos mediates ERK and JNK signals via distinct phosphorylation sites

During Drosophila development Fos acts downstream from the JNK pathway. It can also mediate ERK signaling in wing vein formation and photoreceptor differentiation. Drosophila JNK and ERK phosphorylate D-Fos with overlapping, but distinct, patterns. Analysis of flies expressing phosphorylation site point mutants of D-Fos reveals that the transcription factor responds differentially to JNK and ERK signals. Mutations in the phosphorylation sites for JNK interfere specifically with the biological effects of JNK activation, whereas mutations in ERK phosphorylation sites affect responses to the EGF receptor-Ras-ERK pathway. These results indicate that the distinction between ERK and JNK signals can be made at the level of D-Fos, and that different pathway-specific phosphorylated forms of the protein can elicit different responses (Ciapponi, 2001).

The loss of wing vein tissue on expression of D-Fos^bZIP resembles phenotypes resulting from defects in the Drosophila epidermal growth factor receptor (DER) pathway, caused, for example, by loss-of-function alleles of DER itself or of other genes required for DER signaling, such as rhomboid, vein, ras, and ERK/rolled. Therefore, the above results might indicate that D-Fos acts as a mediator of the DER/ERK signaling pathway during wing vein differentiation. The artificial activation of this RTK pathway, by gain-of-function alleles or by overexpression of downstream effectors, gives rise to ectopic veins in the wing. To establish whether D-Fos might act epistatically to DER, and whether D-Fos^bZIP or a reduced D-fos gene dose (kayak alleles) might suppress such a phenotype was examined (Ciapponi, 2001).

The Ellipse^B1 (Elp^B1) allele of DER represents an activated component of the DER/ERK signaling pathway. In addition to other phenotypes, for example, in the eye, Elp^B1 animals consistently develop wings with ectopic wing vein material. Strikingly, both D-Fos^bZIP expression in the wing imaginal disc (using the 32B Gal4 driver) or the removal of one copy of D-fos in a kay heterozygote, suppresses this phenotype almost completely. To confirm that the observed effect is specific and caused by a reduction of endogenous D-Fos function, add-back experiments were performed in which this reduction was compensated by supplying extra wild-type D-Fos from a transgene, driven by the heat shock promoter (hs D-fos. Significantly, the presence of the D-fos transgene abrogates the suppression of Elp^B1 by kay and reinstates the extra vein phenotype caused by elevated DER activity. This result confirms that the suppression of the activated DER allele is due to a loss of D-fos activity. Hence, D-Fos mediates wing vein patterning downstream from or in parallel with DER (Ciapponi, 2001).

Next, whether D-Fos mediates ERK signaling also during eye morphogenesis was investigated. Defects in photoreceptor differentiation can be induced by the RTK gain-of-function alleles Elp^B1 and sev^S11. The Elp^B1 allele dominantly causes an abnormal eye phenotype that manifests itself in roughness and the occasional lack of outer photoreceptors. This phenotype can be suppressed largely by the removal of one copy of D-fos and restored subsequently by simultaneous transgenic expression of wild-type D-Fos. A gain-of-function transgene of the RTK-coding gene sevenless (sev^S11) causes the characteristic appearance of ectopic R7 photoreceptor cells in nearly all ommatidia. The sev^S11 phenotype can be suppressed by the expression of dominant-negative Fos. In flies carrying sev^S11 in a heterozygous kay² background, the ectopic R7 photoreceptor phenotype is suppressed significantly; the number of normal ommatidia increases from 5% to approximately 20%. Reintroduction of D-fos by a transgene in this double mutant background restores the percentage of ommatidia with extra photoreceptors observed in sev^S11 heterozygous animals. Taken together, these results indicate that D-Fos can act as a rate-limiting component downstream from the RTKs Sev and DER during eye development (Ciapponi, 2001).

Considering that D-Fos is a transcription factor and based on the precedents of D-Jun and c-Fos, the most obvious role for D-Fos in DER and Sev signal transduction would be that of an effector of the Drosophila MAP kinase Rolled. Therefore, the effect of reducing D-fos activity in animals expressing the gain-of-function allele rl^Sem was examined. Expression of Rl^Sem under UAS control in the wing imaginal disc results in an extra-vein phenotype, markedly when the flies are reared at 25°C and milder at 18°C. Simultaneous expression of D-Fos^bZIP along with Rl^Sem causes a striking suppression of ectopic vein formation, whereas additional expression of full-length D-Fos leads to a strong enhancement of the phenotype. To confirm that the observed suppression of the rl^Sem phenotype was not due to an effect of D-Fos on the transgene promoters, a similar genetic interaction experiment was performed using the endogenous rl^Sem gain-of-function allele and kay² allele. kay² heterozygosity suppresses the rl^Sem-induced extra-vein phenotype. This effect is reverted by ubiquitous expression of D-Fos, indicating that the suppression is due specifically to the decreased activity of D-Fos. These genetic interactions confirm that the role of D-Fos in RTK signal is that of an effector of Rolled (Ciapponi, 2001).

The results described above reveal D-Fos as a downstream component of the ERK signal transduction pathway, yet previous genetic analyses have shown that the transcription factor serves as an effector of JNK. This raises the question of whether the function of D-Fos as recipient of ERK or JNK is mutually exclusive and determined by the cellular context, or whether the transcription factor may mediate both JNK and ERK responses in one tissue or one cell. The developing eye provides a system to approach such a question. Biochemical and genetic studies have indicated that the planar polarity pathway downstream from Frizzled (Fz) and Dishevelled (Dsh) leads to the activation of a JNK-type MAPK module. During retinal morphogenesis, this pathway controls the mirror-symmetric arrangement of ommatidial units relative to the dorso-ventral midline. Thus, in the developing eye the activity of JNK and ERK signal transduction can be monitored separately in vivo (by planar polarity and R-cell recruitment, respectively) (Ciapponi, 2001).

To determine whether D-Fos is involved in planar polarity signaling, the effect of D-Fos^bZIP expressed under the control of Gal4 drivers in the developing eye was examined. When D-Fos function is thus reduced, a striking combined phenocopy of defects in ERK and JNK signal transduction ensues. Sections of eyes of the hairy Gal4/UAS D-fos^bZIP or of the sev Gal4/UAS D-fos^bZIP genotypes display both a lack of photoreceptor cells, diagnostic of inadequate ERK signal transduction, and misoriented ommatidia, indicating defects in planar polarity signaling. This mutant phenotype makes it plausible that D-Fos, in addition to its role in photoreceptor cell recruitment downstream from ERK, acts as an effector of JNK signaling in planar polarity determination. Therefore, D-Fos mediates both JNK and ERK responses in a defined group of cells of the developing retina (Ciapponi, 2001).

To investigate the potential regulation of D-Fos by protein phosphorylation, in vitro kinase assays were performed in which recombinant JNK/Bsk and ERK/Rl were used as kinases and different bacterially expressed versions of D-Fos were used as substrates. In this in vitro setting, both Bsk and Rl could phosphorylate full-length D-Fos. To get an initial indication as to which residues might serve as target sites for ERK or/and JNK, the D-Fos amino acid sequence was compared with that of mammalian Jun and Fos proteins. The sequence alignments identified several sequences in D-Fos with similarity to confirmed JNK or ERK phosphorylation sites in the mammalian molecules. T89 and T93 of D-Fos correspond in their sequence context and relative location to established JNK phosphorylation sites in c-Jun. Alignment of the C-terminal parts of D-Fos and c-Fos show a conserved residue (T584) that corresponds to a previously described MAPK phosphorylation site in c-Fos. Moreover, several serine or threonine residues were identified that might serve as target sites for the proline-directed MAPKs. Mutant derivatives of D-Fos were generated in which one or more of these candidate phosphorylation sites was substituted by alanine. JNK/Bsk, but not ERK/Rl, can efficiently phosphorylate a fragment spanning the 170 N-terminal amino acids of D-Fos. When alanine substitutions are introduced in positions T89 and T93, the N-terminal D-Fos fragment is no longer an efficient substrate for JNK phosphorylation (Ciapponi, 2001).

Additional N-terminal residues that conform to the S/TP consensus (T234, S235, T237, and T254) are phosphorylated by neither JNK nor ERK. Similar to the 170-amino-acid N-terminal fragment, a D-Fos fragment covering the N-terminal 285 amino acids is not a substrate for ERK. This indicates that the N-terminal part of D-Fos is a good substrate for JNK/Bsk but not for ERK/Rl, and that the residues T89 and T93 serve as the main N-terminal JNK target sites (Ciapponi, 2001).

Interestingly, a small deletion that removes a sequence with remote similarity to the c-Jun delta-domain (amino acids 28-56), but that does not span the phosphorylation sites T89 and T93, completely abrogates phosphorylation of the D-Fos N-terminal fragment by JNK/Bsk. It is possible that this deletion destroys a delta-domain-like JNK docking site present in D-Fos (Ciapponi, 2001).

Next, the C-terminal part of D-Fos was analyzed; it contains seven potential phosphorylation sites for MAPKs. A fragment spanning the C-terminal 280 amino acids of D-Fos is, in contrast to the N terminus, phosphorylated efficiently by both Bsk and Rl. Alanine substitutions in all seven putative MAPKs target sites causes complete loss of phosphorylation. However, mutating subsets of the seven putative phosphorylation sites does not result in a complete loss of phosphorylation by either Rl or Bsk, indicating the presence of multiple phosphorylation sites in the C-terminal part of D-Fos. These results indicate that, at least in vitro, D-Fos is a direct substrate of both Drosophila JNK and ERK and that it contains overlapping, but distinct, sets of phosphorylation sites for the two kinases (Ciapponi, 2001).

After establishing which residues serve as substrates for JNK and/or ERK in vitro, it was important to determine the regulatory relevance of these sites in vivo. Transgenic fly strains expressing mutant forms of full-length D-Fos were generated in which either the putative N-terminal, JNK-specific phosphorylation sites, or the C-terminally located ERK and JNK substrate sites were replaced by alanine (D-Fos^N-Ala and D-Fos^C-Ala. In the D-Fos^{pan Ala} mutant, all the putative MAPK phosphorylation sites, both N- and C-terminal, were substituted by alanine. When the nonphosphorylatable D-Fos^{pan Ala} was expressed from a UAS-driven transgene under the control of the epidermal driver 69B Gal4, it gave rise to a strong thoracic cleft at the dorsal midline, resembling kay or hep mutants, or phenotypes that result from D-Fos^bZIP expression. Evidently, D-Fos^{pan Ala} represents a dominant-negative mutant that can interfere with JNK-dependent thorax closure (Ciapponi, 2001).

