InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

Misshapen functions upstream of Basket (also known as JNK) in a cascade of interactions relating to dorsal closure in the Drosophila embryo. Before taking a closer look at Misshapen, some background on dorsal closure is offered. Dorsal closure of the Drosophila embryo involves changes in cell shape leading to elongation and migration of the lateral epithelial sheets. This coordinated movement of the lateral epithelia functions to internalize the amnioserosa and connect the two sides of the embryo. An important feature of dorsal closure is the fact that changes in cell shape, sensed in the cytoplasm, trigger a signaling pathway that leads to nuclear changes, in particular the activation of the transcription factor DJun, more properly termed Jun related antigen (Jra). Jra, and its partner Fos related antigen (Fra) are known to target two genes during dorsal closure, dpp and puckered. Dpp may serve to relay signals that trigger cell shape changes (Riesgo-Escovar, 1997), and Fra expression in neighboring cells, while Puckered, a phosphatase, seems to act in a feedback loop (Martin-Blanco, 1998). Puckered expression is upregulated by DJun and in turn, Puckered inactivates the Jra activating kinase Basket, whose function is the activation of Jra (Riesgo-Escovar, 1996; Sluss et al. 1996). A kinase termed Misshapen acts upstream of Basket, in response to cytoskeletal changes, as a signal transducer that leads to the activation of Basket (Su, 1998).

Rac activation is thought to be important for stimulating dorsal closure because expression of dominant negative forms of Rac (DN Rac) or Cdc42 inhibit dorsal closure in the Drosophila embryo (Harden, 1995; Riesgo-Escovar, 1996). The finding that activated Jra rescues the defect in dorsal closure induced by expression of DN Rac indicates that Rac probably functions upstream of Basket/JNK activation to stimulate dorsal closure (Hou, 1997). But between Rac and Basket are a whole chain of kinases whose identities are only partially known. The immediate activator of Basket is Hemipterous, termed a MAPKK (read MAP kinase kinase), because Basket (a MAP kinase) is phosphorylated by Hemipterous. The activator of Hemipterous, which would be termed a MAPKKK, is unknown, but knowledge of kinase pathways suggests that one exists. Misshapen, the subject of this essay, is shown to lie between Rac and the unknown MAPKKK, thus qualifying Misshapen as a MAPKKKK. What kinds of MAPKKKK's have been found in other organisms, and how do they fit into a pathway leading to activation of the JNK?

Genetic epistasis analysis in yeast as well as studies in mammalian cells have indicated that Ste20 related kinases function upstream of MKKKs to regulate the JNK MAPK module: Ste20 kinases have been considered to be MAP kinase kinase kinase kinases (MKKKK). Two families of protein kinases that are closely related to Ste20 in their kinase domains have been identified based on their structure and regulation. The first family includes the mammalian and Drosophila p21-activated protein kinases (PAKs) (see Harden, 1996 for information on the Drosophila PAK). Kinases in this group contain a conserved p21Rac- and Cdc42-binding domain in their amino terminus and are activated by binding GTP-bound Cdc42 and Rac. The second family, to which Misshapen belongs, lacks p21Rac- and Cdc42-binding domains and is named for the yeast SPS1 protein kinase (Friesen, 1994). In contrast to PAKs that contain an amino-terminal regulatory and a carboxy-terminal kinase domain, members of this second family contain an amino-terminal kinase domain and a carboxy-terminal regulatory region. Several SPS1 family kinases have been identified in mammalian cells; these include mammalian Ste20-like kinase 1 (MST1) and MST2, germinal center kinase (GCK), NCK-interacting kinase (NIK), Ste20/oxidant stress response kinase (SOK), hematopoietic progenitor kinase (HPK1), and GCK-like kinase (GLK) (Creasy, 1995, Pombo, 1995, Hu, 1996 and Diener, 1997 and Su, 1997). The ability of several mammalian SPS1 family members, such as GCK, NIK, GLK, and HPK1, to activate JNK when overexpressed transiently in mammalian cells is the strongest evidence that these kinases might activate JNK in response to upstream signals. However, these studies may not be conclusive because JNK activation has been measured under conditions in which the Ste20 kinases are expressed at very high levels and therefore, could have nonphysiological effects. Because of the difficulty in studying Ste20 kinases in mammalian cells, placement of a Ste20 kinase on a genetic pathway in Drosophila would greatly facilitate an understanding of the normal physiological functions of these kinases (Su, 1998).

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 (Harden, 1995 and Riesgo-Escovar, 1996). 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 Msn 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 (Su, 1998).

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 (Teramoto, 1996; Fanger, 1997). 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).

It is intriguing that the C. elegans homolog of msn, mig-15, is also an essential gene in development and, like msn, functions to regulate processes that undoubtedly require changes in the cytoskeleton and cell shape in developing worms (E. Hedgecock, pers. comm. to Su, 1998). mig-15 mutants have a variety of developmental defects including defects in Q-neuroblast migration and muscle arm targeting. Although it is not yet clear whether any or all of the phenotypes apparent in worms lacking mig-15 are attributable to defective activation of the C. elegans JNK, these findings suggest a common theme in which JNK activation plays a central role in a variety of developmental processes by coordinating changes in cell shape and the cytoskeleton. It is likely, however, that some of the phenotypes observed in embryos lacking these Ste20 kinases are independent of their effect on JNK activation. In addition to defects in dorsal closure, some embryos mutant for msn display a ventral defect. Moreover, although msn, like bsk and Jra/Djun, is not required for specifying the fate of photoreceptor cells, clones of msn mutant photoreceptor cells display an abnormal shape (Riesgo-Escovar, 1996; Hou, 1997; Treisman, 1997). These defects are never observed in embryos mutant for bsk and therefore indicate that msn has other essential functions that are independent of JNK activation (Riesgo-Escovar, 1996). The findings reported by Su (1998) also support the idea that the regulation of Ste20 kinases in mammalian cells are likely to be more complex than previously recognized. Several mammalian Ste20 kinases related to msn and NIK that specifically activate the JNK pathway, such as GC kinase and HPK1, have been identified. It has not been clear whether the function served by these kinases is redundant or whether each may function only under specific circumstances. Although the full repertoire of Ste20 kinases in Drosophila is not known, it is clear that members of this family are subject to different modes of regulation and, for at least some functions, are not redundant with other family members. Studying msn and mig-15 in defined genetic systems will be a critical tool in the effort to unravel these complex pathways in mammalian cells (Su, 1998 and references).

REGULATION

Transcriptional Regulation

In the course of a screen designed to identify genes regulated by the photoreceptor transcription factor Glass, a set of lethal non-complementing P-element insertions mapping to 62E6-7 were isolated. Expression of lacZ from these insertions is completely Glass-dependent in photoreceptors. This gene has been named misshapen (Treisman, 1997).

Phosphorylation of Grainy head by ERK is essential for wound-dependent regeneration but not for development of an epidermal barrier

Grainy head (GRH) is a key transcription factor responsible for epidermal barrier formation and repair, whose function is highly conserved across diverse animal species. However, it is not known how GRH function is reactivated to repair differentiated epidermal barriers after wounding. This study shows that GRH is directly regulated by extracellular signal-regulated kinase (ERK) phosphorylation, which is required for wound-dependent expression of GRH target genes in epidermal cells. Serine 91 is the principal residue in GRH that is phosphorylated by ERK. Although mutations of the ERK phosphorylation sites in GRH do not impair its DNA binding function, the ERK sites in GRH are required to activate Dopa decarboxylase (Ddc) and misshapen (msn) epidermal wound enhancers as well as functional regeneration of an epidermal barrier upon wounding. This result indicates that the phosphorylation sites are essential for damaged epidermal barrier repair. However, GRH with mutant ERK phosphorylation sites can still promote barrier formation during embryonic epidermal development, suggesting that ERK sites are dispensable for the GRH function in establishing epidermal barrier integrity. These results provide mechanistic insight into how tissue repair can be initiated by posttranslational modification of a key transcription factor that normally mediates the developmental generation of that tissue (Kim, 2011).

Interestingly, putative ERK phosphorylation sites are also found in the N-terminal domain of Grhl3, a mammalian homolog of GRH. Given that grhl3 mutant mice display defects in both developmental skin barrier formation and wound-induced repair, and that ERK is required for mammalian wound repair, it is plausible that ERK phosphorylation of GRH might be an evolutionarily conserved event in animal epidermal wound repair. Drosophila GRH and mammalian Grhl proteins do not share extended blocks of amino acid sequence homology that include S-P or T-P motifs that are characteristic of ERK consensus sites, but functional phosphorylation sites can show rapid sequence drift during evolution (Kim, 2011).

Mutations of ERK phosphorylation site residues in GRH do not detectably influence its affinity to DNA binding sites in vitro, but the phosphorylation sites are required for GRH functional activity on epidermal wound enhancers in late embryonic epidermal cells. The wound-specific activity of GRH does not appear to involve phosphorylation-dependent nuclear localization, because the GRH protein is constitutively nuclear, either when expressed by endogenous promoters or epidermal GAL4 drivers. In addition, as measured by the strength of overexpressed ERK-site mutants of GRH in activating Ddc expression, the developmental transcriptional activation function of GRH was not significantly altered by the mutation of ERK phosphorylation sites. However, ERK phosphorylation of GRH-binding affinity might enhance its binding to a coactivator or prevent its binding to a corepressor that is specific during wound response. Because phosphomimetic GRH 2E expression did not constitutively activate transcription of GRH target genes in both developmental and wound response contexts, the phosphorylation of GRH alone is not likely to be sufficient for triggering activation of the wound response genes. Given that both GRH and FOS-D are required for the induction of Ddc and msn, and that FOS function can be also activated by ERK phosphorylation, it is believed that wound-induced signaling input through FOS or other transcription factors is also necessary for the transcription of these wound response genes along with the phosphorylation of GRH. Therefore, the ERK-dependent phosphorylation of both GRH and FOS upon injury may activate both transcription factors to synergistically induce many target genes that facilitate epidermal barrier regeneration (Kim, 2011).

Although GRH function in Drosophila embryonic epidermis is critical for both developmental generation and wound-triggered regeneration of epidermal barriers, it was not known whether it is required for the process of reepithelialization (epidermal wound closure) after wounding. Mammalian Grhl3 has been shown to be required for the keratinocyte wound closure in tissue culture, mainly through its activation of RhoGEF19. However, in Drosophila, it was found that the wound closure phenotypes between wild-type and grh^IM homozygous embryos were indistinguishable, indicating that Drosophila GRH is not critical for reepithelialization after wounding (Kim, 2011).

Given the importance of the ERK-GRH axis in transcriptional activation of epidermal wound response genes, the signals and receptors upstream of ERK are of great interest. Mammalian cell culture studies suggest that receptor tyrosine kinases (RTK) are responsible for the wound-dependent activation of ERK. In Drosophila, stitcher (stit) encodes a Ret-family tyrosine kinase that contributes to transmission of an epidermal wound signal, because null mutations in stit result in a partial inhibition of wound-induced ERK phosphorylation, and reduced activation of wound enhancers in embryonic epidermal cells (Wang, 2009). It is very probable that another RTK(s) are responsible for the activation of ERK after epidermal wounding observed in stit null mutant embryos. The PVR RTK is a good candidate because its function has been shown to be required for wound healing in larval epidermal tissue (Kim, 2011).

The RTK-mediated activation of ERK-GRH axis appears to mediate other biological roles in addition to its role in embryonic epidermal barrier repair. GRH has been reported to mediate Torso RTK-dependent repression of the tailless gene in early embryogenesis. Therefore, although the ERK-GRH axis is dispensable for late embryonic epidermal barrier development, it appears to function in other developmental contexts. These distinct functions presumably depend on the context of different transcriptional enhancers with different transcription factor codes (Kim, 2011).

More importantly, the current findings suggest an important mechanism that may underlie injury-induced tissue regeneration. After wounding or amputation, developmental programming must be reinitiated to recover the original structure of the damaged tissue. In the context of the epidermis, GRH is a key transcription factor for generating a normal epidermal barrier during development in a manner independent of ERK phosphorylation. However, GRH is also persistently expressed in terminally differentiated epidermal cells of the embryo, larvae, and adult, and its function in barrier repair must be rapidly and robustly reactivated after wounding. In the current model, a semidormant state of GRH can be overcome by ERK phosphorylation to regain its ability to transcriptionally activate target genes like Ddc, msn, and many others that regenerate epidermal barriers. This model may also apply to c-Jun in mammals, which does not require JNK-dependent phosphorylation sites for developmental eyelid and neural tube closure, but does require those sites for closure of epidermal wounds. Thus, some regeneration processes in tissues or organs of diverse animal species after injury may be initiated through a similar molecular mechanism-posttranslational reactivation of essential transcription factors that are normally involved in developmental morphogenesis (Kim, 2011).