The relevance was investigated of subgroups of the D-Fos MAPK phosphorylation sites during thorax closure. Expression of D-Fos^N-Ala results in a distinctive thorax cleft, although not quite as pronounced as in the case of D-Fos^{pan Ala}. Importantly, this result shows that the N-terminal, JNK-specific phsosphorylation sites in D-Fos are required for a well-defined JNK-dependent developmental mechanism. Expression of D-Fos^C-Ala, which lacks the C-terminal phosphorylation sites, or of D-Fos^wt has no discernible effect. These results indicate that either the C-terminal sites play only an ancillary role and are not essential for JNK signaling in the signal transduction pathway controlling thorax, or that this mutant does not compete well with endogenous D-Fos and therefore has no dominant-negative effect in this context (Ciapponi, 2001).

Next, whether expression of the different phosphorylation mutants of D-Fos might also interfere with ERK responses was investigated. Expression of D-Fos^{pan Ala} in the posterior compartment of wing imaginal disc (using en Gal4) causes loss of wing vein material typical of mutants defective in DER to Rolled signaling. Thus, consistent with the observation that D-Fos^{pan Ala} has lost all substrate sites for both Rolled and Bsk, it acts as a dominant-negative form that interferes with D-Fos function in both the ERK and the JNK pathways (Ciapponi, 2001).

Interestingly, however, the D-Fos^N-Ala and D-Fos^C-Ala mutants influence the ERK and JNK response differently. D-Fos^N-Ala, which interfers dominantly with JNK-mediated thorax closure, has no effect on ERK-dependent wing vein formation. This is consistent with these sites not being substrates for Rolled. The D-Fos^C-Ala mutant, however, which is neutral in thorax development, causes loss-of-vein phenotype (Ciapponi, 2001).

To examine whether differential phosphorylation of D-Fos might also be used in the developing eye to distinguish between ERK and JNK signaling, the effect of the D-Fos^Ala mutants on ERK-dependent photoreceptor cell recruitment and JNK-mediated ommatidial rotation was examined. Different D-Fos^Ala mutants were expressed along with sev^S11 in the eye imaginal disc under the control of the sevenless enhancer. As in the case of wing vein formation, D-Fos^N-Ala does not alter the sev^S11 phenotype, whereas the expression of D-Fos^C-Ala or of D-Fos^{pan Ala} causes a significant suppression of the extra R7-cell recruitment. Thus, ERK signaling, whether it is triggered by DER or by Sev, appears to require only the C-terminal phosphorylation sites of D-Fos (Ciapponi, 2001).

Next, whether the D-Fos^Ala mutants could suppress the ommatidial misrotation phenotype that is elicited by overexpression of Fz and the ensuing activation of JNK was tested. Coexpression of all Ala mutants and Fz under the control of sev Gal4 leads to a significant suppression of the misrotation phenotype observed in flies expressing Fz alone. Wild-type D-Fos does not have this effect. These results indicate that the JNK-phosphorylation sites of D-Fos are required for the manifestation of the Fz gain-of-function phenotype. The Fz-JNK response in the eye is also affected by mutation of the C-terminal sites that do not visibly disturb the thorax closure response. This might be explained by the higher sensitivity of the ommatidial rotation paradigm or a higher relative expression of the Fos^Ala mutant transgene in the photoreceptor cells (Ciapponi, 2001).

The dominant phenotypic effects of D-Fos^Ala expression support the interpretation that Fos is regulated by protein phosphorylation to mediate developmental decisions and indicates that the residues identified by mutagenesis and in vitro kinase assay are required for in vivo function. Moreover, these findings indicate clearly that the function of D-Fos as a mediator of JNK/Bsk and ERK/Rl cascades is in both cases that of a direct kinase substrate (Ciapponi, 2001).

An important question raised by the finding that D-Fos can mediate signaling by both ERK and JNK is how the decision between these distinct cellular responses is made, that is, how the cell 'knows' which program to execute when D-Fos becomes phosphorylated. Several mechanisms have been suggested to contribute to signal specificity in such situations, in which one protein mediates different cellular responses. One model proposes a combinatorial mechanism by which several factors with overlapping broad responsiveness have to cooperate to mediate a defined specific cellular behavior. However, the observation that D-Fos participates in ERK as well as JNK signal transduction in the same group of cells of the developing Drosophila eye, by regulating photoreceptor differentiation and ommatidial rotation, respectively, argues against cell type-specific cofactors that modulate the response to D-Fos phosphorylation. The distinct substrate sites in D-Fos phosphorylated by ERK and JNK raise a novel possibility to explain the signal-specific D-Fos response. It is suggested that D-Fos exists in two different activated forms, depending on whether it is phosphorylated by ERK or JNK. These differentially phosphorylated forms might then selectively trigger either the ERK or the JNK response. This idea is supported by in vivo experiments in which phosphorylation site-specific point mutants of D-Fos were expressed in the developing fly. A mutant that lacks all phosphorylation sites interferes dominantly with both ERK and JNK signaling in thorax closure, the wing, and the eye imaginal disc, supporting further the general relevance of D-Fos phosphorylation in developmental decisions. D-Fos mutants lacking subsets of phosphorylation sites, however, affected JNK and ERK signal responses differentially. An N-terminal cluster of JNK sites that is not phosphorylated by ERK is critical for the JNK response in vivo. A mutant lacking these sites interfers with thorax closure and planar polarity regulations, both bona fide JNK responses, but not with wing vein formation or photoreceptor differentiation, which are regulated by ERK. Conversely, a mutant that removes all ERK substrate sites dominantly suppresses processes normally controlled by this MAP kinase. These data indicate that signal-responsive transcription factors, such as D-Fos, may have different signal-specific functions. It is tempting to speculate that such a mechanism might be used by other signaling proteins that are receptive to different upstream signals (Ciapponi, 2001).

Eyes absent mediates cross-talk between retinal determination genes and the receptor tyrosine kinase signaling pathway: A possible role for the p38 MAPKs in activation of Eya

Drosophila Eyes absent function is positively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation: this regulation extends to developmental contexts independent of eye determination. In vivo genetic analyses, together with in vitro kinase assay results, demonstrate that Eya is a substrate for extracellular signal-regulated kinase, the MAPK acting downstream in the receptor tyrosine kinase (RTK) signaling pathway. Thus, phosphorylation of Eya appears to provide a direct regulatory link between the RTK/Ras/MAPK signaling cascade and the retinal determination gene network (Hsiao, 2001).

To address the possibility that Eya might be a direct downstream target of the RTK pathway, the Eya sequence was examined for potential MAPK phosphorylation consensus sites, defined as P-X-S/T-P (P, proline; X, any amino acid; S/T, serine or threonine). Investigations of alternative possibilities, including direct transcriptional regulation or protein-protein interactions between Yan and Eya, have yielded negative results to date. Two sites matching the consensus were found and will be referred to as 'MAPK sites.' Both MAPK sites are located ~80 residues upstream of the highly conserved 'Eya domain,' which has been shown to mediate interactions with So and Dac proteins. Examination of mammalian Eya protein sequences reveals similarly located MAPK sites in mouse and human Eya1, Eya2, and Eya4, suggesting that MAPK phosphorylation might be important for an evolutionarily conserved aspect of Eya function or regulation. Eya1 contains two MAPK sites, whereas Eya2 and Eya4 retain the second site but have a less stringent two amino acid consensus, S/T-P, at the first. No consensus MAPK sites were found in Eya3, although the less stringent two amino acid consensus is present at the second site (Hsiao, 2001).

To investigate the importance of the two MAPK phosphorylation sites with respect to Drosophila Eya function, the effects of mutating the phosphoacceptor residues were assessed in transgenic flies using an ectopic eye induction assay. First, site-directed mutagenesis was used to replace the serine residues in both sites with alanine (referred to as Eya^S-A), effectively destroying the MAPK sites. Second, the serine residues were mutated to glutamic or aspartic acid (referred to as Eya^S-D/E) to mimic the negative charge associated with phosphorylation. Transgenic lines expressing wild-type Eya (referred to as Eya^WT), Eya^S-A, and Eya^S-D/E under control of the UAS promoter were used to induce tissue-specific expression. For each construct, ten independent lines were assayed, and the data from the strongest and weakest were discarded. For the remaining eight lines, ~300 progeny of the appropriate genotype were scored for each cross, and the percentage of flies exhibiting ectopic eye induction was calculated (Hsiao, 2001).

On average, expression of Eya^WT induced ectopic eye formation in ~50% of the flies assayed. Most ectopic eyes were found on the head near the eye and around the base of the antenna. Patches of red eye pigment were also frequently induced on the wing hinges and underside of the metathoracic legs as well as occasionally on the wings, legs, and antennae. As expected when surveying a collection of independent transgenic insertions, positional effects resulted in a fairly broad distribution of phenotypic strengths. Thus, penetrance of ectopic eye induction ranged from almost 80% in the strongest line to <5% in the weakest. Strikingly, when the nonphosphorylatable Eya^S-A transgene is expressed, the average incidence of ectopic eye formation dropped to ~20% as compared with the 50% observed with Eya^WT. In addition, the size of the ectopic eye patches is noticeably reduced in Eya^S-A. Thus, on average, mutation of the two MAPK sites to a nonphosphorylatable form reduces Eya's effectiveness at inducing ectopic eye formation (Hsiao, 2001).

In the converse experiment in which the MAPK sites were mutated so as to mimic the constitutively phosphorylated state (Eya^S-D/E), the incidence of ectopic eye formation increased to an average value of ~80%. Furthermore, flies expressing Eya^S-D/E exhibit an increase in the size and number of ectopic eye patches relative to flies expressing Eya^WT. For example, in Eya^WT lines, ectopic eyes usually form on only one side of the head, whereas in Eya^S-D/E lines, ectopic eye induction often occurs bilaterally. Thus, mimicking the constitutively phosphorylated state dramatically enhances the efficacy of the Eya transgene in promoting ectopic eye formation. As with Eya^S-A, Eya^S-D/E lines exhibited no obvious changes in expression, stability, or subcellular distribution of the protein product that might provide alternate explanations for the apparent increase in activity (Hsiao, 2001).

To assess whether both MAPK sites are critical for Eya function, transgenes containing single MAPK site mutations were assayed for ability to promote ectopic eye formation. These transgenes are referred to as Eya^1S-A, Eya^2S-A, Eya^1S-D, and Eya^2S-E, where 1 and 2 refer to the specific MAPK site mutated. Expression of these transgenes produces intermediate results compared with the lines in which both MAPK sites were altered. For example, both Eya^1S-A and Eya^2S-A transgenes exhibited an average of ~30% ectopic eye induction, a value that represents an increase relative to Eya^S-A but a decrease relative to Eya^WT. Similarly, both Eya^1S-D and Eya^2S-E transgenes exhibited an intermediate value of ~65%, suggesting reduced activity relative to Eya^S-D/E but increased activity relative to Eya^WT. Together, these results suggest that both MAPK sites additively contribute to Eya function and regulation (Hsiao, 2001).

When interpreting the consequences of overexpressing a transgene, genetic determination of whether the observed phenotype reflects an antimorphic (dominant negative) or a hypermorphic (dominant activated) effect helps in assessing whether the particular transgene is functioning in a positive or negative manner relative to other regulators of the pathway. By genetically reducing the dose of the endogenous gene, it is possible to distinguish between the two possibilities (Hsiao, 2001).

To confirm that the ectopic eye induction associated with expression of the Eya transgenes reflects an increase in Eya activity, it was asked whether a 50% reduction in dosage of endogenous eya could suppress the phenotype. In all three cases (Eya^WT, Eya^S-A, and Eya^S-D/E), flies heterozygous for a null eya allele showsweaker and less penetrant phenotypes. For both Eya^WT and Eya^S-A, a 50% reduction in ectopic eye formation is observed. Suppression is less pronounced with Eya^S-D/E transgenes, consistent with the interpretation that Eya^S-D/E is a hyperactivated protein (Hsiao, 2001).

These results, in conjunction with the finding that the Eya^S-A and Eya^S-D/E products are less and more efficient at promoting ectopic eye induction, respectively, suggest that Eya function is positively regulated via phosphorylation of these two MAPK sites in vivo. The fact that some activity is retained in the Eya^S-A transgenes implies that MAPK phosphorylation does not provide a simple on/off switch, but rather serves to modulate the level of Eya activity (Hsiao, 2001).

The results obtained from overexpressing the Eya^WT, Eya^S-A, and Eya^S-D/E transgenes implicate MAPK phosphorylation as positively regulating Eya activity. Drosophila, like mammals, has multiple MAPK family members, including ERK, that mediates RTK/Ras initiated signals, the Jun N-terminal Kinase (JNK), and the p38 stress-responsive MAPKs. Less is known about the upstream signaling mechanisms that activate JNK and p38 MAPKs, although both families respond to stress and may exhibit some functional redundancy. Although isolation of Eya in an RTK pathway-based genetic screen made ERK the best candidate, other MAPK family members could also potentially phosphorylate Eya. Currently, specific loss-of-function mutations are available for only two members of the MAPK family in Drosophila: ERK and JNK. The gene rolled encodes ERK, whereas the gene basket encodes JNK. Two stress-responsive MAPKs, p38a and p38b, have been cloned and characterized in overexpression assays, although specific mutations have not yet been reported (Hsiao, 2001).

To address whether phosphorylation of Eya by MAPK is physiologically relevant, it was asked whether genetically reducing the dose of either ERK (rolled) or JNK (basket) could suppress the phenotypes associated with Eya overexpression. rolled mutations dramatically suppresses Eya^WT phenotypes, whereas basket mutations have no effect. Strong suppression of Eya^WT phenotypes was also obtained with mutations in the Epidermal growth factor receptor (Egfr), suggesting that activity of the canonical RTK/Ras/MAPK cascade participates in modulating Eya activity. Together, these results suggest that in vivo, ERK, the MAPK responsive to RTK-initiated signals, but not JNK, positively regulates Eya activity. A double mutant between rolled and basket suppresses Eya^WT at the same level as rolled alone, suggesting there are no synergistic effects between these two kinases. In a related experiment, it was found that coexpressing Eya^WT and an activated allele of ERK enhances the penetrance and severity of the phenotypes, whereas coexpression of Eya^WT and JNK do not (Hsiao, 2001).

Interestingly, a reduction in rolled dosage is also able to suppress the phenotypes associated with Eya^S-A transgenes. Because the two MAPK sites are effectively destroyed in this construct, the suppression is unlikely to reflect a direct interaction between ERK and the Eya^S-A product. Two possible interpretations could explain this result: (1) additional sites in Eya could be phosphorylated by ERK, apart from the two MAPK consensus sites identified; (2) the suppression could simply reflect downregulation of endogenous Eya. The fact that reduction in rolled dosage suppresses to a greater extent than reduction in eya dosage is consistent with this interpretation, assuming that the pool of endogenous ERK is limiting. In this case, then the amount of ERK produced in a heterozygous rolled background would be insufficient to activate even 50% of the endogenous Eya, and thus, even stronger suppression would be obtained relative to that seen with a 50% reduction in endogenous eya (Hsiao, 2001).

Consideration of the genetic interaction data obtained with the Eya^S-D/E transgenes increases the likelihood of the second scenario. A 2-fold reduction of either endogenous eya or endogenous rolled led to a comparable ~15% reduction in penetrance of ectopic eye induction in the Eya^S-D/E background. If Eya function were regulated via MAPK phosphorylation at additional sites, reducing the dose of rolled should have a greater impact on the phenotype than reducing the dose of eya, even in the hyper-activated Eya^S-D/E background. Thus, the explanation is favored that suppression of Eya^S-A and Eya^S-D/E upon reduction of endogenous ERK is primarily attributable to the dose sensitivity of the phenotype relative to endogenous Eya activity. The fact that suppression of Eya^S-D/E is less striking than that observed in either the Eya^WT or the Eya^S-A backgrounds is consistent with the hypermorphic nature of the Eya^S-D/E product (Hsiao, 2001).

To complement the in vivo genetic experiments and to determine whether the interaction between Eya and MAPK is direct, kinase assays were performed to assess which MAPK phosphorylates Eya preferentially in vitro. Activated ERK or JNK were immunoprecipitated from Drosophila S2 cultured cells transfected with appropriate cDNA constructs. Both kinases were active, as demonstrated by their ability to phosphorylate myelin basic protein and GST-Yan. GST-Eya^WT and GST-Eya^S-A fusion proteins were generated and tested for the ability to serve as ERK or JNK substrates. Consistent with the in vivo genetic results, ERK phosphorylates GST-Eya^WT, whereas JNK, although clearly active, does not (Hsiao, 2001).

The p38a and p38b MAPKs were also tested for ability to phosphorylate Eya using the in vitro kinase assay. Both p38a and p38b phosphorylated GST-Eya^WT at levels comparable to those obtained with ERK. These results suggest a possible role for the p38 MAPKs in activation of Eya, although in vivo validation of these predictions must await the isolation of specific mutations in p38a and p38b. However, given the recent report suggesting that p38b MAPK functions downstream of dpp signaling, it is tempting to speculate that the kinase assay results may provide a mechanistic basis underlying the genetic synergy that has been demonstrated between dpp and the retinal determination genes (Hsiao, 2001).

Over the course of the experiments looking at frequency of ectopic eye induction, several phenotypes associated with Eya overexpression were noticed that had not been previously reported. Specifically, Eya overexpression results in wing defects, increased number of scutellar macrochaetae, and problems with thoracic closure. Additional phenotypes, including rough eyes and arista to leg transformations, were observed at low penetrance and have not been extensively characterized (Hsiao, 2001).

Although eya loss-of-function phenotypes have not been reported in adult tissues other than the eye and germ line, it was found that all of the phenotypes associated with Eya overexpression can be suppressed by a 50% reduction in endogenous eya. This argues strongly that eya is expressed and could play a role in the normal development of these tissues. Furthermore, similarly strong suppression is achieved upon reducing the dose of endogenous dac or so, suggesting that part or all of retinal determination gene network may be redeployed in developmental contexts independent of eye formation (Hsiao, 2001).

Genetic tests to determine which MAPK might be responsible for modulating Eya activity in these contexts were performed as described for the ectopic eye phenotype. Because the thoracic closure and macrochaetae phenotypes were more variable and therefore less conducive to quantitative analyses, focus was placed on Eya regulation in the wing. A reduction in rolled dosage strongly suppresses whereas a reduction in basket dosage has little effect on the observed wing phenotypes. Conversely, coexpression of Eya^WT and activated ERK enhances the severity of the wing defects, whereas coexpression of JNK has little effect. Because expression of activated ERK alone results in ectopic wing vein formation, it is formally possible that the enhancement is additive rather than synergistic. However, within the same wing, the two phenotypes are easily distinguished, and the best interpretation is that the Eya-associated defects are enhanced whereas the ERK-associated ones are not. As was found in the ectopic eye formation assay, mutations in Egfr also suppressed the wing phenotypes. Thus, in the contexts of both eye and wing development, Eya activity appears to be modulated by ERK, the MAPK functioning downstream of the RTK signaling pathway (Hsiao, 2001).

These results suggest RTK signaling plays dual and opposing roles during eye development. Previous work has indicated that an early, MAPK-independent aspect of RTK signaling antagonizes the decision to become an eye. The current study shows that a MAPK-dependent signal positively promotes eye morphogenesis later in development by directly modulating Eya activity. The fact that the same pathway can exert such apparently opposing effects is not unusual; depending on the specific context, the same signal may be interpreted in strikingly different ways (Hsiao, 2001 and references therein).