Targets of Activity

To determine whether Msn activates Basket/JNK, cultured cells were transfected with msn together with an epitope-tagged JNK, and kinase activity assays were performed on JNK precipitates. Overexpression of either msn or mammalian NIK leads to about a four- to fivefold increase in JNK kinase activity as assessed by in vitro kinase reaction. In agreement with previous studies using NIK, a mutation abolishing Msn's kinase activity markedly reduces Msn's ability to activate JNK. The ability of Msn to activate JNK was confirmed by examining its effect on an activated transcription factor 2 (ATF2)-stimulated luciferase reporter gene; JNK has been shown to phosphorylate and activate ATF2. Overexpression of Msn in cultured cells leads to an approximately 10-fold increase in the transcriptional activity of ATF2. These findings indicate that msn and NIK are both structurally and functionally similar and suggests that msn may function to activate JNK in Drosophila (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)

The conserved Misshapen-Warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila

Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. This study shows that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP (Li, 2014).

Previous studies have shown that endothelial cells (ECs) produce regulatory factors in response to infection and damage and function as part of the niche to regulate intestinal stem cell (ISC)-mediated regeneration. Meanwhile, recent reports show that enteroblasts (EBs) can also produce growth factors including EGF receptor ligands, Wingless and Upd3, although the pathways that regulate their production are not known. The current results demonstrate that differentiating EBs also function as an important part of the niche to regulate ISC division via the Msn pathway. EB-specific knockdown of msn leads to highly increased Upd3 expression and midgut proliferation. A previous report suggests that undifferentiated EBs if remain in contact with the mother ISC can inhibit proliferation. Although the hyperproliferating midguts after loss of Msn contain many EBs, these EBs do go into normal differentiation and express high level of Upd3, which may overcome any inhibitory effect of undifferentiated EBs on ISC proliferation (Li, 2014).

Msn is known to regulate a number of biological processes. During embryonic dorsal closure the MAP kinase pathway Slipper-Hemipterous-JNK is downstream of Msn, and Slipper is able to bind to Msn in vitro. In the adult midgut, JNK is a mediator of aging-related intestinal dysplasia and is a stress-activated kinase in ECs to positively regulate ISC division. While the current RNAi experiments show that JNK has a function in EBs to negatively regulate ISC proliferation, this phenotype is not dependent on Upd3 or Yki. No change of JNK phosphorylation was detected after loss of Msn. Mammalian MAP4K4 has also been shown to function independently of JNK in some biological contexts. Therefore, Msn and JNK probably have independent functions in the midgut (Li, 2014).

This study has instead uncovered an interaction of Msn with Wts and subsequently regulation of Yki. Hpo-Wts-Yki has been demonstrated to have a function in ECs for stress and damage-induced response. Gal4 driven experiments have many caveats including cell-type specificity, differences in promoter strengths, and knockdown efficiency in different cell types. Nonetheless, the results of many parallel experiments that this study conducted strongly suggest that Msn and Hpo independently regulate Wts-Yki in EBs and ECs, respectively. How the Msn and Hpo pathways in the two cell types are coordinately regulated to produce an appropriate amount of Upd3 to achieve desirable intestinal growth under different circumstances remains an important question to be answered (Li, 2014).

Previous experiments in developing discs suggest that Wts and Yki but not Hpo act downstream of cytoskeleton regulators. Similarly, the mammalian Hpo homologs MST1/2 appear not to be involved in LATS regulation after cytoskeletal perturbation in some cell types. In vivo assay in midgut suggests a function for Msn, Yki and Upd3 downstream of actin capping proteins in EBs. Similarly, the Latrunculin B effect on MEFs suggests that MAP4K4 is required for cytoskeleton-regulated LATS and YAP phosphorylation. The situation in mammalian cells may be more complicated because the Msn/MAP4K4 subfamily also includes two other closely related kinases TNIK and MINK1. Proper regulation of Wts by the cytoskeleton may require both positive and negative regulators, because recent work in flies identified the LIM-domain protein Jub as a negative regulator of Wts in response to cytoskeletal tension. It will be interesting in future studies to determine how positive and negative regulators of Wts act in a coordinated manner to regulate cell fate and proliferation in response to cytoskeletal tension (Li, 2014).

Protein Interactions

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).

Recent studies suggest that the SH2/SH3 adaptor Dock/Nck transduces tyrosine phosphorylation signals to the actin cytoskeleton in regulating growth cone motility. The signaling cascade linking the action of Dock/Nck to the reorganization of cytoskeleton is poorly understood. Dock is shown to interact with the Ste20-like kinase Misshapen (Msn) in the Drosophila photoreceptor (R cell) growth cones. Loss of msn causes a failure of growth cones to stop at the target, a phenotype similar to loss of dock, whereas overexpression of msn induces pretarget growth cone termination. Physical and genetic interactions between Msn and Dock indicate a role for Msn in the Dock signaling pathway. It is proposed that Msn functions as a key controller of growth cone cytoskeleton in response to Dock-mediated signals (Ruan, 1999).

To investigate the potential role of msn in R cell growth cones, the effect of msn mutations on R cell projections was assessed. As strong loss-of-function alleles of msn are embryonic lethal, R cell projections were examined in third-instar larvae homozygous for a hypomorphic allele of msn [msn^l(3)03349]. While in msn mutants R cell growth cones are able to extend into the developing optic lobe, their innervation patterns within the lamina and the medulla are altered. The msn phenotype exhibits a certain similarity to that of dock loss-of-function mutants. In dock mutants, many R1-R6 growth cones pass over their normal target (i.e., lamina) and extend further into the medulla layer, generating gaps in the lamina R1-R6 termination site (a smooth continuous line of immunoreactivity in wild type). In addition, dock affects R cell fasciculation and growth cone morphology. Similarly, it has been found that loss of msn function causes defects in R cell targeting and fasciculation; gaps are observed in the R1-R6 termination site, coincident with projections of abnormal, large bundles into the medulla. R cell growth cone morphology is also altered in msn mutants. Unlike in dock, however, in msn, R cell growth cones are able to expand upon reaching the target. While all msn mutants examined exhibited defects in R cell innervation pattern, the severity of the phenotype varies from individual to individual (Ruan, 1999).

To determine whether msn is required in the developing eye for R cell projections, genetic mosaic analysis was carried out. Mutant eye clones homozygous for msn¹⁰², a strong loss of function allele, were generated in an otherwise wild-type fly by eye-specific mitotic recombination. R cell projections in mosaic larvae were visualized with mAb 24B10. Similar defects in R cell innervation pattern are observed. The percentage (~46%) of larvae showing obvious defects was close to the percentage (~50%) of individuals with relatively large mutant eye patches (identified as white eye tissue in adult). To specifically assess the role of Msn in R1-R6 growth cones, msn mutant eye patches were generated in msn heterozygous flies carrying the adult R1-R6-specific marker Rh1-lacZ. In wild-type adult flies, all R1-R6 axons terminate in the lamina, as assessed by lacZ staining. In contrast, in all mosaic adults examined, R1-R6 axons from msn mutant patches pass over the lamina and terminate abnormally in the medulla. These results indicate that msn, like dock, is genetically required in the eye for R1-R6 growth cone targeting. Similarly, no obvious defects are detected in the differentiation of the R1-R6 targeting region (i.e., lamina) in msn mutants, as assessed by anti-Dachshund staining. Moreover, eye-specific expression of a msn transgene rescues R cell projection defects in homozygous msn mutants (Ruan, 1999).

Dock protein is enriched in R cell axons and growth cones. If Msn has a functional relationship with Dock in R cell growth cones, it would be expected that Msn protein is expressed in R cells and is localized to growth cones. The expression pattern of Msn in third-instar larval eye-brain complexes was determined with a rabbit anti-Msn serum. Msn staining is seen in R cell axons along the path of projections (from the developing eye disc to the lamina) in wild-type whole-mount preparations. The lamina plexus is strongly stained as a continuous layer of immunoreactivity, a pattern that is indistinguishable from that stained with anti-Dock antibody. Since at this stage the vast majority of axonal processes in the lamina neuropil are expanded R1-R6 growth cones, the uniform staining of Msn and Dock in the lamina neuropil suggests strongly that Msn and Dock colocalize to R1-R6 growth cones.The localization of Msn in R1-R6 growth cones is consistent with a role for Msn in coordinating the response to target-derived signals (Ruan, 1999).

The fact that loss of msn caused the failure of R1-R6 growth cones to stop at their target lamina suggests a role for msn in the shutdown of growth cone motility when axons reach their target. Target-derived stop signals may activate Msn, which in turn coordinates cytoskeletal reorganization in decelerating growth cone motility. If this model is correct, one may predict that ectopic activation of Msn should induce abnormal termination of R cell growth cones. To test this, the endogenous msn gene was overexpressed in differentiating R cells using the eye-specific promoter GMR. Overexpression of Msn in R cell axons was confirmed by immunohistochemical staining. Compared with wild-type, overexpression of msn causes a large number of R1-R6 growth cones to stop before they reached their normal target lamina. Overexpression of msn also causes defects in the medulla terminal field. In contrast, neither the shape of R cells nor their localization on the developing eye disc is affected. Overexpression of msn from a transgene containing msn cDNA under control of the GMR promoter causes a similar early stop phenotype. The severity of the phenotype is dose dependent, because the increase in the copies of msn transgene enhanced the phenotype (Ruan, 1999).

One possible explanation for the gain-of-function phenotype is that overexpression of Msn activates the Msn pathway prematurely: this sends a terminating signal to growth cone cytoskeleton to induce pretarget termination. Alternatively, the hyperactivation of the Msn pathway may cause some general defects in the reorganization of growth cone cytoskeleton, leading to the arrest of growth cones before they reach the target. The former interpretation, that Msn plays an instructive role in terminating R1-R6 growth cones, is favored for the following reasons: (1) in msn gain-of-function mutants, the early stop growth cones expand, similar to growth cones that terminate correctly in the lamina. In wild type, R cell growth cones expand only when they terminate in the target; (2) the fact that the early stop R cell growth cones are still able to expand in the lamina argues against a general defect in the reorganization of growth cone cytoskeleton (Ruan, 1999).

To examine whether Msn interacts with Dock physically, a glutathione S-transferase (GST) fusion protein was generated containing a fragment of Msn that encompasses multiple consensus PXXP motifs for SH3 domain-binding. The immobilized GST-Msn fusion protein precipitates Dock from adult fly lysates in a dose-dependent manner, indicating the direct association of Msn with Dock. To test whether Msn associates with Dock in intact flies, coimmunoprecipitation experiments were carried out. Fly lysates were prepared from third-instar larval eye-brain complexes or adult heads. Anti-Dock antibody was used to precipitate Dock and its interacting proteins from the lysates. Msn protein is detected in anti-Dock precipitates but not in control serum precipitates, indicating an in vivo association of Msn with Dock in flies at both developmental and adult stages (Ruan, 1999).

To define the domains of Dock and Msn that mediate the binding, the yeast two-hybrid system was used to analyze their interactions. Consistent with binding experiments using GST-Msn fusion protein, the PXXP fragment of Msn binds to Dock in yeast. The binding of Dock to Msn is mediated mainly by its SH3-1 and SH3-2 domains. Mutations in either SH3-1 or SH3-2 inhibit the association of Dock with Msn, indicating that a stable association requires the simultaneous binding of SH3-1 and SH3-2 to the PXXP sequence in the polypeptide of Msn, whereas SH3-3 is less necessary for the binding (Ruan, 1999).

To determine the biological relevance of the physical association of Msn with Dock, a test was performed to see whether dock and msn interact genetically. The dosage of dock gene was reduced in larvae homozygous for the hypomorphic msn allele [msn^l(3)03349]. The reduction by half of dock gene dosage dramatically enhances the msn phenotype. The R1-R6 termination site at the lamina becomes more disorganized. R cell growth cones are much less expanded and appear more similar to those of dock mutants. This enhanced phenotype is completely penetrant. It is estimated that in each hemisphere, ~70%-100% of growth cones are less expanded, as compared with those in controls. In dock and msn double mutants, R cell projections are indistinguishable from those in dock mutants. These results, together with the physical association of Msn with Dock, strongly suggest that Msn and Dock function in the same signaling pathway controlling R cell projections (Ruan, 1999).

That Dock/Nck is capable of binding activated receptor tyrosine kinases via its SH2 domain, together with the above phenotypic analysis of dock and msn mutants, suggests that Msn is activated by Dock-mediated stop signals in terminating R1-R6 growth cones in the lamina. This model makes the simple prediction that gain of function in msn should suppress the R1-R6 nonstop phenotype in dock mutants. To assess this possibility, the endogenous msn gene was overexpressed in homozygous dock mutants. In dock mutants, the medulla layer is hyperinnervated, as many R1-R6 axons fail to stop at the lamina termination site. Overexpression of Msn in dock mutants largely suppresses the R1-R6 nonstop phenotype ; R cell axons in the medulla are dramatically reduced in all larvae examined. The fact that gain of function in msn is capable of terminating R1-R6 growth cones in dock null mutants is consistent with the prediction that Dock functions upstream of Msn activation in decelerating R1-R6 growth cone motility. Surprisingly, overexpression of msn in the absence of dock also causes the premature termination of many R cell growth cones within the optic stalk, a phenotype that is not observed in wild-type flies overexpressing msn. This result raises the intriguing possibility that Dock is also able to negatively regulate the function of Msn at certain stages of axonal projections (Ruan, 1999).