JNK signaling pathway is required for efficient wound healing in Drosophila

Efficient wound healing including clotting and subsequent reepithelization is essential for animals ranging from insects to mammals to recover from epithelial injury. It is likely that genes involved in wound healing are conserved through the phylogeny and therefore, Drosophila may be a useful in vivo model system to identify genes necessary during this process. Furthermore, epithelial movement during specific developmental processes, such as dorsal closure (DC), resembles that seen in mammalian wound healing. Since puckered (puc) gene is a target of the JUN N-terminal kinase signaling pathway during DC, puc gene expression was investigated during wound healing in Drosophila. puc expression is induced at the edge of the wound in epithelial cells and Jun kinase is phosphorylated in wounded epidermal tissues, suggesting that the JUN N-terminal kinase signaling pathway is activated by a signal produced by an epidermal wound. In the absence of the Drosophila c-Fos homologue, puc gene expression is no longer induced. Finally, impaired epithelial repair in JUN N-terminal kinase deficient flies demonstrates that the JUN N-terminal kinase signaling is required to initiate the cell shape change at the onset of the epithelial wound healing. It is concluded that the embryonic JUN N-terminal kinase gene cassette is induced at the edge of the wound. In addition, Drosophila appears as a good in vivo model to study morphogenetic processes requiring epithelial regeneration, such as wound healing in vertebrates (Ramet, 2002).

In most cases, flies were anesthetized and then mechanically wounded with iridectomy scissors to cut adult abdomen vertically between the third and the seventh tergites. Semi-thin sections were used to examine the histology of wound healing at the cellular level. The first response to epithelial wound is the formation of a clot at the initial site. Subsequently, the clot becomes melanized making the location of the wound clearly visible. The clot appears to consist of an accumulation of melanin and by hemocytes that aggregated at the site of injury. Hemocytes may be involved also in the clearance of cellular debris and invading microbes (Ramet, 2002).

During the first 2 h after wounding no sign of epithelial cell movement can be seen. In most cases, the edges of the cut epidermis are found far away from the broken cuticle. As for the wounded embryonic epidermis, the adult epidermal layer may be submitted to an intrinsic isotropic epidermal tension that retracts it upon any break injury. By 4 h, the epithelial cells of the edge of the wound seem to shed from the disrupted cuticle. These cells appear larger than the epithelial cells lining the normal cuticle, and exhibit cytoplasmic protrusions. By 12 h, the protrusive cytoplasmic extensions extend from the cells of the edge of the wound and 'migrate' toward each other under the melanin clot, Subsequently, they cause the epidermis to form a suture. These cytoplasmic extensions suggest that adult epidermis is healed by the activity of dynamic lamellipodia or filipodia. Correspondingly, cytoskeleton reorganization has been previously described in wound healing model of cultured Madin-Darby canine kidney cell (MDCK) and during Drosophila DC. At this point, the epithelial cells are still enlarged but start to return to their initial shape. The suture of the epithelium is normally achieved within 18 h after injury. By this time, the wounded epithelium has healed, and cells have returned to their original shape (Ramet, 2002).

To ascertain the importance of melanin production in wound healing, the survival rate of Black cells (Bc) homozygous flies was measured after a transversal wound of the adult abdomen cuticle. Bc/Bc flies lack hemolymphatic phenoloxydase activity and therefore, do not produce melanin. By 24 h after wounding, wild type flies and Bc/+ heterozygous flies, present a dominant melanized crystal cell phenotype, but have wild-type phenoloxidase activity, both of which have about 20% mortality, suggesting an efficient wound repair. In contrast, the vast majority (91%) of wounded Bc/Bc mutants died. 50% mortality of Bc flies was already seen by 6 h, suggesting that phenoloxydase activity is essential early in the wound healing process. Similarly, lozenge (lz) mutants, which lack crystal cells and hence present a weak hemolymphatic phenoloxydase activity, exhibit a poor ability to recover from the injury (Ramet, 2002).

The wound clots differently in Bc flies compared to wild type. In wild type flies, a melanin deposit is observed as early as 10 min after wounding and it is still visible 6 h after injury. In contrast, there is no evidence of melanin formation in the wounded integument of Bc flies, indicating that the latter is of hemolymphatic origin. Furthermore, the two edges of the wound are found apart in Bc flies. This failure to keep the edge of the wound in close proximity leads to death due to bleeding. These results underlie the essential role of the phenoloxydase activity, or an associated phenomenon, when it comes to efficient clot formation and the prevention of bleeding (Ramet, 2002).

To ascertain that the Drosophila JNK pathway is activated in wounded epidermis, DJun N-terminal kinase activity was assayed using anti-phospho-JNK antibodies. Protein extracts from adult abdominal integument from control and wounded flies were assayed for anti-phospho-JNK reactivity. Phospho-JNK can be detected only in the protein extracts from the wounded epidermis, whereas the unphosphorylated form of DJNK is present in all of the samples. This indicates that DJNK is phosphorylated in response to wounding in epidermal cells, and therefore, suggests that the JNK signaling pathway is activated (Ramet, 2002).

In contrast to embryonic DC where only the most dorsal cell row at the leading edge is expressing puc gene, several rows of adult epidermal cells show a strong ß-galactosidase activity during wound healing. This result is consistent with high DJNK activity in the vicinity of the wound. Indeed, the extent of the area expressing puc-lacZ clearly depends on the size of the wound (up to 8 cell rows). Furthermore, puc gene expression showed a decreasing gradient from the edge of the wound towards healthy epithelium. This suggests that a newly formed signal emerges from the wound and diffuses through the epidermal layer (Ramet, 2002).

During embryonic DC, the DJNK pathway activates puc expression in the LE and as a negative feedback loop, puc itself down-regulates the DJNK. To further test if the DJNK pathway also mediates puc induction during wound healing, puc expression pattern in adult epidermis was analyzed in fly mutants of this pathway. In hep, a hypomorphic allele of hemipterous, (encoding the MAPKK), puc-lacZ expression is almost completely normal. This result is not totally unexpected since hep flies present no epidermal defects. Stronger hep allele mutations are lethal and could not be studied. However, a heteroallelic combination of kay mutations leads to viable flies. Since the incidence of dorsal thoracic cleft phenotype of this mutant is higher than that in hep, it is more likely to affect the regulation of puc expression. In kay¹/kay² animals, puc gene induction is drastically reduced at the site of injury compared to wild-type. This result demonstrates that puc regulation is dependent on the transcriptional activator DFos during wound healing. Interestingly, still 18 h after wounding, the mutant cells are separated from the edge of the wound comparably to that observed with wild type at 3 h after wounding. This suggests that the epidermal sheet has been unable to spread under the wound and that the process is blocked at the initiation stage. Since DFos, together with DJun, is the target of the DJNK pathway, and forms the transcription factor AP-1, it is likely that the DJNK pathway is switched on by an integument injury (Ramet, 2002).

To find out if DFos mutation has a cell autonomous effect, the UAS/GAL4 system was used to express a dominant negative form of DFos in the pannier (pnr) expression domain. In the pnr-Gal4 line, Gal4 protein is expressed in a large dorsal band of adult epidermis. A continuous wound was done to overlap this dorsal epidermal expression domain and the dorso-lateral and ventral epidermal domain. puc-lacZ expression was then assayed 12 h after wounding. In control flies, puc is expressed at the wounded epidermis independent of the location. When DFos^bZip dominant negative form of DFos is expressed in the dorsal band, X-Gal staining shows a clear, albeit not total, reduction of puc expression at the expected places. In contrast, puc expression is induced normally outside of the pnr expression domain. This demonstrates that the puc gene induction is under the control of the DFos transcriptional factor in a cell-autonomous manner similar to that observed during dorsal and thorax closure (Ramet, 2002).

To ascertain the importance of the DJNK pathway in wound healing, the phenotype of kay mutant was investigated during the course of wound healing. As expected, the wounded epidermis from kay deficient flies fails to recover. The epithelial cells at the edge of the wound also fail to undergo any evident cell shape change or show any cytoplasmic protrusive extensions. Even at 18 h after injury, the wound is not repaired. Therefore, the transcriptional activator DFos appears necessary for a normal epithelial repair in adult Drosophila. Interestingly, over a period of 6 days, wounded mutant flies did not suffer any higher mortality compared to wounded wild type flies, suggesting that epithelial repair is not crucial for early survival (Ramet, 2002).

JNK, targeting Foxo, extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling

Aging of a eukaryotic organism is affected by its nutrition state and by its ability to prevent or repair oxidative damage. Consequently, signal transduction systems that control metabolism and oxidative stress responses influence life span. When nutrients are abundant, the insulin/IGF signaling pathway promotes growth and energy storage but shortens life span. The transcription factor Foxo, which is inhibited by insulin/IGF signaling, extends life span in conditions of low insulin/IGF signaling activity. Life span can also be increased by activating the stress-responsive Jun-N-terminal kinase (JNK) pathway. JNK requires Foxo to extend life span in Drosophila. JNK antagonizes insulin/IGF signaling, causing nuclear localization of Foxo and inducing its targets, including growth control and stress defense genes. JNK and Foxo also restrict insulin/IGF signaling activity systemically by repressing insulin/IGF ligand expression in neuroendocrine cells. The convergence of JNK signaling and IIS on Foxo provides a model to explain the effects of stress and nutrition on longevity (Wang, 2005).

These data suggest Foxo is a convergence point for insulin/IGF signaling (IIS) and JNK signaling. Through its responsiveness to these two pathways, Foxo is well positioned to integrate information about environmental stress and nutrient availability and to elicit appropriate biological responses. Such a system would ensure that growth could proceed in an unrestrained manner when energy resources are available and the cell is not exposed to external insults (IIS is active, JNK is off, and Foxo is repressed). However, in situations of low food availability or an adverse environment, IIS would cease to signal, or JNK would be activated, resulting in translocation of Foxo to the nucleus. The ensuing Foxo-induced gene expression has several effects at the cell as well as the organism level and is likely to counteract premature senescence. The induction of genes such as thor can reduce cell growth, presumably to limit the cell’s anabolic expenses in adverse situations. Other target genes, such as the small heat shock protein l(2)efl, are expected to have a direct role in allaying damage inflicted by environmental insults and may prevent the accumulation of toxic protein aggregates. The suppression of dilp2 expression by JNK and Foxo in insulin-producing cells, in contrast, is likely to control growth, metabolism, and stress responses systemically by downregulating IIS in all responsive tissues in a coordinated fashion (Wang, 2005).