To further investigate the relationship between msn and dock in the control of growth cone motility, the effect of overexpressing Dock on the msn gain-of-function phenotype was examined. Dock was overproduced in R cells under control of the GMR promoter. In wild type, overexpression of Dock has no effect on R cell projections, suggesting that Dock is not rate limiting in the termination of growth cones. Overexpression of Dock in msn gain-of-function mutants largely suppresses the pretarget termination phenotype, confirming that Dock also negatively regulates the function of msn. SH3 mutants incapable of binding Msn in yeast either completely fail to suppress the phenotype or only weakly suppress the phenotype. In contrast, the SH3-3 mutant, displaying Msn-binding activity, suppresses the phenotype as efficiently as wild-type Dock. These results argue that the physical association of Dock with Msn is essential for the regulation of Msn by Dock. Interestingly, although the R336Q mutation (eliminating phosphotyrosine-binding activity of the SH2 domain) does not affect the binding of Dock to Msn, it completely abolishes the ability of Dock to suppress the msn gain-of-function phenotype. These data suggest that the negative regulation of Msn function by Dock involves an SH2-dependent tyrosine phosphorylation signal (Ruan, 1999).

It is proposed that Dock couples different signals to Msn at different stages of axonal projection. At an early stage, signals promoting growth cone extension may induce tyrosine phosphorylation on specific proteins (e.g., docking protein), which then recruit Msn through Dock (via the SH2 domain) to specific regions within the growth cone. Consequently, this may segregate Msn from its substrates, thus preventing the premature activation of the Msn pathway. In growth cones overexpressing Msn, however, excessive Msn that cannot be recruited by a limited amount of endogenous Dock may diffuse freely into certain regions to activate its (Msn's) substrates, which then induce pretarget growth cone termination. Similarly, the pretarget termination phenotype is enhanced by loss of dock and is suppressed by overexpression of dock. Once the growth cone reaches the target, upregulation of Msn may be accomplished in two steps through the combination of reducing the extension signal and increasing the stop signal. (1) The Dock-Msn complex needs to be released from those docking sites, which would be achieved by dephosphorylation through the activation of some protein tyrosine phosphatases. One such candidate is the receptor tyrosine phosphatase PTP69D, which has recently been shown to be required for the proper targeting of R1-R6 growth cones. (2) The stop signal activates the function of Msn through Dock by either positioning Msn close to its substrate or directly stimulating its activity, leading to the termination of the growth cone in the target. In the absence of Dock, endogenous Msn may not reach a threshold local concentration or activity required for growth cone termination. The observation that reduction of dock gene dosage enhances the hypomorphic msn loss-of-function phenotype is consistent with this view. While the above model fits with the results, understanding of the exact biochemical mechanism underlying the regulation of Msn by Dock awaits identification of upstream regulators of Dock in R cell growth cones (Ruan, 1999).

Recent studies suggest that Dock/Nck plays a highly conserved role in growth cone signaling. Nck can be recruited into signaling complexes in response to the activation of the vertebrate guidance receptors EphB1 and EphB2, two Eph receptor tyrosine kinase family members (see Drosophila Eph receptor tyrosine kinase). Moreover, Nck can functionally replace Dock in R cell growth cones. Furthermore, Dock, like Nck, is capable of binding ligand-activated EphB1. Given the extraordinary sequence conservation between Msn and NIK, it is highly likely that in vertebrate growth cones, NIK plays a similar role in response to Nck-mediated signals. Hence, the interaction between Dock/Nck and Msn/NIK may represent an evolutionarily conserved mechanism linking tyrosine phosphorylation to changes in growth cone behavior (Ruan, 1999 and references therein).

While extensive studies in several systems have made considerable progress in defining the general mechanisms that direct growth cone extension, much less is known of the mechanism that makes growth cones stop at a specific target layer underlying the formation of layer-specific connections. Misshapen (Msn) has been proposed to shut down Drosophila photoreceptor (R cell) growth cone motility in response to targeting signals linked to Msn by the SH2/SH3 adaptor protein Dock. To identify downstream targets of Msn in R cell growth cones, a genetic dissection was undertaken to search for second-site mutations that modify a Msn hyperactivation phenotype. bifocal (bif), a gene encoding a putative cytoskeletal regulator, shows strong interaction with msn. Bif binds to F-actin in vitro and colocalizes with F-actin during development. Bifocal is a component of the Msn pathway for regulating R cell growth cone targeting. Phenotypic analysis indicates a specific role for Bif to terminate R1-R6 growth cones. Biochemical studies show that Msn associates directly with Bif and phosphorylates Bif in vitro. Cell culture studies demonstrate that Msn interacts with Bif to regulate F-actin structure and filopodium formation. It is proposed that Bif functions downstream of Msn to reorganize actin cytoskeleton in decelerating R cell growth cone motility at the target region (Ruan, 2002).

A genetic approach was undertaken to search for genes encoding other components of the Dreadlocks-Msn signaling pathway. A msn gain-of-function phenotype was generated by overexpressing Msn in R cell growth cones. In wild-type, after exiting the optic stalk, R1–R6 growth cones migrate over a distance of ~20 µm within the lamina, then stop extension and expand significantly in size to form the lamina plexus, while R7 and R8 growth cones migrate through the lamina into the medulla. In larvae overexpressing msn, however, many R1–R6 growth cones terminate before reaching the lamina plexus, a phenotype in marked contrast to that in msn loss-of-function mutants in which many R1–R6 growth cones failed to stop at the lamina layer. Overexpression of msn also disrupts the regular array of R7 and R8 growth cones in the medulla. It was reasoned that if a gene functions downstream of msn, then reducing the dosage of this gene by half would decrease the level of signaling through the Msn pathway, thereby suppressing the Msn hyperactivation phenotype. Thus, a screen for modifiers of this msn hyperactivation phenotype might lead to the identification of other components of the Dock-Msn signaling pathway (Ruan, 2002).

To determine the feasibility of this approach, an examination was carried out to see if reducing the dosage of other genes in the genome would dominantly modify the pretarget termination phenotype in flies overexpressing msn. Analysis of deficiency lines shows that reducing the dosage of a number of cytological regions could dominantly modify the msn overexpression phenotype, indicating that this msn gain-of-function genetic background is indeed sensitive to the dosage of other genes. This approach was undertaken to examine the potential interaction between msn and a set of genes that had been previously implicated in regulating cytoskeletal changes in Drosophila. Interestingly, it was found that reducing the dosage of bif largely suppresses the pretarget msn hyperactivation phenotype. Suppression was observed using two different bif alleles R38 (~56%, n = 34) and R47 (~75%, n = 20). In contrast, reducing the dosage of bsk, a gene that encodes the fly homolog of C-Jun N-terminal kinase and has been shown previously to function downstream of Msn to regulate dorsal closure in early embryos, showed no effect. This result argues against the idea that msn and bsk interact similarly in R cell growth cones (Ruan, 2002).

Among other genes examined, reducing the dosage of cdc42 (~10%, n = 34 hemispheres) or disabled (dab) (~30%, n = 12 hemispheres) enhances the msn overexpression phenotype. In eye-brain complexes showing enhanced phenotype, R cell axons terminate within the optic stalk or the eye disc, which is never observed in wild-type larvae overexpressing msn. No modification of the msn overexpression phenotype is observed by reducing the dosage of either dpak, Rho1, all three Rac genes Rac1-Rac2-Mtl, or chickadee (chic), which encodes the fly homolog of profilin (n = 26 hemispheres) (Ruan, 2002).

The observation that Msn hyperactivation phenotype is sensitive to the dosage of bif, together with previous reports showing the link between Bif and the actin cytoskeleton, raises the interesting possibility that Bif functions downstream of Msn to regulate cytoskeletal changes in R cell growth cones (Ruan, 2002).

How could upstream stop signals be relayed through Dock and Msn to Bif? While the molecular nature of upstream regulators of Dock in R cell growth cones remains unknown, it has been shown that Dock functions downstream of the guidance receptor Dscam, a member of immunoglobulin superfamily, in larval photoreceptor growth cones. Dock mediates growth cone signaling through recruiting Pak to activated Dscam. It is speculated that Dock relays stop signals to Msn similarly in the adult visual system to regulate R1–R6 growth cone targeting. Dock and Msn form an in vivo complex at both larval and adult stages. It is speculated that when R1–R6 growth cones reach the lamina, stop signals produced by the intermediate target (i.e., lamina marginal glia) activate their growth cone receptors, which subsequently recruit the Dock-Msn complex. Consequently, this might bring Msn in proximity to Bif or directly stimulate the activity of Msn through a conformational change, leading to increased phosphorylation on Bif (Ruan, 2002).

While Msn associates directly with Bif and phosphorylates Bif in vitro, it is not known if Msn and Bif constitutively associate in R cell growth cones or if the association between them is transient and dependent on stop signals from the target region. Given that Bif is predominantly associated with the plasma membrane, it is speculated that the recruitment of Dock-Msn complex by activated growth cone receptors might relocate Msn from cytoplasm to plasma membrane, thus bringing Msn and Bif together. The formation of such a signaling complex could allow Msn to regulate the function of Bif through phosphorylation, or relocate Bif into a specific region within the growth cone to initiate downstream events. Testing these speculations awaits the identification of upstream stop signals and R cell growth cone receptors (Ruan, 2002).

How does the interaction between Msn and Bif regulate the changes in growth cone cytoskeleton? One possible scenario is that Bif, activated by Msn, induces the redistribution of F-actin within the growth cone, leading to the withdrawal of the growth cone leading edge. Consistently, cell culture studies show that Msn can reorganize Bif-induced actin fibers and reduce the number and the length of filopodia-like structures. Several studies have also demonstrated that the arrest of growth cone extension in vitro could be achieved through growth cone collapse, which is due at least in part to the loss of actin bundles at the leading edge of the growth cone. While the fact that R cell growth cones expand significantly in size upon reaching the target region argues against a mechanism involving the collapse of the whole growth cone, it remains possible that the initial termination involves partial growth cone collapse at the leading edge after exposure to stop signals. Since R cell growth cone morphology remains normal in msn and bif mutants, the view is favored that the interaction between Msn and Bif regulates the reorganization of actin filaments in spatially restricted domains within the growth cone without affecting the general structure of growth cone cytoskeleton (Ruan, 2002).

That Bif colocalizes with F-actin and can promote actin polymerization in cultured cells, together with a report that immobilized F-actin could pull down Bif from fly lysates, suggest strongly that Bif associates either directly or indirectly with F-actin filaments. Such association may stabilize actin filaments, thus contributing to the dramatic increase in the level of F-actin observed in cultured cells. Whether Bif also plays a similar role in promoting actin polymerization in R cells remains unclear since loss of bif affects neither growth cone outgrowth nor the amount of F-actin in R cell bodies and growth cones. One possible explanation is that other functionally redundant proteins maintain the level of F-actin in the absence of Bif. It is speculated that Bif may have at least two activities in R cell growth cones. Bif may be functionally redundant with other proteins to promote actin polymerization. Additionally, it may also play a role in restructuring F-actin in terminating R cell growth cones. The latter activity of Bif may resemble that of the Dictyostelium actin binding protein Severin. Severin and its mammalian homolog Gelsolin can bind to F-actin and fragment actin filaments. Interestingly, both Severin and Gelsolin are also phosphorylated by members of the GCK family of Ste20-like kinases in vitro. It is speculated that phosphorylation of Bif by Msn might directly increase such activity of Bif, thus inducing the shortening and aggregation of F-actin filaments leading to growth cone termination (Ruan, 2002).

DEVELOPMENTAL BIOLOGY

Embryonic

In early embryos, there is a low level of homogeneous MSN mRNA expression, presumably maternally contributed, with a concentration of transcript in the pole plasm. At gastrulation, strong expression is seen in the invaginating mesoderm. Later the strongest expression is seen in the visceral mesoderm, with weak expression also present in the central nervous system. The msn expression pattern during embryogenesis includes many cells that undergo migration or shape changes. Pole cells migrate around the embryo, at first passively and then actively. Mesodermal cell elongate and invaginate during gastrulation. The sections of the gut elongate to meet each other, and the endoderm then migrates along the visceral mesoderm. Cells in the CNS undergo both migration and exon outgrowth. A role for msn in these cell movements has yet to be demonstrated, since msn is maternally expressed and is essential for oogenesis. MSN mRNA is expressed in ovarian follicle cells, and especially strongly in the border cells, which migrate from the anterior tip of the egg chamber through the nurse cells to the oocytes (Treisman, 1997).

misshapen was identified during an enhancer trap expression pattern survey as one of a collection of loci expressed in the ring gland (Harvie, 1998).

misshapen was also identified in an enhancer trap screen designed to discover genes involved in the cellular aspects of defense mechanisms, as well as in melanotic tumor formation processes linked to blood cell dysregulation (Braun, 1997).

The Ste20-like kinase Misshapen functions together with Bicaudal-D and dynein in driving nuclear migration in the developing Drosophila eye

Nuclear translocation, driven by the motility apparatus consisting of the cytoplasmic dynein motor and microtubules, is essential for cell migration during embryonic development. Bicaudal-D (Bic-D), an evolutionarily conserved dynein-interacting protein, is required for developmental control of nuclear migration in Drosophila. Nothing is known about the signaling events that coordinate the function of Bic-D and dynein during development. This study shows that Misshapen (Msn), the fly homolog of the vertebrate Nck-interacting kinase is a component of a novel signaling pathway that regulates photoreceptor (R-cell) nuclear migration in the developing Drosophila compound eye. Msn, like Bic-D, is required for the apical migration of differentiating R-cell precursor nuclei. msn displays strong genetic interaction with Bic-D. Biochemical studies demonstrate that Msn increases the phosphorylation of Bic-D, which appears to be necessary for the apical accumulation of both Bic-D and dynein in developing R-cell precursor cells. It is proposed that Msn functions together with Bic-D to regulate the apical localization of dynein in generating directed nuclear migration within differentiating R-cell precursor cells (Houalla, 2005).

EFFECTS OF MUTATION

Clones of cells in the eye homozygous for misshapen are externally rough; in tangential sections, mutant rhabdomeres appear elongated instead of having a round cross-section. Since few rhabdomeres are visible in more basal sections, it is likely that this change is due to abnormalities in either cell shape or cell orientaion. Many of these photoreceptor nuclei are displaced from their normal positions near the apical and basal surfaces of the retina. The number of photoreceptors within each ommatidium is generally normal, although occasional examples of either extra or missing photoreceptors are seen (Treisman, 1997).

Drosophila embryos mutant for JNK (basket), JNK kinase (hemipterous), or Djun display a dorsal open phenotype, indicating that activation of this pathway is essential for normal embryonic development. To test whether Msn activates the JNK MAPK module in Drosophila, it was necessary to determine if embryos zygotically mutant for msn also display a dorsal open phenotype. Two inversion alleles of msn (msn¹⁰² and msn¹⁷²) were used in this analysis. Embryos homozygous for either msn allele or transheterozygous for the two alleles display a defect in dorsal closure, resembling embryos zygotically mutant for bsk. The observed defect in dorsal closure is observed at a frequency of ~15%, which is similar to that found for bsk¹. Expression of a msn cDNA in the epidermis rescues the dorsal closure defect in msn mutant embryos, allowing survival of all homozygotes to the pupal stage; this demonstrates that the phenotype is attributable to loss of msn function (Su, 1998).

Evidence that msn and basket function in the same pathway comes from the observation that some embryos doubly heterozygous for msn and bsk display a dorsal open phenotype. About 10% of embryos derived from a cross between msn/+ and bsk/+ flies exhibit a dorsal open phenotype. The defect in dorsal closure in embryos doubly heterozygous for msn and bsk is not a dominant effect of either gene; a defect in dorsal closure was very rarely observed when msn/+ or bsk/+ flies are crossed with wild-type flies. The presence of such defects in doubly heterozygous flies strongly suggests that the genes function in the same pathway. Moreover, the severity of the phenotype correlates with the strength of the bsk allele with which the msn/+ flies were crossed. To confirm genetically that msn also functions upstream of hemipterous, embryos doubly heterozygous for msn and hep were examined. hep is on the X chromosome, and both maternal and zygotic hep contribute to dorsal closure. To obtain flies doubly heterozygous for msn and hep, msn/+ males were crossed with hep^r75/+ females. About 35% of the flies obtained from this cross display a defect in dorsal closure. This finding is very close to the predicted frequency of 37% for embryos with a reduction in both the maternal and zygotic dosage of hep and the zygotic dosage of msn. A defect in dorsal closure is not observed when hep/+ females are crossed with +/Y males, indicating that embryos with the dorsal closure defect carry the msn mutation. Reduced dosage of both zygotic and maternal hep is required for the zygotic lethality of heterozygous msn/+ embryos. The viability of msn/+ flies lacking one copy of zygotic hep is reduced by >80% (Su, 1998).

A test of a constitutively active form of Jra (DJun) determined it could rescue the dorsal open phenotype in msn mutant embryos. Previous studies have shown that activated Djun rescues the bsk phenotype, indicating that one of the main functions of JNK is to phosphorylate and activate Jun. A constitutively active form of Jra was made by replacing the JNK phosphorylation sites with acidic residues. To test whether this activated Djun rescues the dorsal open phenotype in msn mutant embryos, it was expressed under the control of the hsp70 heat shock promoter in the msn mutant background. Expression of activated Djun rescues the dorsal open phenotype in most of the msn mutant embryos; heat shock decreased the number of embryos with a dorsal open phenotype from about 50%. In addition, expression of an activated form of thick veins, tkv^Q253D, also rescues the dorsal open phenotype in msn mutant embryos. GAL4 driven by the ectoderm-specific promoter at 69B was used to direct the expression of UAS-tkv^Q253D in msn mutant embryos. This expression of activated tkv partially rescues the dorsal open phenotype caused by msn; it also has a dorsalizing effect on the ventral ectoderm of the embryos related to the earlier function of dpp in establishing the dorsoventral axis, which served to mark embryos expressing activated tkv. Thus, these findings provide genetic evidence that msn functions upstream of the JNK MAP kinase module in leading edge cells (Su, 1998).

Germline clones mutant for msn fail to develop into eggs. msn must therefore function in the oocyte or the nurse cells; both cell types require a functional cytoskeleton in order to transport and localize determinants of positional information (Treisman, 1998).

Misshapen acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function mutations result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, evidence is provided for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn (Paricio, 1999).

In the Drosophila eye, EPP is reflected in the mirror-symmetric arrangement of ommatidial units relative to the dorso-ventral midline (the equator). This pattern is generated posterior to the morphogenetic furrow when ommatidial preclusters rotate 90° toward the equator, adopting opposite chirality depending on their dorsal or ventral positions. Polarity defects are manifested in the loss of mirror-image symmetry, with the ommatidia misrotating and adopting random chirality or remaining symmetrical. The gain-of-function dsh phenotype (sev-Dsh) has been successfully used in previous reports to identify new components of the Fz/Dsh planar polarity pathway. This same assay, dominant genetic modification of the sev-Dsh phenotype, was used to screen through a large number of known genes. Among the few mutants that show a specific interaction are two msn alleles. msn¹⁰² and msn¹⁷² are X-ray-induced inversions with breakpoints in the msn gene. Both loss-of-function alleles of msn act as dominant suppressors of sev-Dsh, comparable to other planar polarity-specific Dsh effectors (Paricio, 1999).

In addition, msn has been isolated in a gain-of-function screen for genes involved in planar polarity generation. Overexpression of genes required in planar polarity signaling at the relevant time often results in defects that are similar to the loss-of-function mutant phenotypes, e.g. with Fz and Dsh. In such a screen, ap-GAL4 flies (ap-GAL4 induces overexpression of the corresponding gene in the notum and the dorsal part of the wing), were crossed to the collection of 2200 E/P lines and the progeny were scored for disarranged microchaetae on the notum. One of the lines isolated in this screen, ep(3)0549, shows an abnormal orientation of the microchaetae similar to phenotypes obtained with ap driven Fz overexpression. Similarly, ap-GAL4, ep(3)0549 flies show typical polarity phenotypes on the dorsal surface of the wing where these are manifest in the presence of multiple wing hairs. In situ hybridization experiments to polytene chromosomes and complementation analyses reveal that the EP-element insertion in line ep(3)0549 is in the msn locus and represents a msn allele. Subsequent sequence analyses confirm that the EP insertion is located 24 bp upstream of the 5'-end of a msn cDNA. Taken together, these results suggest that msn is involved in EPP signaling and possibly acts downstream of Dsh (Paricio, 1999).

To gain further confirmation of the role of Msn in Fz/Dsh-mediated polarity signaling, an in vitro assay was used to determine whether Msn acts downstream of Dsh in JNK pathway activation. Previous experiments have shown that expression of Dsh in NIH 3T3 cells activates JNK and Jun phosphorylation, indicating that Dsh is a potent activator of a Jun-kinase pathway. Using the same assay, it was asked whether co-expression of a dominant-negative (kinase-inactive) Msn protein (DN-Msn) has an effect on Dsh-induced Jun phosphorylation. Significantly, co-expression of DN-Msn in this context causes a dramatic concentration-dependent inhibition of Jun phosphorylation. Taken together with the genetic interactions, these experiments confirm that Msn is acting downstream of Fz/Dsh in planar polarity signaling (Paricio, 1999).

msn mutations affect the morphology of the rhabdomeres in photoreceptors, causing malformed, 'misshapen' rhabdomeres, and also, at lower frequency, the number of photoreceptors. In addition, msn is required for the process of dorsal closure, and embryos mutant for msn display a typical dorsal open phenotype. To analyze its requirements in polarity generation, msn mutant clones in the eye and the wing were examined in detail. A phenotypic analysis of eye clones reveals that msn is required for the generation of planar polarity. msn mutant ommatidia containing the normal complement of photoreceptors are often misrotated and display the wrong chiral form or are symmetrical (non-chiral). To confirm that the polarity defects of msn mutant ommatidia are primary defects, and thus implicate msn in polarity generation, ommatidial polarity was examined in msn mutant clones at the earliest possible stage in third instar larval imaginal discs (when tissue polarity genes are required). Spalt is expressed in the R3/R4 precursor pair for about two columns at this stage. In wild type this reflects the regular arrangement and direction of rotation of the preclusters. In msn mutant tissue, ommatidial rotation, and thus polarity, is randomized (e.g. ommatidia rotate in the opposite direction as their wild-type neighbors) showing that these defects result from an early failure in polarity establishment. Thus in the eye, the msn phenotype (defects in polarity, malformed, misshapen and missing photoreceptors) is very reminiscent of other genes involved in both polarity and terminal photoreceptor differentiation (Paricio, 1999).

The fz gene has been implicated in the specification of the R3 cell within the R3/R4 pair in the process of chirality generation. The mosaic analysis of both loss-of-function and gain-of-function fz alleles has shown that Fz signaling is required in R3 for correct ommatidial chirality generation and also induces R3 fate. The genetic interactions and cell culture experiments have shown that msn acts downstream of Fz/Dsh, and thus it was asked whether msn is also involved in the selection of R3 in analogy to the fz requirement. The genotypic composition of mosaic ommatidial clusters were examined within the R3/R4 pair. This analysis revealed that, as is the case for fz, the msn⁺ cell has a strong preference for adopting the R3 photoreceptor fate. This can often lead to chirality inversions, where the msn⁺ R4 precursor adopts the R3 position and displaces the original msn^- R3 precursor. In summary, the genetic requirements of msn in single photoreceptors, in particular the R3/R4 pair, are very similar to those of fz (Paricio, 1999).

msn mutant clones in the wing affect the process of hair development and polarity. Phalloidin stainings of msn clones in pupal wings reveal that cells mutant for msn show defects in prehair initiation. These range from a complete failure of actin polymerization in the prehair to approximately wild-type levels of actin. Loss of Misshapen activity specifically affects wing hair actin organization, since adherens junction actin in msn clones appears normal. Although in some mutant cells an actin 'hair' is detected in the pupal wing, often at abnormal positions within the cell, the adult hairs in msn^- tissue are either missing, branched or stunted. Some cells that generate stunted hairs initiate them at multiple sites (a typical planar polarity phenotype. These phenotypes are reminiscent of the defects observed either when prehair actin organization is disrupted by dominant-negative Cdc42 or after cytochalasin D treatment of cultured pupal wing discs (Paricio, 1999).

To test whether overexpression of Msn in the eye can cause polarity defects comparable to sev-Fz or sev-Dsh, the msn E/P-line ep(3)0549 and a UAS-msn strain were used, and these were crossed to sev-GAL4. The eyes of the resulting flies (sev>msn) are externally rough and reveal typical polarity defects in tangential sections. Taken together with the genetic requirements and the loss-of-function phenotypes, the suppression of the sev-Dsh genotype and the cell culture experiments, these data demonstrate that Msn acts in the Fz/Dsh-mediated polarity signaling downstream of Dsh (Paricio, 1999).

During Drosophila embryogenesis msn acts upstream of the JNK-type MAPK module in dorsal closure signaling. Several lines of evidence support a function of JNK cascade components in the generation of planar polarity. To confirm the cell culture experiments and to determine whether components of JNK signaling act downstream of msn in polarity signaling in vivo, the gain-of-function eye polarity phenotype of msn (sev>msn) was used to test for dominant interactions with mutations in JNK signaling components. In sev>msn eyes, only 62.6% of the ommatidia are correctly oriented (compared with 100% in wild-type eyes) with the remaining ommatidia showing polarity defects as well as defects in photoreceptor shape and differentiation. Reducing the dosage of known components of the JNK cascade causes a strong dominant suppression of sev>msn. These results are consistent with msn acting upstream of the JNK module in polarity signaling and with the notion that Msn generally acts upstream of JNK-like cascades in higher eukaryotes (Paricio, 1999).