The interaction between JNK and Foxo is thus expected to influence stress tolerance and life span at two levels. In peripheral tissues, JNK activates Foxo and prevents senescence cell-autonomously. Such a mechanism is exemplified by the recent finding that Foxo overexpression prevents age-dependent decline of cardiac performance. Systemic control of IIS by JNK-mediated activation of Foxo in IPCs, in contrast, would serve to coordinate cellular responses to changes in the environment throughout the organism. These data indicate that this latter mechanism plays a significant role in the regulation of life span by JNK and Foxo. The identification of this endocrine function of JNK/Foxo signaling supports and extends the proposed role of JNK signaling on longevity and demonstrates a role for IPCs in life span regulation. In addition to controlling growth and metabolism, IPCs may thus act as a coordination point for the organism’s stress response by downregulating Dilp production in response to oxidative stress and JNK activation. In target tissues, such a mechanism would induce protective gene expression by the second, cell-autonomous tier of Foxo signaling. Interestingly, the effects of IPC-specific JNK activation on longevity and growth are separable. Life span can be extended by moderately increased JNK activity in IPCs when growth effects are yet not evident. This finding is consistent with observations by others who showed that the extension of life span in IIS loss-of-function situations is not a mere consequence of small body size (Wang, 2005).

How did such a multilayered regulation of IIS activity by JNK evolve? It is tempting to speculate that localized activation of Foxo is required to prevent cellular damage and ultimately senescence in conditions in which stressful insults are confined to specific tissues. Such localized insults could, for example, be inflicted by reactive oxygen species that are produced in the environment of amyloid deposits in Alzheimer’s disease as well as by mechanical and oxidative stress experienced by particularly active tissues such as the heart. Systemic regulation of Foxo activity, in contrast, is expected to be an important response mechanism to coordinate metabolism and stress defenses throughout the organism upon changes in the environment. A good example for such a mechanism is the induction of diapause in invertebrates in response to environmental stress or food deprivation. Accordingly, sensory neurons expressing the insulin-like peptide DAF-28 are required for the induction of the dauer larval stage in response to environmental cues in C. elegans (Wang, 2005).

Systemic and tissue-autonomous effects of JNK/Foxo signaling may be connected in multiple ways. The data indicate that JNK and Foxo interact in IPCs to repress dilp2 expression, ultimately activating Foxo in Dilp2 target tissues in a coordinated fashion. Since JNK is be activated in IPCs even under normal culture conditions, it is likely that this systemic control of IIS activity by JNK and Foxo plays a critical role in life span regulation. It is, however, also possible that the cell-autonomous protective function of JNK/Foxo signaling is most critical for the survival of specific tissues as the organism ages, thus extending life of the organism by preventing the loss of indispensable cells or tissues. In addition, stress and the JNK-mediated activation of Foxo in peripheral tissues may signal back to IPCs to initiate a systemic response. In Drosophila, such a mechanism has been documented in the case of the fatbody. Activation of Foxo in this tissue relays a signal to the IPCs, causing them to curb Dilp2 production, a process that has been proposed to require Foxo activity. The exact nature of this feedback signaling mechanism in flies is unclear, but it is reminiscent of the complex signaling interactions between β cells and insulin target tissues in mammals. Further studies are required to shed light on the relative contributions of JNK/Foxo signaling in IPCs or Dilp target tissues to life span regulation (Wang, 2005).

JNK-mediated modulation of IIS activity is likely to be evolutionarily conserved. Inhibitory crosstalk from JNK to IIS in mammalian cells has been found to occur by JNK-mediated phosphorylation and inhibition of IRS-1. This interaction is responsible for obesity-induced insulin resistance in mice. Whether mammalian homologs of Foxo take part in this pathology remains to be determined. A second possible mechanism for JNK/IIS pathway interaction is the direct phosphorylation and activation of Foxo by JNK. Such a mechanism is supported by the observation that in mouse cells JNK can phosphorylate the DFoxo homolog Foxo4 in response to oxidative stress. The physiological relevance of this phosphorylation event has not yet been addressed. The JNK target residues on IRS-1 and Foxo4 are not conserved in the Drosophila homologs Chico and DFoxo, and further studies are thus required to determine whether JNK-Foxo crosstalk in Drosophila is mediated via homologous mechanisms (Wang, 2005).

The systemic regulation of IIS activity by JNK and Foxo appears to be conserved as well. It has been suggested that C. elegans Daf16/Foxo regulates life span (at least in part) by reducing the expression of insulin-like peptides. In mammals, pancreatic β cells (the counterparts of IPCs) reduce their production of insulin in response to oxidative stress-mediated JNK activation. Conversely, dephosphorylation of JNK by MAPK phosphatase 1 can induce insulin expression in these cells. Reducing circulating insulin levels by JNK-mediated Foxo activation may thus be a general mechanism that balances growth and metabolism with stress defense and damage repair (Wang, 2005).

Chameau HAT and DRpd3 HDAC function as antagonistic cofactors of JNK/AP-1-dependent transcription during Drosophila metamorphosis

Gene regulation by AP-1 transcription factors in response to Jun N-terminal kinase (JNK) signaling controls essential cellular processes during development and in pathological situations. The histone acetyltransferase (HAT) Chameau and the histone deacetylase DRpd3 act as antagonistic cofactors of DJun and DFos to modulate JNK-dependent transcription during pupal thorax metamorphosis and JNK-induced apoptosis in Drosophila. It has been demonstrated, in cultured cells, that DFos phosphorylation mediated by JNK signaling plays a central role in coordinating the dynamics of Chameau and DRpd3 recruitment and function at AP-1-responsive promoters. Activating the pathway stimulates the HAT function of Chameau, promoting histone H4 acetylation and target gene transcription. Conversely, in response to JNK signaling inactivation, DRpd3 is recruited and suppresses histone acetylation and transcription. This study establishes a direct link among JNK signaling, DFos phosphorylation, chromatin modification, and AP-1-dependent transcription and its importance in a developing organism (Miotto, 2006).

Insights into the mechanism by which Chm supports JNK target gene transcription were initially obtained from observations made in the developing animal. (1) Chm improves the activation of a lexA-acZ reporter by the LexA-DFos fusion protein, revealing that Chm stimulates the transactivating potential of DFos when it is tethered to a promoter. (2) The thorax cleft phenotype caused by DFos^NAla, a protein deficient for the JNK phosophorylation sites, is not rescued upon simultaneous overexpression of Chm. Since the DFos^NAla variant recruits Chm as efficiently as the wild-type protein, this suggests that transcriptional improvement by Chm requires DFos phosphorylation, providing an attractive link between Chm function and JNK signaling (Miotto, 2006).

Study of the mode of action of the antagonistic cofactors Chm and DRpd3 in cultured cells has provided further insights into a chromatin-based mechanism that executes a modulation of the transcriptional response to JNK signaling. The following model is proposed, based on the dynamics of cofactor recruitment and activity, chromatin modification and transcriptional status related to reversible activation of the pathway. In response to JNK signaling, Chm HAT activity sets up a histone modification pattern that is instructive for transcriptional enhancement. Consistent with this notion Chm acetylates H4, with a marked preference for K16, and facilitates H3K4 trimethylation. However, AP-1 likely also engages HATs of different substrate specificity, since H4K8 acetylation, a modification required for the recruitment of the SWI/SNF-activating complex, is directed by DFos in the absence of Chm. After JNK signaling has ceased, DRpd3 gets recruited to promoters occupied by unphosphorylated DFos and counteracts the effects of Chm by reversing histone modifications, which results in transcriptional down-regulation. Strikingly, the recruitment of DRpd3 seems not to result from the displacement of Chm from the promoter, since invariant levels of Chm are associated with the promoter in sorbitol experiments, whereas DRpd3 starts to be recruited only once the signal has been eliminated. Thus, as opposed to an exchange of a HAT coactivator complex for an HDAC corepressor complex, which occurs for instance between Pcaf/NF-kappab and DRpd3/AP-1 complexes at the attacin promoter (Kim, 2005), a complex containing both Chm and DRpd3 could then form at the target promoter whose activity changes the histone modification pattern back to a pattern less permissive to transcription. Thus, DRpd3 most likely functions during a transient phase from a transcriptionally active to silent status. Its absence from the promoter at the inactive steady state in nonstimulated cells, suggests that unphosphorylated DFos then lies in a conformational environment that prevents DRpd3 recruitment by the ZIP domain (Miotto, 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}faf^EP381, 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}faf^EP381. 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}faf^EP381 in suppressing UAS-Aplip1-induced axonal accumulation of synaptic proteins. Equivalent expression of a 'kinase-dead' mutant transgene (UAS-wnd^KD) 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, the 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).

Molecular identification and functional characterization of a Drosophila dual-specificity phosphatase DMKP-4 which is involved in PGN-induced activation of the JNK pathway

MAP (Mitogen-activated protein) kinases play an important role in regulating many critical cellular processes. The inactivation of MAP kinases is always accomplished by a family of dual-specificity phosphatases, termed MAPK phosphatases (MKPs). This study identified a novel MKP-like protein, designated DMKP-4, from the Drosophila genome. DMKP-4 is a protein of 387 amino acids, with a dual-specificity phosphatase (DSP) catalytic domain. Recombinant protein DMKP-4 retains intrinsic phosphatase activity against chromogenic substrate pNPP. Overexpression of DMKP-4 inhibited the activation of ERK, JNK and p38 by H₂O₂, sorbitol and heat shock in HEK293-T cells, and JNK activation in Drosophila S2* cells under PGN (Gram-negative diaminopinelic acid-type peptidoglycan) stimuli. 'Knockdown' of DMKP-4 expression by RNAi significantly enhanced the PGN-stimulated activation of JNK, but not ERK nor p38. Further study revealed that DMKP-4 interacted specifically with JNK via its DSP domain. Mutation of Cys-126 to serine in the DSP domain of DMKP-4 not only eliminated its interaction with JNK, but also markedly reduced its phosphatase activity. Thus, DMKP-4 is a Drosophila homologue of mammalian MKPs, and may play important roles in the regulation of various developmental processes (Sun, 2008).