Although there is accumulating evidence that JNK-type MAPK modules are involved in planar polarity signaling, the analysis of mutant clones of either hep or bsk alleles shows no or weak phenotypes in imaginal discs. These observations suggest a high degree of redundancy at this level in the polarity signaling pathway. To address this issue further, a potential involvement of related kinases that could account for the proposed redundancy was examined. The recently described Drosophila kinases, belonging to the JNK/p38 class within the MAPK modules were examined for genetic interactions with the planar polarity phenotypes of sev-Dsh and sev-msn. These are obvious candidates to be cooperating with Hep and Bsk in polarity generation. At the level of Hep/JNKK (an MKK7 homolog), two other MKKs have been reported (DMKK3 and DMKK4). Similarly, at the level of Bsk/JNK, two p38-like kinases were isolated (Dp38a and Dp38b). Since no mutants have yet been isolated for these genes, whether deficiencies removing these kinases would show an interaction with sev-Dsh was examined. DMKK3 maps in the vicinity of hep: deficiencies removing DMKK3, Df(X)G24 and Df(X)H6, also remove hep. These deficiencies show externally a very strong suppression of sev-Dsh with a marked decrease of misrotated ommatidia as observed in tangential sections. Deficiency Df(3R)p13 removes the DMKK4 locus and also dominantly suppresses sev-Dsh. Similarly, deficiencies removing either Dp38a, Df(3L)crb^87-4 and Df(3L)crb^F89-4, or Dp38b, Df(2L)b80e3 and Df(2L)b87e25, are suppressors of sev-Dsh. Whether the respective deficiencies showed an interaction with sev>msn was also examined, and it was found that all of them act as dominant suppressors of this genotype as well. It is interesting to mention that the Msn-induced defects in rhabdomere morphology are also suppressed by those deficiencies. These interactions suggest that the p38 kinases are redundant with JNK in the context of planar polarity signaling (Paricio, 1999).

The effects of Misshapen overexpression and loss-of-function in the wing suggest that it may represent a branchpoint in the eye and wing polarization pathways. Misshapen overexpression in the wing produces a multiple wing hair phenotype similar to that of the wing-specific tissue polarity genes inturned and fuzzy. In contrast, msn loss-of-function clones have defects similar to those of dominant-negative CDC42 expressing cells; hair actin polymerization is defective and adult hairs are missing or stunted. One explanation of these data is that Misshapen acts through the JNK pathway, as it does in the eye, but that the targets of transcriptional activation are the components needed for hair formation. Excess production of these components may lead to multiple hairs and their loss to missing hairs. This model is not favored because Dishevelled, which acts upstream of Misshapen in this pathway, affects only hair polarity and is not required for hair formation. Furthermore, expression in the wing of a kinase-inactive version of JNK, which acts as a dominant negative, has no effect on hair formation or polarity. The data are more consistent with a model in which localized Misshapen activity directly promotes polarized cytoskeletal reorganization leading to hair formation; excess Misshapen might then expand the region of the cell competent for hair outgrowth. According to this model, activation of the Fz/Dsh signal transduction pathway in the wing would not necessarily increase the absolute level of Misshapen activity, but rather serve to localize Misshapen activity within the cell. It will be interesting to determine whether molecules such as CDC42, Inturned or Fuzzy represent wing-specific targets of Misshapen. Taken together with the phenotypes in the eye and its role in dorsal closure, these observations indicate that Msn has a function in both nuclear signaling and cytoskeletal rearrangements (Paricio, 1999).

Several studies in yeast and mammalian cells indicate that STE20 kinases function upstream of MKKKs regulating JNK. Msn is the Drosophila homolog of mammalian NIK, belonging to the SPS1 family of STE20-like kinases. Based on differences in structure and possible regulation, two subfamilies of STE20 kinases have been described: mammalian and Drosophila PAKs (p21 Activated Kinases), which are activated by binding GTP-bound Cdc42 and Rac and contain N-terminal regulatory and a C-terminal kinase domain. Members of the second subfamily, containing the SPS1 kinase in yeast, do not interact physically with Cdc42 or Rac and contain an N-terminal kinase and a C-terminal regulatory domain. Several members of the SPS1 subfamily have been described in mammals, but only a subset of them, such as GCK, GLK and NIK have been shown to activate JNK. Genetic and biochemical studies have recently demonstrated that Msn can activate the JNK module and is required during dorsal closure. Msn also acts upstream of the JNK module in polarity signaling in the eye and the results reported here support this as a general mechanism. It remains unclear how Msn is linked to the Rho/Rac GTPases. Genetic data argue for Msn acting downstream of RhoA/Rac. However, it has also been suggested that Rac and Msn act in parallel pathways (Paricio, 1999).

Although genetic evidence suggests an involvement of bsk (JNK) and hep (JNKK) in polarity signaling, phenotypic analyses suggest that the JNK module components are highly redundant in this process. In the search for other kinases involved, it was found that deficiencies uncovering the genes encoding the recently described p38 MAP kinases (Dp38a and Dp38b), and the MAP kinase kinases (DMKK3 and DMKK4) dominantly suppress the sev>msn and sev-Dsh phenotypes, suggesting that these proteins also function downstream of Dsh and Msn in polarity signaling. It is interesting to note that all phenotypic defects of sev>Msn were dominantly suppressed by mutations in both components of the JNK and the p38 kinase module. In contrast to these interactions, tissue culture experiments in mammalian cells have shown that NIK overexpression leads to JNK phosphorylation, but no detectable p38 activation was observed. This difference can be explained by cell- and tissue-specific requirements, e.g. in Drosophila during dorsal closure, JNK activation downstream of Msn is not redundant, while redundancy and p38 interactions are observed in polarity signaling. Thus, it is tempting to speculate that both JNK and p38 kinases cooperate in polarity generation (Paricio, 1999).

The reported promiscuity of the kinases at both the MKK and the MAPK levels could account for the redundancy. The Drosophila MKKs and JNK/p38 MAPKs also appear to act (at least partially) on overlapping downstream targets. Whereas DMKK3 appears rather specific for p38 activation (although it activates both p38s), DMKK4 and Hep (the MKK7 cognate) both activate Bsk/JNK. Similarly, Bsk/JNK and both Dp38s can phosphorylate the downstream targets dJun and ATF2. Thus, a potential downstream target can still be phosphorylated when one of the upstream kinases is removed, and likewise for their upstream activators. An even more complicated picture may emerge when all relevant kinases are identified. Other examples of redundancy are described in yeast MAP kinases. Although KSS1 and FUS3 normally have specific roles in different pathways, it has been shown that they are redundant in the process of mating and in this case KSS1 replaces Fus3 when the latter is not present. The isolation and analysis of all the respective kinases and their mutants will be necessary to understand fully the contribution of each single kinase in planar polarity signaling (Paricio, 1999).

misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. A structure-function analysis of Msn has been performed in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct upstream signaling systems (Su, 2000).

While a role for Ste20 kinases in promoting JNK activation has been previously identified, little is known about their regulation or about the specific in vivo function of these kinases. Msn has been shown to function upstream of the Drosophila JNK, Bsk, to stimulate dorsal closure of the Drosophila embryo. It is now shown that Msn requires both intact kinase activity and a C-terminal regulatory domain conserved in a number of Ste20 kinases of the GCK family in order to activate JNK in vivo in flies. The previous finding that the C-terminal regulatory domain of mammalian NIK binds the N-terminal regulatory domain of the mammalian Ste11 kinase MEKK1 led the authors to propose that the interaction of the C-terminal domain of NIK with downstream Ste11 kinases (DMKKK) is critical for NIK and other GCK family members to activate the JNK MAP kinase module. However, studies on NIK were performed in assays in which NIK protein was expressed at high levels, and under these circumstances, NIK is able to mediate JNK activation independent of an upstream activating signal. The requirement for both the C-terminal domain and the kinase activity of Msn to promote dorsal closure indicates that these domains are required in order for GCK family members to activate JNK in a physiologically relevant setting and suggests that an unknown Drosophila Ste11 kinase also couples Msn to JNK activation and dorsal closure (Su, 2000).

In addition to its role in JNK activation and dorsal closure, Msn is critical for the correct targeting of photoreceptor axons in Drosophila. Thus, the data indicate that msn is important in vivo for regulating at least two distinct biological events: dorsal closure and photoreceptor axon pathfinding. Interestingly, the upstream molecules that regulate msn in these two pathways are distinct, since a mutation eliminating the function of Msn in axon guidance does not affect its activity in dorsal closure. One molecule that may act upstream of Msn in the pathway leading to JNK activation and dorsal closure is a DTRAF; DTRAF1 can interact with Msn to activate the JNK pathway in cell lines (Liu, 1999). Mutation of a PXXP motif in Msn prevents it from binding to Dock and from rescuing photoreceptor axon pathfinding, indicating that Dock and/or related SH3 domain-containing molecules may act in concert with Msn in this process (Su, 2000).

The mechanism by which upstream factors regulate Msn is not known. A common requirement for Msn activation may involve its increased local concentration. This could occur either by the recruitment of Msn to phosphotyrosine-containing proteins or by DTRAF1-induced aggregation of Msn, thereby allowing juxtaposed Msn molecules present in the complex to transphosphorylate and activate each other. Alternatively, the finding that deletion of the region between the kinase and C-terminal domains of Msn leads to its constitutive activation raises the possibility that upstream signals activate Msn by inducing a conformational change and/or displacing a negative regulator bound to this region (Su, 2000).

The ability of axons to make precise connections during development requires the axonal growth cone, localized to the leading edge of projecting axons, to interpret multiple guidance cues that ultimately navigate axons to their destinations. Changes in the growth cone's actin cytoskeleton and/or the affinity for binding of the integrins to the matrix are thought to be the key elements whereby guidance cues regulate the path taken by developing axons. The finding that dock is required for Drosophila photoreceptor axon guidance and targeting has provided a starting point for beginning to dissect the intracellular signaling pathways that are activated at the growth cone to mediate these guidance cues. Dock is a member of a large family of adapter proteins consisting essentially of SH2 and SH3 domains, of which the prototypic member is Grb2. SH2-containing adapter molecules regulates signaling pathways by coupling catalytic molecules bound to their SH3 domains to phosphotyrosine-containing proteins (Su, 2000).

While a number of proteins that bind the SH3 domains of Nck and Dock have been identified, which of these serve as targets in vivo has been difficult to resolve. In contrast to the SH2-SH3 adapter molecule Grb2, for which interaction with the downstream SH3 binding partner Sos has been demonstrated using genetic evidence, the physiologically relevant binding partners for Nck and Dock and the downstream signaling pathways have only recently begun to be defined. In this regard, the Ste20 kinase Pak has been shown to interact with Dock, and expression of a myristylated form of Pak can partially rescue the dock mutant phenotype (Hing, 1999). It is shown here that Msn also binds to the SH3 domains of Dock and the amino acids that mediate this binding are required for the correct targeting of photoreceptor axons (Su, 2000).

However, these findings do not provide conclusive evidence that msn functions downstream of dock in photoreceptor targeting. Rather, they highlight the complex role of msn in photoreceptor targeting and suggest that unraveling the exact functions of msn in this process is unlikely to be simple. For example, it is likely that Msn functions in both photoreceptor cells and the brain. The severe photoreceptor axon guidance defects observed when msn mutants are rescued with UAS-msn(P656A, P659A), defective in binding Dock, are stronger than those caused by either the absence of msn in the eye or the complete loss of function of dock. Interaction between Msn and an SH3 domain-containing protein or proteins other than Dock in nonphotoreceptor cells, such as those in the brain, is a likely explanation. Although photoreceptor development in most of the eye disc is normal when rescue is carried out with UAS-msn(P656A, P659A), defects in brain development in these larvae may contribute to the axon guidance phenotype; an enhancer trap insertion in msn shows expression in the optic lobes as well as in the eye. This hypothesis is difficult to test directly, as many aspects of optic lobe development are directly dependent on retinal innervation. Because the defects in photoreceptor axonal targeting are specific to a mutation in a proline motif (proline appears at positions 656 and 650) that matches consensus SH3 binding motifs, this phenotype is probably due, at least in part, to the loss of interaction of Msn with an SH3 domain-containing protein (Su, 2000).

The finding that the dock phenotype is enhanced by the presence of msn suggests that the signaling pathways regulated by msn, which are critical for the correct targeting of R-cell axons, intersect with the signaling pathways regulated by dock. However, this interaction does not clarify whether msn functions on the same pathway as dock or on a parallel pathway. In addition, expression of a form of Msn that is constitutively active is not sufficient to rescue the dock phenotype. One possible explanation for these data is that msn acts downstream of dock but is not the only downstream mediator of its function. The Ste20 kinase Pak has been shown to interact with Dock, and expression of a myristylated form of Pak can partially rescue the dock mutant phenotype. Interestingly, this form of Pak predominantly rescues the expansion of growth cones in the medulla, a process that does not appear to require msn function in the photoreceptor axons. It is possible that msn and Pak mediate separable functions of dock in photoreceptor cells. An alternative possibility is that the function of Msn expressed in photoreceptor cells is mediated by the binding of Msn to an SH3 domain-containing protein other than Dock. The difference in the phenotypes caused by loss of msn and loss of dock in the photoreceptor axons would support this hypothesis. Mutations in the gene encoding such a hypothetical protein, which would function on a pathway parallel to the dock pathway, have yet to be identified (Su, 2000).

While this report was under review, Ruan (1999) reported a role for msn in photoreceptor axonal targeting and Dock signaling. However, in contrast to the findings reported here that msn mutant R1 to R6 axons terminate prematurely, Ruan reported that the R1 to R6 axons overshoot the lamina and terminate in the medulla. In addition, they found that overexpression of Msn in photoreceptor cells in dock mutants reversed the overshoot of the R1 to R6 axons. These findings and other data led them to conclude that dock and msn act in the same pathway. The reason for the discrepancy between the findings reported here and their results is not clear at present. One possibility is that the expression of Msn was much higher in the studies by Ruan, enabling them to see rescue of the dock mutant phenotype; they used an enhancer promoter line containing a UAS element inserted in the 5' promoter region of msn to overexpress msn. However, Ruan also found that overexpression of msn in a wild-type background led to the premature termination of many R-cell growth cones, essentially the same phenotype as they observed when msn was overexpressed in dock mutants; thus, it is not clear that this in fact constitutes rescue of the dock phenotype. In contrast, expression of a myristylated form of Pak largely rescues the dock mutant phenotype without inducing additional defects (Su, 2000).

An attractive hypothesis is that Dock and/or related SH3 domain-containing molecules function as adapters to couple Msn to tyrosine-phosphorylated proteins in response to signaling by a receptor tyrosine kinase localized at the axonal growth cone. Eph receptors, which constitute the largest family of receptor tyrosine kinases, are good candidates for receptors that may function at the axonal growth cone to regulate changes in the actin cytoskeleton and/or adhesion of integrins to the matrix that ultimately facilitate the correct targeting of retinal axons. NIK kinase activity is activated in mammalian cells by the EphB1 and EphB2 receptors and NIK couples EphB1 to both JNK and integrin activation. However, although a Drosophila Eph receptor kinase (DEK) is expressed on retinal axons, misexpression and overexpression of wild-type DEK or a kinase-defective form of DEK do not affect axonal pathfinding in Drosophila (Su, 2000 and references therein).

The intracellular signals activated downstream of Msn that mediate the correct pathfinding of photoreceptor axons are not yet known. The finding that regulation of the actin cytoskeleton is critical for growth cones to navigate correctly suggests that Msn may control the targeting of photoreceptor axons by regulating the actin cytoskeleton. The downstream pathways regulated by Msn are likely to be diverse and will not be limited to the activation of JNK. This is suggested by the finding that msn is required for oogenesis, while bsk and hep are not, and that ventral defects can be induced by a kinase-defective form of Msn, although maternal and zygotic bsk mutants do not show such a phenotype. It is not thought that msn directs axonal guidance via activation of the JNK MAP kinase pathway, because photoreceptor axonal targeting shows only minor defects (including occasional overshooting of R1 to R6 axons), in bsk¹ mutant clones made in a Minute background in the eye disc. However, because bsk¹ is not a complete loss-of-function mutant, these studies cannot definitively rule out a role for JNK. Small clones with mutations in both hep and the other Drosophila p38 MAPK kinase encoded by licorne also show an apparent overshoot of R1 to R6 axons, resembling the dock phenotype but not the msn phenotype. However, the dock phenotype could not be rescued with an activated allele of hep, indicating that activation of the JNK pathway is not sufficient to rescue the dock phenotype. While a direct link between Pak family Ste20 kinases and the actin cytoskeleton has been shown, a direct link between GCK family Ste20 kinases and the actin cytoskeleton has not yet been demonstrated. Thus, the ability to use genetics to identify and validate potential targets of Msn should provide a valuable tool to uncover not only the relevant biological functions regulated by Ste20 kinases but also their physiological downstream targets (Su, 2000).

Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the Ras1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of ras (Ksr) functions genetically downstream of Ras1 in this signal transduction cascade. Expression of dominant-negative Ksr (KDN) in the developing eye blocks Ras pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate Ras signaling, a screened was carried out for genes that alter Ras1/Ksr signaling efficiency when misexpressed. In this screen, three known genes, Lk6, misshapen, and Akap200, were recovered. Seven previously undescribed genes were recovered; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS (Huang, 2000).

Overexpression of msn in an sE-KDN background enhances the rough eye phenotype. msn encodes the Drosophila homolog of Nck interacting kinase (NIK), a member of the mammalian SPS1 subfamily of the STE20 kinase family. msn is an essential gene involved in dorsal closure during embryogenesis in Drosophila and mutant clones result in misshapen rhabdomeres (due to defects in polarity, malformed and missing photoreceptors) in the adult eye. STE20, the founding member of the family in yeast, acts in the pheromone signaling pathway and activates the yeast MAPK module. However, the upstream activators of the STE20 pathway are not known. msn, the STE20 homolog in Drosophila, acts upstream of the c-Jun amino-terminal kinase (JNK) MAPK cascade required for dorsal closure and has recently been implicated to act downstream of the Frizzled receptor in the epithelial planar polarity pathway. Published results have suggested that msn, when overexpressed in the eye in an otherwise wild-type background, can generate a rough eye. However, in the experiments described here, no effect on eye morphology was found using sE-GAL4 to drive either UAS-msn or EP(3)0609 and EP(3)0549, the two EP lines upstream of the msn gene. Drosophila Jun has been implicated as a downstream target of both the JNK MAPK and RAS1/MAPK signal transduction pathways by overexpression analysis of dominant-negative mutations; however, no other components of either pathway are shared. If the JNK pathway can partially compensate for the RAS1 pathway in eye development, one would expect that msn overexpression would suppress sE-KDN. The overexpression results from this screen suggest that as a misexpression suppressor of RAS1, msn decreases signaling in the pathway. msn may independently inhibit neuronal cell fate, although there is no previous evidence for this. It is possible that the JNK signaling pathway may compete with the RAS pathway for common components. Alternatively, this interaction may be tissue specific and only uncovered in the eye with overexpression. There is also the possibility that misexpression of msn causes promiscuous signaling through an independent pathway that also affects eye morphology. Although no phenotype is seen when msn is overexpressed in the eye, this situation may sensitize the eye and nonspecifically enhance the sE-KDN phenotype (Huang, 2000 and references therein).

Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death, was identified in a large-scale gain-of-function screen. Eiger is a type II transmembrane protein with a C-terminal TNF homology domain. It is predominantly expressed in the nervous system. Genetic evidence shows that Eiger induces cell death by activating the Drosophila JNK pathway. Although this cell death process is blocked by Drosophila inhibitor-of-apoptosis protein 1 (DIAP1, Thread), it does not require caspase activity. Genetically, Eiger has been shown to be a physiological ligand for the Drosophila JNK pathway. These findings demonstrate that Eiger can initiate cell death through an IAP-sensitive cell death pathway via JNK signaling (Igaki, 2002).

Many mammalian TNF superfamily proteins activate both the NF-kappaB and the JNK pathway, and activation of the latter pathway facilitates cell death (Davis, 2000). To examine whether Eiger activates the JNK pathway, the genetic interactions of Eiger with the components of the Drosophila JNK cascade were examined. The reduced-eye phenotype induced by Eiger is strongly suppressed in basket (bsk), a heterozygous mutant of Drosophila JNK. In addition, overexpression of a dominant-negative form of Bsk almost completely suppresses the eye phenotype. Moreover, heterozygosity at the hemipterous (hep) locus, which encodes Drosophila JNKK, suppresses the reduced-eye phenotype much as does bsk, and its hemizygosity (null background) rescues the phenotype almost completely. Furthermore, the co-expression of a dominant-negative form of dTAK1 TGF-ß activated kinase 1; Drosophila JNKKK) also rescues the Eiger-induced phenotype completely. Misshapen (Msn) is a MAPKKKK that is genetically upstream of the JNK pathway in Drosophila. A half dosage of the msn gene strongly suppresses the Eiger-induced phenotype. Heterozygosity of Drosophila jun, a target of the JNK pathway, did not show any genetic interaction with Eiger, raising the possibility that the Eiger-stimulated death-inducing JNK pathway may not require new transcripts that are controlled by Jun (Igaki, 2002).

Puckered (Puc) is a dual-specificity phosphatase, the expression of which is induced by the Drosophila JNK pathway to inactivate Bsk, so that puc expression can be used to monitor the extent of activation of the JNK pathway. To confirm that the JNK pathway is actually activated by Eiger, puc expression level was assessed in the eye disc of GMR>regg1^GS9830 flies using a puc-LacZ enhancer-trap allele. The strong induction of puc-LacZ was observed in the region posterior to the morphogenetic furrow of the eye disc compared with the control eye disc. Furthermore, Western blot analysis with an anti-phospho-JNK antibody has revealed that Bsk is phosphorylated by Eiger overexpression. These genetic and biochemical data led to a model in which Eiger activates Msn, thereby triggering the JNK signaling pathway, sequentially stimulating dTAK1, Hep and Bsk. Using RT-PCR analysis, whether Eiger could stimulate the NF-kappaB pathway was tested; however, no obvious upregulation of the antimicrobial peptide genes, the target genes of the Drosophila NF-kappaB pathway, was detected (Igaki, 2002).

Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution. Consistent with this interpretation, several components of the Drosophila JNK pathway are rate limiting in mediating or preventing Eiger-induced apoptosis. The removal of one wild-type copy of either DTRAF1 (encoding the homolog of human TRAF2), misshapen (encoding a Ste20 kinase that binds to DTRAF1), or basket (encodes Drosophila JNK) suppresses Eiger-induced apoptosis. Conversely, animals heterozygous for a mutation in puc display an enhanced phenotype (Moreno, 2002).

The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).

The integrin effector PINCH regulates JNK activity and epithelial migration in concert with Ras suppressor 1: Genetic interaction with msn

Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. PINCH (Steamer duck), a protein required for integrin-dependent cell adhesion and actin-membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, RSU-1, a regulator of Ras signaling in mammalian cells, has been identified as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding Drosophila RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. These results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development (Kadrmas, 2004).

To determine if PINCH contributes to DC, its localization was examined in stage 14 embryos. PINCH and ß-PS integrin colocalize in both the LE and the amnioserosa, consistent with PINCH's established role as an integrin effector. The amnioserosa is an extraembryonic tissue present on the dorsal surface of the embryo. Since it has been established that coordinated signaling between the amnioserosa and migrating epithelium is key to formation of LE focal complexes, PINCH could exert an effect in the LE epithelium, the amnioserosa, or both tissues. stck homozygous mutant embryos rescued with a PINCH:GFP transgene under the control of the endogenous PINCH promoter display PINCH-GFP at the LE of the advancing epithelial sheets. Within the LE, PINCH is precisely localized to areas of active phosphotyrosine signaling at triangular nodes corresponding to apical adherens junctions (Kadrmas, 2004).

Zygotic stck mutants proceed normally through DC with complete lethality arising at the embryo-to-larval transition. When maternal PINCH contribution is eliminated, only 12% of cuticles have wild-type morphology. Dorsal puckers and dorsal holes characteristic of aberrant DC are observed at a 36% and 23% frequency, respectively, indicating that maternally inherited PINCH is a key contributor to the process of DC. Moreover, in the absence of maternal PINCH, epithelial defects are observed in ventral patterning and head involution, indicating that PINCH may have additional functions in the developing embryo. Cuticles from embryos lacking both maternal and zygotic PINCH have the same array of phenotypes (Kadrmas, 2004).

PINCH is composed of five LIM domains, each of which could serve as a protein-binding interface. The SH2-SH3 adaptor protein, Nck2, has been reported to interact with mammalian PINCH. This association is intriguing because the Drosophila homologue of Nck2, Dreadlocks, interacts directly with Misshapen (Msn), a MAP4K in the JNK signaling cascade. As with other components of the JNK pathway, null mutations in msn result in embryonic lethality due to failure of DC. Although no direct binding of PINCH to Dreadlocks was observed in Drosophila, this study uncovered a link between PINCH's role in DC and the JNK cascade by testing for genetic interaction between stck and msn. Reduction of PINCH protein levels by introduction of a single copy of the loss-of-function allele, stck¹⁸, into the msn¹⁰² homozygous null background allows partial restoration of DC, suggesting that PINCH functions as a negative regulator of JNK signaling (Kadrmas, 2004).

Puckered (Puc) is a JNK phosphatase whose expression is up-regulated in response to JNK activation, setting up a negative feedback loop. During DC, JNK-regulated expression of a Puc-LacZ fusion reporter is restricted to the LE cells. In embryos lacking maternal PINCH, expression of the Puc-LacZ fusion protein is disorganized and present in an expanded number of cells, including those beyond the LE border. This phenotype is similar to Puc-LacZ expression observed in puc loss-of-function mutants and further supports a role for PINCH in the negative regulation of the JNK cascade (Kadrmas, 2004).

Thorax closure is a post-embryonic developmental process with features common to DC, including migration of epithelial sheets and a dependence on JNK signaling. Within the wing disc, cells of the stalk region are functionally similar to LE cells during DC. These cells comprise the eventual fusion site for adjacent imaginal discs and are active in JNK signaling. Spatially restricted JNK signaling in the stalk of wing disc can be visualized via a Puc-LacZ reporter, and PINCH expression overlaps with Puc-LacZ in this area of active JNK signaling. Therefore, as in DC, PINCH is properly positioned to act as a regulator of the JNK cascade (Kadrmas, 2004).