Activation of JNK signaling mediates amyloid-β-dependent cell death

Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimer's neuropathology is the generation of large aggregates of Aβ42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood. This study employed a Drosophila eye model of AD to study how Aβ42 causes cell death. Misexpression of higher levels of Aβ42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. Blocking caspase-dependent cell death initially blocked cell death but did not lead to a significant rescue in the adult eye. However, blocking the levels of c-Jun NH(2)-terminal kinase (JNK) signaling pathway significantly rescued the neurodegeneration phenotype of Aβ42 misexpression both in eye imaginal disc as well as the adult eye. Misexpression of Aβ42 induced transcriptional upregulation of puckered (puc), a downstream target and functional read out of JNK signaling. Moreover, a three-fold increase in phospho-Jun (activated Jun) protein levels was seen in Aβ42 retina as compared to the wild-type retina. When both caspases and JNK signaling were blocked simultaneously in the fly retina, the rescue of the neurodegenerative phenotype is comparable to that caused by blocking JNK signaling pathway alone. These data suggests that (1) accumulation of Aβ42 plaques induces JNK signaling in neurons and (2) induction of JNK contributes to Aβ42 mediated cell death. Therefore, inappropriate JNK activation may indeed be relevant to the AD neuropathology, thus making JNK a key target for AD therapies (Tare, 2011).

One of the characteristic features of neurodegenerative disorders like AD and Parkinson disease (PD) is the late onset of neuropathology due to aberrant cellular homeostasis probably due to misregulation of several signaling pathways involved in growth, patterning and survival. Thus, it is apparent that these neurodegenerative disorders are not due to a single gene mutation but a cumulative outcome of impairment of a large spectrum of signaling pathways. Therefore, in order to understand the complexity of the human disorders and to develop therapeutic approaches, it is important to discern the role of various signaling pathways in the neuropathology caused by Aβ42-plaques. This evident complexity is one of the reasons why neurodegenerative diseases are so difficult to understand and treat. The goal of this study was to tease out the role of the cell death pathways in Aβ42 neurotoxicity. It has been known for some time that high levels of Aβ42 result in small and disorganized phenotypes of eyes that contain thin retinas with poorly differentiated photoreceptors. This small eye suggests that Aβ42 induces extensive cell death in the developing eye. To understand when the cell death occurs, how the maturation of photorecepotors is affected by the presence of Aβ42 was studied (Tare, 2011).

The highly versatile model of Drosophila eye was employed to understand the role of signaling pathways involved in cell death in Aβ42-plaque mediated neuropathology. Since the eye is dispensible for the survival of fly, the transgenic Drosophila eye model is ideal for these studies since it is possible to assay the effects throughout eye development without killing the fly. The data suggest that neurodegeneration in the fly retina can be triggered as early as third instar eye imaginal disc using GMR-Gal4 driver mediated misexpression of Aβ42 (GMR>Aβ42), which is only a few hours after Aβ42 expression starts in the developing eye field. It was also found that even though cell death is induced as early as the third instar eye imaginal disc, the morphology of the developing eye field does not dramatically differ between the wild type eye versus the GMR>Aβ42. At this time the toxicity of Aβ42 is only apparent at the level of cell membranes, which shows minor effects on cell arrangement. However, the number of the dying cells shows dramatic increase in GMR>Aβ42 eye imaginal disc as compared to the wild-type eye imaginal disc. Thus, genetic programming that triggers the onset of Aβ42-plaque mediated neurodegeneration is activated soon after the onset of misexpression of Aβ42 in the developing retina. Therefore, the experiments to demonstrate rescue of neurodegeneration phenotype should take this time window into consideration (Tare, 2011).

The larval eye imaginal disc metamorphose into the prepupal retina, which shows clumping of photoreceptor clusters, an indication that photoreceptor specification and signaling are aberrant. The clumping phenotype is caused by fusion of photopreceptor neurons and results in loss of ommatidial cluster integrity. Despite these changes at the photoreceptor neurons level, the outline of the pupal retina shows subtle effects. In the late pupal retina, the size of the retina begins to reduce as the severity of the phenotypes increases at this stage. In the late pupal stage, the retina contains holes due to loss of photoreceptors. The outcome of this cellular aberrations in the eye leads to a small adult eye with glazed appearance and fused ommatidia. Thus, extensive cell death is responsible for some of the phenotypes observed in the adult eye expressing Aβ42. Not surprisingly, the neurodegenerative phenotypes exhibited by Aβ42-plaque are age and dose dependent. Since the Gal4-UAS system is temperature sensitive, it serves as an excellent source to test the dose dependence. The cultures reared at 25°C showed less severe phenotypes as compared to the ones reared at 29°C. Furthermore, the severity of phenotypes increased with the age (Tare, 2011).

It was asked which pathways mediate the extensive cell death induced by Aβ42. The idea was to test the caspase-dependent pathway since the majority of cell death is triggered by activation of caspase-dependent cell death in tissues. To demonstrate the role of caspases in Aβ42-mediated cell death, it was shown that the misexpression of baculovirus P35 protein, significantly reduce the number of TUNEL-positive cells in the larval eye disc. Interestingly, unlike the larval eye disc, the adult eyes did not show comparable strong rescues. It seems there is block in cell death mainly during the larval eye imaginal disc development but the adult eye exhibits a weaker rescue of GMR>Aβ42 neurodegenerative phenotype. This reduction in cell death supports the possible role of caspase-mediated cell death in the small eye induced by Aβ42. However, the eye of GMR>Aβ42+P35 is reduced and disorganized (partial rescue), suggesting that other pathways contribute to Aβ42 neurotoxicity in the eye (Tare, 2011).

JNK-mediated caspase-independent cell death also plays an important role in tissue homeostasis during development. JNK signaling, a family of multifunctional signaling molecules, is activated in response to a range of cellular stress signals and is a potent inducer of cell death. Consistent with this, Aβ42 activates JNK signaling in the eye imaginal disc as indicated by the transcriptional regulation of puc and Jun phosphorylation. Moreover, JNK signaling upregulation increases cell death, supporting the role of JNK in Aβ42 neurotoxicity. Conversely, blocking JNK signaling dramatically reduces cell death in larval eye imaginal disc and the resulting flies from blocking JNK signaling exhibit large and well organized eyes. Thus, it was possible to identify the JNK signaling pathway as a major contributor to cell death observed in the Aβ42 eyes. These studies also highlight that cell death response to misexpression of Aβ42-plaques is way earlier before its affect can be discernible at the morphological level. Since neurons are post-mitotic cells, they can not be replaced. Therefore, early detection of the onset of neurodegeneration is crucial. If the disease is detected later, it may only be possible to block the further loss of healthy neurons. However, the neurons lost prior to block of cell death will not be replaced. It is possible that JNK signaling activation may serve as an early bio-marker for Aβ42 plaque mediated neuropathology. Thus, members of JNK signaling pathway can serve as excellent biomarkers or targets for the therapeutic approaches (Tare, 2011).

Blocking JNK signaling significantly rescued the neurodegenerative phenotypes but the eyes still show subtle signs of Aβ42 in the disorganization of the lattice. Therefore, both caspase dependent cell death and JNK signaling were blocked in fly retina misexpressing Aβ42. Blocking both caspase and JNK pathways simultaneously produced the protection against Aβ42, suggesting that Aβ42 induces cell death by several mechanisms. The results suggest that blocking multiple pathways may result in significant protection against Aβ42 neurotoxicity, an important consideration for potential AD therapies (Tare, 2011).

JNK signaling pathway has been known to be involved in different processes of ageing and development, including tissue homeostasis, cell proliferation, cell survival and innate immune response. Interestingly, evidence collected in several models of AD supports the involvement of JNK signaling in AD. Consistent with the observations of this study, Aβ42 induces JNK activation in primary cultures of rat cortical neurons. Also, the kinase activity of JNK phosphorylates Tau in vitro, thus contributing to the production of hyperphosphorylated Tau, one of the key toxic molecules in AD. Moreover, inhibition of JNK with peptides prevented cell loss in an Tg2576; PS1^M146L brain slice model. Additionally, it has been shown that the neuroprotective effect of the diabetes drug rosiglitazone inhibits JNK and results in reduced Tau phosphorylation in rats and mice. The current results support these findings in mammalian models of AD, and provide the first evidence that direct manipulation of JNK activity modulates Aβ42 neurotoxicity in vivo. Despite this evidence, JNK is currently not a major pathway in AD research. These results suggest that more attention should be paid to the role of JNK in AD pathogenesis and its potential as a therapeutic target and biomarker. In fact, the protective activity of JNK may not be limited to AD, as JNK inhibition may show beneficial effects in other diseases, including PD, stroke and others (Tare, 2011).

Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but understanding of the in vivo function and regulation of these enzymes is limited. This study investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42^RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described in this study identifies crucial roles for Tap42 phosphatase complexes in governing imaginal disc and fly development (Wang, 2012).

Understanding about the in vivo function of α4/Tap42, especially in development, is limited in part because global knockout of this gene in mice and flies leads to early embryonic death (see Cygnar, 2005 and Kong, 2004). Cellular studies have also revealed that depletion of α4/Tap42 causes death in embryonic stem cells, mouse embryonic fibroblasts, adipocytes, hepatocytes, B and T cells of the spleen and thymus, and Drosophila S2 cells (Bielinski, 2007; Kong, 2004; Yamashita, 2006). Although studies of a conditional (Cre-LoxP) α4 knockout in mouse hepatocytes and a mosaic assay of Tap42 in Drosophila wing disc have provided insights into the cellular biology of α4 and Tap42 (Cygnar, 2005; Kong, 2004), the impact of these gene products on the development of tissues and host have not yet been described. This report utilized Tap42-targeted RNAi and the Gal4/UAS system to investigate the biological effects of silencing Tap42 expression in specific Drosophila tissues. Suppressing the Tap42 gene using two tissue-specific drivers (pnr-Gal4 and ap-Gal4) led to a pleiotropic fly phenotype, which included major deformities in the adult thorax and wings as well as decreased survival rates. The experimental platform described in this study has allowed exploration of the role of Tap42 and Tap42-regulated phosphatases in the control of cellular signaling, tissue development, and Drosophila viability (Wang, 2012).