Although msn null mutations are embryonic lethal due to DC failure, flies homozygous for the hypomorphic allele msn³³⁴⁹ are semi-viable and a large proportion of the eclosing adults have thorax closure defects. These observations underscore the similarities between thorax closure and DC. In a stck¹⁸ heterozygous background, a greater percentage of msn³³⁴⁹ homozygotes are able to eclose, supporting the hypothesis that PINCH is a negative regulator of the JNK pathway in both dorsal and thorax closure (Kadrmas, 2004).

Drosophila PINCH was purified in complex with its binding partners using tandem affinity purification (TAP)–tagged PINCH (TAP-PINCH). stck homozygous mutant embryos rescued with a TAP:PINCH transgene driven by the endogenous stck promoter to wild-type levels afford material for purification of soluble, cytoplasmic TAP-PINCH complexes in the absence of endogenous PINCH protein. Three partners that copurified stoichiometrically with TAP-PINCH from embryos, as well as in complex with TAP-PINCH from cultured Drosophila S2R⁺ cells, were identified by mass spectrometric analysis. Consistent with what is observed in mammalian cells, ILK copurified with PINCH. The Drosophila homologue of the parvin/actopaxin family of proteins, Parvin, is also present in PINCH protein complexes. Parvin is known to bind ILK and actin in mammalian systems, but the isolated Parvin/ILK/PINCH complexes are the first to be described in Drosophila. Additionally, a novel 31-kD protein was identified as Drosophila CG9031. The CG9031 protein is 55% identical and 74% similar to human RSU-1, a leucine-rich repeat containing protein first identified as a suppressor of cell transformation by v-Ras and subsequently implicated in regulation of MAP kinase signaling, specifically the JNK and ERK cascades, when overexpressed in cultured cells. Despite its potent ability to act as a tumor suppressor, little is known about the mechanism of action of RSU-1. Its partnership with the PINCH protein allows placement of RSU-1 in a molecular pathway that is linked to integrins (Kadrmas, 2004).

To assess the specificity and nature of the interaction between PINCH and RSU-1, domain-mapping studies were performed in cell culture and in yeast two-hybrid assays. Drosophila RSU-1 copurifies with full-length His-tagged PINCH, but not with a truncated His-tagged PINCH containing only LIM1–3, confirming the specificity of the interaction and suggesting LIM4 and/or 5 is the site of binding. ILK, which binds LIM1 of PINCH, copurifies with both full-length and the truncated LIM1–3 version of His-tagged PINCH, serving as a positive control. Both PINCH and ILK are copurified with His-tagged RSU-1. Moreover, endogenous PINCH and RSU-1 can be coimmunoprecipitated. The site of RSU-1 binding to PINCH was further mapped using yeast two-hybrid analysis. Only cells expressing LIM5 bait/RSU-1 prey activated all three reporters, indicating LIM5 is the site of RSU-1 binding. Consistent with the view that they interact in vivo, PINCH:GFP and RSU-1 are prominently colocalized at integrin-rich muscle attachment sites in Drosophila embryos (Kadrmas, 2004). Drosophila RSU-1, which displays seven leucine-rich repeats with high sequence similarity to small GTPase regulators, is encoded by the CG9031 locus. A P-element insertion allele was characterized that disrupts the RSU-1 coding sequence. Flies homozygous for this mutation within CG9031 are viable and fertile, and lack RSU-1 protein as indicated by Western analysis with multiple anti-RSU-1 antibodies. PINCH and RSU-1 are both expressed in larval wing discs and similar to stck wing clones, the mutation within CG9031 produces flies with wing blisters at 60% penetrance. These data are consistent with PINCH and RSU-1 acting in concert to support integrin-dependent adhesion. The CG9031 gene was named icarus (ics) after the son of Daedalus who had unstable wings (Kadrmas, 2004).

Although elimination of RSU-1 function does not result in dorsal or thorax closure defects, the role of RSU-1 in these processes was evaluated by testing for genetic interactions between ics and msn. Similar to what occurs with reduction of stck dosage, homozygous mutation of ics suppresses DC defects observed in msn¹⁰² mutant embryos. Absence of RSU-1 also increases eclosure rates of msn³³⁴⁹ hypomorphs and completely suppresses the thorax defects present in msn³³⁴⁹ animals, suggesting that like PINCH, RSU-1 can function as a negative regulator of JNK signaling. To confirm that the suppression of msn DC defects by ics mutation is mediated by the JNK signaling cascade, RSU-1 was eliminated in basket (bsk) embryos that lack zygotic JNK, the terminal kinase in this cascade. Homozygous ics mutation suppresses the DC defects of bsk¹ mutants, confirming that ics loss-of-function mutations affect DC by influencing the JNK cascade. Moreover, wing discs isolated from ics mutants display a 30% increase in active phospho-JNK relative to wild type, providing direct biochemical confirmation that RSU-1 influences JNK activation state in vivo. Although no localized accumulation of RSU-1 during DC was detected, RSU-1 is readily detected by Western analysis in stage 13 embryos that are undergoing DC. Thus, the temporal pattern of RSU-1 expression is consistent with genetic results that highlight its role in regulation of JNK-dependent morphogenesis (Kadrmas, 2004).

Analysis of PINCH and RSU-1 levels in wild-type versus stck or ics mutant embryos provided insight into the physiological significance of their association. In embryos mutant for both maternal and zygotic stck, RSU-1 is dramatically reduced relative to wild-type levels. Likewise, in ics embryos, PINCH levels are also decreased. These observations suggest that PINCH and RSU-1 are reciprocally dependent on each other for maximal expression and/or stability. The mechanism for coordinate regulation of RSU-1 and PINCH remains to be determined. Because the phenotypes associated with loss of RSU-1 represent a subset of stck phenotypes, the processes disturbed in ics mutants may be exquisitely sensitive to PINCH levels. Alternately, RSU-1 may have functions that are independent of its role in PINCH stabilization (Kadrmas, 2004).

The data are consistent with a model in which PINCH could modulate JNK signaling in two distinct ways. (1) PINCH is present at areas where JNK is active and antagonizes JNK signaling. This behavior is reminiscent of Drosophila Puc, a phosphatase regulator of the JNK cascade that establishes a negative feedback loop. PINCH has no intrinsic catalytic activity, but might recruit proteins that could alter the availability or activity of JNK signaling components. Like Puc, PINCH expression is up-regulated in response to constitutive JNK signaling. Availability of RSU-1 at these sites of active JNK signaling could independently regulate JNK signaling or modulate the effects of PINCH on JNK through regulation of PINCH stability. (2) PINCH and RSU-1 are required for integrin-dependent adhesion. PINCH links integrins to the actin cytoskeleton via ILK and Parvin, and these connections could influence both integrin-dependent adhesion and signaling. Integrin signaling, through a variety of tyrosine kinases and Rac, stimulates the JNK cascade; therefore, PINCH may also exert an influence on JNK signaling via integrin. The current findings illustrate that the cellular concentration of PINCH affects the level of RSU-1 and vice versa. Thus, modulation of the ratio of RSU-1 to PINCH could provide a mechanism to regulate JNK signaling during DC and thorax closure in Drosophila. It is hypothesized that PINCH/RSU-1 complexes fine-tune and integrate the JNK and integrin signaling cascades required during morphogenesis, highlighting the potential role of integrin-associated apical junctional complexes as signal coordination points for epithelial morphogenesis (Kadrmas, 2004).

The Ral/exocyst effector complex counters c-Jun N-terminal kinase-dependent apoptosis in Drosophila melanogaster

Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, null and hypomorph alleles of the Drosophila Ral gene have been generated. Ral null animals are not viable. Reduced Ral expression in cells of the sensory organ lineage has no effect on cell division but leads to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggest that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst, a protein complex involved in vesicles trafficking, specifically the tethering and spatial targeting of post-Golgi vesicles to the plasma membrane prior to vesicle fusion, is a Ral effector. Epistasis between mutants of Ral and of misshapen (msn), the fly ortholog of HGK, suggests the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. It is conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. It is proposed that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK (Balakireva, 2006).

The Ral pathway is an essential component of physiological Ras signaling as well as Ras-driven oncogenesis. It can be instrumental in oncogenic transformation, and an activated form of a Ral exchange factor, Rlf, recapitulates the capacity of Ras to transform immortalized human cell cultures, either alone or together with other Ras effectors. Reciprocally, the lack of RalGDS, another Ral exchange factor, reduces tumorigenesis in a multistage skin carcinogenesis model and transformation by Ras in tissue culture. The molecular basis of the Ral contribution to oncogenesis remains to be elucidated (Balakireva, 2006).

None of the Ral effectors and their attributed cellular functions are obvious actors in oncogenesis. One of the two well-documented Ral effectors, RLIP76/RalBP1, is involved in endocytosis. The other, the exocyst complex, is involved in secretion, polarized exocytosis, and migration and can be found at the tip of filopods and at tight junctions. The exocyst complex is composed of eight proteins, which have been initially identified via mutants of secretion in the budding yeast. Exocyst complexes are bound to vesicles and are supposed to participate in vesicle trafficking and tethering to the plasma membrane. Globally, Ral appears to be a regulator of vesicle trafficking with consequences on cell proliferation, cell fate, and cell signaling (Balakireva, 2006).

In order to gain insight into Ral function, a genetic and cell biology approach was undertaken using Drosophila, which has a single Ral gene. Null and hypomorph alleles of Ral were generated, and Ral was shown to be an essential gene. Ral loss-of-function has dramatic effects on the differentiation of sensory organ precursor cells and leads to caspase-8-independent cell death by releasing ectopic tumor necrosis factor (TNF) receptor-associated factor 1-c-Jun N-terminal kinase (TRAF1-JNK) signaling. Sensory organ cell survival in Ral mutants is rescued by an activation of p38 mitogen-activated protein (MAP) kinase, revealing an antiapoptotic function of this latter. The influence of Ral on sensory organ cell fate is directly mediated by the exocyst complex together with a novel interaction partner, the MAP4K4 (also known as hepatocyte progenitor kinase-like/germinal center kinase-like kinase [HGK] in mammals and Misshapen [MSN] in flies). This suggests that a Ral/exocyst/JNK regulatory axis may represent a key component of developmental regulatory programs (Balakireva, 2006).

Hypomorph mutations of Ral displayed a loss-of-bristle phenotype with sockets without shafts, as do flies expressing dominant negative alleles of Ral). Whereas Ral is expressed in many if not all tissues, the only situation where a decreased level of Ral appears compatible with adult viability leads to a developmental phenotype in the bristle sensory organs. In Ral mutants, the pI precursor cells undergo the right number of divisions with a correct timing, but afterward shaft cells die by apoptosis, showing that death hits after cell division and determination has taken place, during the subsequent differentiation stage (Balakireva, 2006).

The various pathways that lead to apoptosis for their interactions with Ral have been explored. The caspase-8-mediated pathway did not contribute to the Ral phenotype, as opposed to a caspase-9-mediated pathway. The JNK pathway, a cascade of four kinases starting with MSN (MAP4K4 or HGK in human), which requires formation of a complex with TRAF1 for its full activity, and ending at the Jun N-terminal kinase, was tested. Puckered is a phosphatase that dephosphorylates and deactivates JNK (Balakireva, 2006).

Loss-of bristle and apoptosis phenotypes due to decrease of Ral signaling were suppressed by down-regulation of the JNK pathway and enhanced by its up-regulation. Symmetrically, a phenotype due to a hyperactivation of the Ral pathway by the overexpression of Ral^G20V was suppressed and enhanced by enhancing or decreasing JNK signaling, respectively (Balakireva, 2006).

The fact that the enhancement and suppression can be induced by genetic alterations of TRAF and MSN as well as of JNK proteins suggests that Ral is a general negative regulator of this cascade. Dominant negative alleles of transcriptional effectors of the JNK, Jun itself but also Fos, suppress the Ral phenotype, suggesting that Ral regulates transcriptional events involved positively or negatively in apoptosis (Balakireva, 2006).

Down-regulating the JNK pathway is not only suppresses apoptosis in Ral-defective cells but also rescues normal bristle development. Together with data in S2 cells, where Ral behaves also as a negative regulator of JNK in the absence of any cell death (Sawamoto, 1999), the results suggest a functional relationship between Ral and the JNK pathway wherein Ral activation keeps JNK down. Data using activated and dominant negative alleles of Ral in mammalian cell culture support a positive effect of Ral on JNK activation. The source of this discrepancy, which might be due to cell- and/or context-specific interactions of Ral with the JNK pathway, is not understood. However, the current data obtained by RNA interference in HeLa cells are consistent with the fly model (Balakireva, 2006).

Epistatic relationships between Ral and p38 MAP kinase mutants revealed another actor in Ral-dependent apoptosis: the p38 MAP kinase behaves as an antiapoptotic kinase, which could be positively regulated by Ral (Balakireva, 2006).