Analyses of Tap42^RNAi wing discs revealed significant alterations in multiple signal transduction pathways including JNK, DPP, and HH. Marked increases in p-JNK signals were found in ap-Gal4>Tap42^RNAi wing discs. This observation, together with previous studies showing increased c-Jun phosphorylation in α4-null mouse embryonic fibroblasts (Kong, 2004) and activated JNK in Tap42-depleted clones of fly wing discs (Cygnar, 2005), indicate that α4/Tap42 likely plays a negative role in regulation of JNK signaling. Silencing the Tap42 gene in the ap gene domain also changed DPP and HH signaling in the wing discs. Although ap-Gal4-mediated silencing of Tap42 had a profound effect on JNK, DPP, and HH signaling, these pathways were unaffected in pnr-Gal4>Tap42^RNAi wing discs, thus demonstrating that the thorax cleft phenotype seen in the pnr-Gal4>Tap42^RNAi flies is not due to alterations in these pathways. Collectively, these findings indicate that Tap42 plays a crucial role in the modulation of JNK, DPP, and HH signaling, but the effects of Tap42 on these pathways appear to play a minimal role in normal thorax development (Wang, 2012).

The HH pathway is one of the major guiding signals for imaginal disc development. Recent investigations have revealed that the phosphorylation state of Ci and Smo, two components of the HH signaling pathway, are controlled by Drosophila PP2A (Mts) and PP4 (Jia, 2009). Additional studies implicate a role for specific Mts complexes in the control of HH signaling, whereby holoenzyme forms of Mts containing the Wdb and Tws regulatory B subunits act at the level of Smo and Ci, respectively (Su, 2011). Together, these findings point to key roles for Mts and PP4 in HH signaling and suggest that a common subunit of these phosphatases, namely Tap42, may also be involved in HH signaling. Indeed, the current data clearly show that Tap42 plays an important regulatory role in this pathway as silencing of Tap42 within the wing discs leads to an elimination of both Smo and Ci expression. Although the precise role(s) of Tap42 in the control of HH signaling remains unclear, it likely involves Tap42-dependent regulation of one or more phosphatase catalytic subunits (e.g., Mts, PP4, and possibly PPV) or specific holoenzymes forms of these phosphatases (e.g., Wdb/Mts, Tws/Mts). The pleiotropic effects of Tap42^RNAi on JNK, DPP, and HH signaling could be due to loss of Tap42's regulation of phosphatase activity, cellular levels, holoenzyme assembly, or subcellular localization (Wang, 2012).

Depletion of α4 in mouse embryonic fibroblasts caused an increase in phosphorylation of a variety of established PP2A substrates, which was attributed to a 'generalized defect in PP2A activity.' Instead of the expected unidirectional increase in protein phosphorylation, the current findings demonstrate a dual role for Tap42 in the control of JNK activation as hyperphosphorylation and hypophosphorylation of JNK were observed in the dorsal and ventral sides of the Tap42^RNAi wing disc, respectively, relative to control wing discs. Silencing of Tap42 in the ap domain also impacted DPP in a bi-directional fashion; these flies exhibited significantly decreased DPP expression in the scutellum but augmented expression around the wing blade. Consistent with previous studies showing that PP2A functions at different levels within the Ras1 and HH pathways, the current data indicate that Tap42-regulated phosphatases likely target multiple substrates within the JNK and DPP pathways in different regions of wing discs (Wang, 2012).

Close examination of the PE cells in the wing disc revealed that Tap42 expression occurs in only a fraction of these cells. It is noteworthy that the majority of Tap42 localized in rows of cells delineating the PE/DP (peripodial epithelium/disc proper) boundary. These cells are commonly referred to as 'medial edge' cells, which represent a subpopulation of PE cells that play a crucial role in thorax closure during metamorphosis. Interestingly, α4-PP2A complexes appear to play a major role in the control of cell spreading, migration, and cytoskeletal architecture, presumably via their ability to modulate the activity of the small G-protein Rac. Yeast Tap42 has also been implicated in the cell cycle-dependent and polarized distribution of actin via a Rho GTPase-dependent mechanism. Therefore, it is hypothesized that the wing disc structural deformities and thorax cleft phenotype of Tap42^RNAi flies are a result of unregulated phosphatases leading to defective spreading and migration of the medial edge cells during metamorphosis. The thorax cleft phenotype provides an opportunity to delineate the precise roles of Tap42-phosphatase complexes in processes controlling thoracic closure (e.g., cell spreading and migration) (Wang, 2012).

α4/Tap42 appears to function as an essential anti-apoptotic factor as cells lacking this common regulatory subunit of PP2A family members undergo rapid death. These studies implicate a role for α4/Tap42-dependent regulation of PP2A-like enzymes, and presumably the phosphorylation state of multiple pro- and anti-apoptotic proteins, in the maintenance of cell survival. The current findings reveal that silencing Tap42 in wing discs triggers apoptosis, thus providing supportive in vivo evidence that depletion of Tap42 (α4) leads to deregulated phosphatase action, which switches these enzymes from pro-survival to pro-apoptotic mediators. Because JNK activation is a hallmark feature of apoptosis, the overlap of apoptotic cells and hyperphosphorylated JNK indicates that the Tap42^RNAi-induced apoptosis may be dependent on JNK activation (Wang, 2012).

Since α4 is required for maintaining the normal function of PP2A, PP4, and PP6, it is suspected that misregulation of these phosphatases could be responsible for the pleiotrophic phenotypes observed in Tap42^RNAi flies. Consistent with this idea, introduction of the mts^XE2258 heterozygous allele into ap-Gal4>UAS-Tap42^RNAi flies partially rescued the thorax and wing defects, and significantly improved fly survival rates. The partial rescue by mts^XE2258 suggests that the defects seen in the Tap42^RNAi flies are due, in part, to unregulated Mts activity, possibly as a result of increased Mts levels or enzymatic activity. Indeed, previous studies have demonstrated an accumulation of Mts in Tap42-depleted clones of the fly wing disc. Thus, mts^XE2258 appears to function as a mild mutant that partially restores misregulated Mts function following depletion of Tap42. However, given the biochemical findings showing that Tap42 also interacts with PP4 and PPV, additional studies will be needed to determine the relative contribution of these phosphatases to the Tap42^RNAi-induced defects (Wang, 2012).

The phenotypes observed in flies expressing Tap42^RNAi could also be attributed to loss of a phosphatase-independent function(s) of Tap42 that controls normal fly development. However, introduction of a phosphatase binding-defective mutant of Tap42 (Tap42^ED) into the Tap42^RNAi background failed to rescue the phenotypes and lethality associated with Tap42 depletion. In contrast to Tap42^ED, introduction of Tap42^WT fully rescued the phenotypes and lethality of the Tap42^RNAi flies. These findings indicate that the Tap42^RNAi-induced phenotypes are entirely due to the impaired interactions between Tap42 and PP2A family members, and provide compelling support for the hypothesis that Tap42-dependent regulation of the functions of these enzymes is crucial for normal wing disc development and Drosophila viability (Wang, 2012).

Although understanding the exact molecular mechanisms underlying Tap42's regulation of PP2A family members is still incomplete, these studies clearly demonstrate that Tap42-phosphatase interactions play crucial roles in the control of multiple signaling pathways governing cell growth and survival. The experimental platform described in this report will undoubtedly serve as a valuable system to further explore the in vivo function and regulation of Tap42-phosphatase complexes. Furthermore, given the remarkable phenotypes seen in the Tap42^RNAi flies (e.g., thorax cleft and deformed wings), it is anticipated that this model system will drive future studies (e.g., phenotype-based suppressor/enhancer screens) aimed at identifying direct targets of Tap42-regulated phosphatases, as well as additional pathways under the control of these phosphatase complexes (Wang, 2012).

De-regulation of JNK and JAK/STAT signaling in ESCRT-II mutant tissues cooperatively contributes to neoplastic tumorigenesis

Multiple genes involved in endocytosis and endosomal protein trafficking in Drosophila have been shown to function as neoplastic tumor suppressor genes (nTSGs), including Endosomal Sorting Complex Required for Transport-II (ESCRT-II) components vacuolar protein sorting 22 (vps22), vps25, and vps36. However, most studies of endocytic nTSGs have been done in mosaic tissues containing both mutant and non-mutant populations of cells, and interactions among mutant and non-mutant cells greatly influence the final phenotype. Thus, the true autonomous phenotype of tissues mutant for endocytic nTSGs remains unclear. This study shows that tissues predominantly mutant for ESCRT-II components display characteristics of neoplastic transformation and then undergo apoptosis. These neoplastic tissues show upregulation of JNK, Notch, and JAK/STAT signaling. Significantly, while inhibition of JNK signaling in mutant tissues partially inhibits proliferation, inhibition of JAK/STAT signaling rescues other aspects of the neoplastic phenotype. This is the first rigorous study of tissues predominantly mutant for endocytic nTSGs and provides clear evidence for cooperation among de-regulated signaling pathways leading to tumorigenesis (Woodfield, 2013).

While it is well established how de-regulated signaling pathways in ESCRT-II mutant clones mediate non-cell autonomous interactions with neighboring non-mutant cells to contribute to hyperplastic overgrowth and increased cell survival, it was largely unknown which signaling pathways trigger neoplastic transformation autonomously. To address this question, predominantly mutant eye-antennal imaginal discs were generated in which competitive interactions are eliminated so that it was possible to examine the autonomous results of de-regulated signaling (Woodfield, 2013).

Overall, it appears that the same signaling pathways that are induced in mosaic clones are also activated in predominantly mutant tissues. However, two results of this study are noteworthy. First, it is surprising that JNK activity is strongly induced in tissues predominantly mutant for ESCRT-II genes. This is surprising because JNK signaling was believed to be induced by cell competition from neighboring non-mutant cells in mosaic tissues. However, non-mutant tissue is largely eliminated by the ey-FLP/cl method and thus competitive interactions are eliminated. Therefore, it is not known how JNK signaling is induced in these tissues. Nevertheless, JNK signaling is critical for the overgrowth phenotype of predominantly ESCRT-II mutant eye discs as inhibition of this pathway partially blocks cell proliferation. Second, de-regulation of the JAK/STAT signaling pathway is critical for the neoplastic transformation of vps22 mutant discs. Loss of JAK/STAT signaling dramatically normalizes the neoplastic phenotype of vps22 mutant cells. In addition to JNK and JAK/STAT activity, Notch activity was also found to be increased in discs predominantly mutant for ESCRT-II genes. Therefore, a genetic requirement of Notch signaling was tested for neoplastic transformation of ESCRT-II mutant cells. However, loss of Notch was inconclusive because even the wild-type control discs did not grow when Notch was inhibited (Woodfield, 2013).

Interestingly, although ESCRT-II mutant tissues undergo neoplastic transformation, they also show high levels of apoptosis. Animals with predominantly mutant eye-antennal imaginal discs die as headless pharate pupae, a phenotype likely caused by the apoptosis of the imaginal discs before the adult stage. Reduction of JNK signaling in vps22, vps25, or vps36 mutant discs leads to lower levels of apoptosis, supporting a role for JNK signaling in the cell death of the predominantly mutant tissues. More excitingly, JNK also controls proliferation in these tissues, as shown by the reduction of proliferation seen when JNK signaling was down-regulated. This observation is consistent with previous findings that JNK can induce non-cell autonomous proliferation and that apoptosis-induced proliferation is mediated by JNK activity. While inhibition of JNK signaling reduces proliferation in predominantly mutant ESCRT-II mutant discs, it does not affect other aspects of the neoplastic phenotype (Woodfield, 2013).

The role of JAK/STAT signaling in these mutants is complex. In mutant clones of ESCRT-II mosaic discs, Notch-induced secretion of the JAK/STAT ligand Upd triggers non-cell autonomous proliferation. However, autonomous de-regulated JAK/STAT signaling observed in predominately mutant discs is critical for the neoplastic transformation of vps22 mutants. In vps22 Stat92E double mutant discs, organization of cellular architecture is definitively rescued with the layout of the tissue closely resembling that of a wild-type eye-antennal imaginal disc. In addition, apical-basal polarity markers are localized more-or-less correctly in these tissues, indicating that epithelial polarity is more intact. Finally, differentiation in the posterior portion of the eye disc is preserved when JAK/STAT signaling is inhibited. Thus, de-regulation of JAK/STAT signaling in vps22 mutant discs contributes to the cellular disorganization and the lack of differentiation seen in the tissues, which is consistent with a previous study that implicated JAK/STAT signaling in cell cycle control, cell size, and epithelial organization in tsg101 mutant tissues (Woodfield, 2013).

It was recently shown that cells with strong gain of JAK/STAT activity transform into supercompetitors and eliminate neighboring cells with normal JAK/STAT activity by cell competition. However, in mosaic discs, a supercompetitive behavior of ESCRT-II mutant cells has not been observed. In fact, these mutant cells are eliminated by apoptosis. Only if apoptosis is blocked in these cells, is a strong overgrowth phenotype with neoplastic characteristics observed. Thus, apoptosis can serve as a tumor suppressor mechanism to remove cells with potentially malignant JAK/STAT activity (Woodfield, 2013).

How endosomal trafficking specifically regulates JAK/STAT signaling and, thus, how blocking trafficking leads to increases in signaling pathway activity are interesting questions to answer in the future. It is possible that, like endocytic regulation of the Notch receptor, the endosomal pathway tightly regulates Domeless (Dome), the JAK/STAT pathway receptor. It has been shown previously that Dome is trafficked through the endocytic machinery and that this trafficking of Dome can affect the downstream output of the JAK/STAT signaling pathway. It is also possible that Notch-induced Upd secretion causes autocrine JAK/STAT signaling in these mutants. However, technical problems (knocking down Notch function both in wild-type and mutant tissue causes general problems in tissue growth) prevented examination of this possibility (Woodfield, 2013).

It will be important to examine how de-regulated JAK/STAT signaling in ESCRT-II mutants causes neoplastic transformation. JAK/STAT signaling is known to be an oncogenic pathway in Drosophila and in humans but its downstream targets that promote tumorigenesis are not yet clear. JAK/STAT signaling may be feeding into other pathways that promote tumorigenesis, such as dpp signaling, or may be targeting other proteins involved in transformation, such as Cyclin D (Woodfield, 2013).

A number of studies have implicated genes that function in endocytosis and endosomal protein sorting as tumor suppressors in human cancers. Most well known is Tsg101, as early studies showed that downregulation of Tsg101 (see Drosophila TSG101) promotes the growth of mouse 3T3 fibroblasts in soft aga. When these cells were injected into nude mice, they formed metastatic tumors. However, later studies have shown conflicting results, and it is still unclear if Tsg101 functions as a tumor suppressor in metazoans. Importantly, a number of studies have shown changes in expression of ESCRT components in human cancer cells, including changes in expression of ESCRT-I components Tsg101 and Vps37A and ESCRT-III components Chmp1A and CHMP3. Since the primary proteins that function in endocytosis and endosomal trafficking are conserved from yeast to humans, it is likely that these findings in Drosophila may have important implications for human disease (Woodfield, 2013).

Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction

This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment (Wong, 2015).

Mucolipidosis type IV (MLIV) and Batten disease are untreatable lysosomal storage diseases (LSDs) that cause childhood neurodegeneration. MLIV arises from loss-of-function mutations in the gene encoding TRPML1, an endolysosomal cation channel belonging to the TRP superfamily. The absence of TRPML1 leads to defective lysosomal storage and autophagy, mitochondrial damage, and macromolecular aggregation, which together initiate the protracted neurodegeneration observed in MLIV). Batten disease arises from the absence of a lysosomal protein, CLN3), and results in psychomotor retardation. Both diseases cause early alterations in neuronal function. For instance, brain imaging studies revealed that MLIV and Batten patients display diminished axonal development in the cortex and corpus callosum, the causes of which remain unknown (Wong, 2015).

To better understand the etiology of MLIV in a genetically tractable model, flies were generated lacking the TRPML1 ortholog. The trpml-deficient (trpml¹) flies have led to insight into the mechanisms of neurodegeneration and lysosomal storage (Wong, 2015).

This study reports that trpml¹ larvae exhibit diminished synaptic growth at the NMJ, a well-studied model synapse. Lysosomal function supports Rag GTPases and MTORC1 activation, and this is essential for JNK phosphorylation and synapse development (Wong, 2015).

Drosophila larvae and mice lacking CLN3 also exhibit diminished Rag/ MTORC1 and JNK activation, suggesting that alterations in neuronal signaling are similar in different LSDs and are evolution- arily conserved. Interestingly, the NMJ defects in the two fly LSD models were suppressed by the administration of a high-protein diet and a drug that is currently in clinical trials to treat certain forms of cancer. These findings inform a pharmacotherapeutic strategy that may suppress the neurodevelopmental defects observed in LSD patients (Wong, 2015).

This study shows that lysosomal dysfunction in Drosophila MNs results in diminished bouton numbers at the larval NMJ. Evidence is presented that lysosomal dysfunction results in decreased activation of the amino-acid-responsive cascade involving Rag/MTORC1, which are critical for normal NMJ development (Wong, 2015).

Despite the requirement for MTORC1 in NMJ synapse development, previous studies and the current findings show that bouton numbers are independent of S6K and 4E-BP1. Rather, MTORC1 promotes NMJ growth via a MAP kinase cascade culminating in JNK activation. Therefore, decreasing lysosomal function or Rag/MTORC1 activation in hiw^ND8 suppressed the associated synaptic overgrowth. However, the 'small-bouton' phenotype of hiw^ND8 was independent of MTORC1. Thus, MTORC1 is required for JNK-dependent regulation of bouton numbers, whereas bouton morphology is independent of MTORC1. Furthermore, although both rheb expression and hiw loss result in Wnd-dependent elevation in bouton numbers, the supernumerary boutons in each case show distinct morphological features. Additional studies are needed for deciphering the complex interplay between MTORC1-JNK in regulating the NMJ morphology (Wong, 2015).

Biochemical analyses revealed that both JNK phosphorylation and its transcriptional output correlated with the activity of MTORC1, which are consistent with prior observations that cln³ overexpression promotes JNK activation and that tsc1/tsc2 deletion in flies result in increased JNK-dependent transcription. These findings point to the remarkable versatility of MTORC1 in controlling both protein trans lation and gene transcription (Wong, 2015).

Using an in vitro kinase assay, this study demonstrates that Wnd is a target of MTORC1. Because axonal injury activates both MTORC1 and DLK/JNK, these findings imply a functional connection between these two pathways. Interestingly, the data also suggest that MTORC1 contains additional kinases besides MTOR that can phosphorylate Wnd. One possibility is that ULK1/Atg1, which associates with MTORC1, could be the kinase that phosphorylates Wnd. Consistent with this notion, overexpression of Atg1 in the Drosophila neurons has been shown to promote JNK signaling and NMJ synapse overgrowth via Wnd) (Wong, 2015).

This study also found that developmental JNK activation in axonal tracts of the CC and pJNK levels in cortical neurons were compromised in a mouse model of Batten disease. Thus, the signaling deficits identified in Drosophila are also conserved in mammals. The activity of DLK (the mouse homolog of Wnd) and JNK signaling are critical for axonal development in the mouse CNS. Therefore, decreased neuronal JNK activation during development might underlie the thinning of the axonal tracts observed in many LSDs (Wong, 2015).

Although the findings of this study demonstrate a role for an amino-acid- responsive cascade in the synaptic defects associated with lysosomal dysfunction, simply elevating the dietary protein content was not sufficient to rescue these defects. These findings were reminiscent of an elegant study that showed that the growth of Drosophila neuroblasts is uncoupled from dietary amino acids owing to the function of ALK, which suppresses the uptake of amino acids into the neuroblasts (Cheng, 2011). Indeed, simultaneous administration of an ALK inhibitor and a high-protein diet partially rescued the synaptic growth defects associated with the lysosomal dysfunction, and improved the rescue of pupal lethality associated with trpml¹. Although these studies do not causally link the defects in synapse development with pupal lethality, they do raise the intriguing possibility that multiple phenotypes associated with LSDs could be targeted using ALK inhibitors along with a protein-rich diet (Wong, 2015).

Although LSDs result in lysosomal dysfunction throughout the body, neurons are exceptionally sensitive to these alterations. The cause for this sensitivity remains incompletely understood. Given the findings of this study that mature neurons do not efficiently take up amino acids from the extracellular medium, lysosomal degradation of proteins serves as a major source of free amino acids in these cells. Therefore, disruption of lysosomal degradation leads to severe shortage of free amino acids in neurons, regardless of the quantity of dietary proteins, thus explaining the exquisite sensitivity of neurons to lysosomal dysfunction (Wong, 2015).

A Drosophila ABC transporter regulates lifespan

MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. This study investigated the biological functions of a Drosophila homolog of human MRP4, dMRP4. dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, it was demonstrated that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. Impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling (Huang, 2014: PubMed).

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