A control of the basic JNK activity might serve two purposes: (1) it minimizes JNK activity and avoids undesirable cell death in normal conditions; (2) a low level of basal JNK activity allows better differential in activation of JNK when this activation happens in response to stresses that lead eventually to apoptosis (Balakireva, 2006).

The molecular basis of Ral action on the JNK pathway was addressed genetically and biochemically. The model that emerges is that the exocyst complex is the matchmaker between Ral and the JNK pathway, and the simplest interpretation of genetic data is that the exocyst works like a negative regulator of HGK activity. Finally, the exocyst complex was found to bind in vivo to HGK, providing a biochemical basis for the functional effect of Ral on JNK (Balakireva, 2006).

Decreasing the JNK pathway seems to favor the oncogenic capacity of Ras in mouse primary fibroblasts. The current results can explain one of the contributions of the Ral pathway to oncogenesi: cancer cells have to sustain proliferative signals and relieve proapoptotic signals, and Ral via the exocyst complex might be in charge, at least, of this latter task in oncogenesis. Finally, it has been recently shown that the exocyst complex carries enzymatic activities working in the NF-kappaB pathway. These data together with the present report widen the role of the exocyst to functions other than directing vesicle traffic and contributing to exocytosis (Balakireva, 2006).

EVOLUTIONARY HOMOLOGS

Yeast SPS1

During sporulation of Saccharomyces cerevisiae, meiosis is followed by encapsulation of haploid nuclei within multilayered spore walls. Completion of the late events of the sporulation program requires the SPS1 gene. This developmentally regulated gene, which is expressed as cells are nearing the end of meiosis, encodes a protein with homology to serine/threonine protein kinases. The catalytic domain of Sps1 is 44% identical to the kinase domain of yeast Ste20, a protein involved in the pheromone-induced signal transduction pathway. Cells of a MATa/MAT alpha sps1/sps1 strain arrest after meiosis and fail to activate genes that are normally expressed at a late time of sporulation. The mutant cells do not form refractile spores as assessed by phase-contrast microscopy and do not display the natural fluorescence and ether resistance that is characteristic of mature spores. Examination by electron microscopy reveals, however, that prospore-like compartments form in some of the mutant cells. These immature spores lack the cross-linked surface layer that surrounds wild-type spores and are more variable in size and number than are the spores of wild-type cells. Despite their inability to complete spore formation, sps1-arrested cells are able to resume mitotic growth on transfer to rich medium, generating haploid progeny. These results suggest that the developmentally regulated Sps1 kinase is required for normal progression of transcriptional, biochemical, and morphological events during the later portion of the sporulation program (Friesen, 1994).

NCK-interacting kinase

The Nck-interacting kinase (NIK: Drosophila homolog Misshapen), a member of the STE20/germinal center kinase (GCK) family, has been identified as a partner for the beta1A integrin cytoplasmic domain. NIK is expressed in the nervous system and other tissues in mouse embryos and colocalizes with actin and beta1 integrin in cellular protrusions in transfected cells. To demonstrate the functional significance of this interaction, Caenorhabditis elegans was used, since it has only one beta (PAT-3) integrin chain, two alpha (INA-1 and PAT-2) integrin chains, and a well-conserved NIK ortholog (MIG-15). Using three methods, it has been shown that reducing mig-15 activity results in premature branching of commissures. A significant aggravation of this defect is observed when mig-15 activity is compromised in a weak ina-1 background. Neuronal-specific RNA interference against mig-15 or pat-3 leads to similar axonal defects, thus showing that both mig-15 and pat-3 act cell autonomously in neurons. A genetic interaction occurs between mig-15, ina-1, and genes that encode Rac GTPases. This study provides the first evidence that the kinase NIK and integrins interact in vitro and in vivo. This interaction is required for proper axonal navigation in C. elegans (Poinat, 2002).

Nck, an adaptor protein composed of one SH2 and three SH3 domains, is a common target for a variety of cell surface receptors. A novel mammalian serine/threonine kinase has been identified that interacts with the SH3 domains of Nck, termed Nck interacting kinase (NIK). This kinase is most homologous to the Sterile 20 (Ste20) family of protein kinases. Of the members of this family, GCK and MSST1 are most similar to NIK in that they bind neither Cdc42 nor Rac and contain an N-terminal kinase domain with a putative C-terminal regulatory domain. Transient overexpression of NIK specifically activates the stress-activated protein kinase (SAPK) pathway. Both the kinase domain and C-terminal regulatory region of NIK are required for full activation of SAPK. NIK likely functions upstream of MEKK1 to activate this pathway; a dominant-negative MEK kinase 1 (MEKK1) blocks activation of SAPK by NIK. MEKK1 and NIK also associate in cells and this interaction is mediated by regulatory domains on both proteins. Two other members of this kinase family, GCK and HPK1, contain C-terminal regulatory domains with homology to that of NIK. These findings indicate that the C-terminal domain of these proteins encodes a new protein domain family and suggests that this domain couples these kinases to the SAPK pathway, possibly by interacting with MEKK1 or related kinases (Su, 1997).

The Drosophila Ste20 kinase encoded by misshapen (msn) is an essential gene in Drosophila development. msn function is required to activate the Drosophila c-Jun N-terminal kinase (JNK), Basket (Bsk), to promote dorsal closure of the Drosophila embryo. Later in development, msn expression is required in photoreceptors in order for their axons to project normally. A mammalian homolog of Msn, the NCK-interacting kinase (NIK) (recently renamed to mitogen-activated protein kinase kinase kinase kinase 4; Map4k4), has been shown to activate JNK and to bind the SH3 domains of the SH2/SH3 adapter NCK. To determine whether NIK also plays an essential role in mammalian development, mice deficient in NIK were created by homologous recombination at the Nik gene. Nik^-/- mice die postgastrulation between embryonic day (E) 9.5 and E10.5. The most striking phenotype in Nik^-/- embryos is the failure of mesodermal and endodermal cells that arise from the anterior end of the primitive streak (PS) to migrate to their correct location. As a result Nik^-/- embryos fail to develop somites or a hindgut and are truncated posteriorly. Interestingly, chimeric analysis demonstrates that NIK has a cell nonautonomous function in stimulating migration of presomitic mesodermal cells away from the PS and a second cell autonomous function in stimulating the differentiation of presomitic mesoderm into dermomyotome. These findings indicate that despite the large number of Ste20 kinases in mammalian cells, members of this family play essential nonredundant roles in regulating specific signaling pathways. In addition, these studies provide evidence that the signaling pathways regulated by these kinases are diverse and not limited to the activation of JNK because mesodermal and somite development are not perturbed in JNK1-, and JNK2-deficient mice (Xue, 2001).

Misshapen/NIKs-related kinase (MINK) is a member of the germinal center family of kinases that are homologous to the yeast sterile 20 (Ste20) kinases and regulate a wide variety of cellular processes, including cell morphology, cytoskeletal rearrangement, and survival. A novel human Misshapen/NIKs-related kinase beta (hMINK beta) encodes a polypeptide of 1312 amino acids. hMINK beta is ubiquitously expressed in most tissues with at least five alternatively spliced isoforms. Similar to Nck interacting kinase (NIK) and Traf2 and Nck-interacting kinase (TNIK), hMINK beta moderately activates c-Jun N-terminal kinase (JNK) and associates with Nck via the intermediate domain in the yeast two-hybrid system and in a glutathione S-transferase (GST) pull-down assay. Interestingly, overexpression of the kinase domain deleted and kinase-inactive mutants of hMINK beta in human fibrosarcoma HT1080 cells enhances cell spreading, actin stress fiber formation, and adhesion to extracellular matrix, as well as decreased cell motility and cell invasion. Furthermore, these mutants also promote cell-cell adhesion in human breast carcinoma MCF7 cells, evidenced by cell growth in clusters and increased membrane localization of beta-catenin, a multifunctional protein involved in E-cadherin-mediated cell adhesion. Finally, hMINK beta protein colocalizes with the Golgi apparatus, implicating that hMINK beta might exert its functions, at least in part, through the modulation of intracellular protein transport. Taken together, these results suggest that hMINK beta plays an important role in cytoskeleton reorganization, cell adhesion, and cell motility (Hu, 2004).

MIG-15 and ERM-1 promote growth cone directional migration in parallel to UNC-116 and WVE-1

Neurons require precise targeting of their axons to form a connected network and a functional nervous system. Although many guidance receptors have been identified, much less is known about how these receptors signal to direct growth cone migration. This study used C. elegans motoneurons to study growth cone directional migration in response to a repellent UNC-6 (netrin homolog) guidance cue. The evolutionarily conserved kinase MIG-15 (NIK; Nck-interacting kinase - Drosophila homolog Misshapen) regulates motoneuron UNC-6-dependent repulsion through unknown mechanisms. Using genetics and live imaging techniques, it was shown that motoneuron commissural axon morphology defects in mig-15 mutants result from impaired growth cone motility and subsequent failure to migrate across longitudinal obstacles or retract extra processes. To identify new genes acting with mig-15, a screen was performed for genetic enhancers of the mig-15 commissural phenotype, and the ezrin/radixin/moesin ortholog ERM-1, the kinesin-1 motor UNC-116 and the actin regulator WVE-1 complex, were identified. Genetic analysis indicates that mig-15 and erm-1 act in the same genetic pathway to regulate growth cone migration and that this pathway functions in parallel to the UNC-116/WVE-1 pathway. Further, time-lapse imaging of growth cones in mutants suggests that UNC-116 might be required to stimulate protrusive activity at the leading edge, whereas MIG-15 and ERM-1 maintain low activity at the rear edge. Together, these results support a model in which the MIG-15 kinase and the UNC-116-WVE-1 complex act on opposite sides of the growth cone to promote robust directional migration (Teuliere, 2011).

The Nck-interacting kinase phosphorylates ERM proteins for formation of lamellipodium by growth factors

The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. Two previously unrecognized actions of NIK are reported in this study: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressing a kinase-inactive NIK, suppressed for NIK expression with siRNA oligonucleotides, or expressing ezrin T567A that cannot be phosphorylated. These data suggest that direct phosphorylation of ERM proteins by NIK constitutes a signaling mechanism controlling growth factor-induced membrane protrusion and cell morphology (Baumgartner, 2006; full text of article).

Because activation of ERM proteins promotes F-actin anchoring to the plasma membrane, their phosphorylation by NIK likely stabilizes extending lamellipodia. However, it is predicted that NIK also regulates membrane dynamics through mechanisms independent of ERM proteins. MTLn3 cells expressing NIK-D152N, but not ezrin T567A, had constitutive, albeit small, ruffles. Substrates, including NHE1, and possibly gelsolin or cofilin, which are phosphorylated by the closely related kinases TNIK and NRK, respectively, might contribute to NIK-dependent membrane protrusion. Additionally, NIK phosphorylation of ERM proteins or other substrates might act coordinately with Nck to promote or stabilize membrane protrusions. The finding that NIK activity is necessary to phosphorylate ERM proteins in response to EGF and PDGF, but not to thrombin, is consistent with NIK binding to the Src homology 3 domain of Nck, an adaptor protein associated with receptor tyrosine kinases, and with Msn binding to DOCK, the Drosophila ortholog of Nck. Nck also binds and activates the Wiskott-Aldrich syndrome protein WASP and the WASP family verprolin homologous protein WAVE, which promote actin assembly by the Arp2/3 complex and membrane protrusion. Although NIK may act coordinately with Nck to regulate membrane dynamics, its phosphorylation of ERM proteins can occur independently of Nck because truncated NIK 1-321 lacking the C-terminal Nck-binding domain was sufficient to increase phosphorylation of ERM proteins in quiescent cells. Additionally, kinase inactive NIK-D152N did not block activation of ERK1/ERK2 by PDGF, suggesting that NIK regulates ERM protein phosphorylation downstream or independently of an ERK-mediated pathway, the latter possibility being consistent with NIK acting independently of Nck (Baumgartner, 2006).

These findings indicate that activation of ERM proteins by NIK is a cellular mechanism to promote local alterations in cell morphology in response to growth factors. This mechanism is likely important in migrating cells because NIK activity is necessary for growth factor-induced phosphorylation of ERM proteins in lamellipodia. Because activation of NIK and ezrin is implicated in processes related to tumor cell dissemination with aberrant growth factor signaling, a functional interaction between NIK and ERM proteins might play a previously unrecognized role in tumor cell metastasis (Baumgartner, 2006).

Hematopoietic progenitor kinase 1 (HPK1), an SPS1 family member

In mammalian cells, a specific stress-activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. A PCR-based subtraction strategy has been developed and applied to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. One such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen-activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3-containing mixed lineage kinase MLK-3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type-specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation (Kiefer, 1996).

Adapter proteins function by mediating the rapid and specific assembly of multi-protein complexes during the signal transduction that guards proliferation, differentiation and many functions of higher eukaryotic cells. To understand their functional roles in different cells it is important to identify the selectively interacting proteins in these cells. Two novel candidates for signaling partners of Crk family adapter proteins, the hematopoietic progenitor kinase 1 (HPK1) and the kinase homologous to SPS1/STE20 (KHS), bind with great selectivity to the first SH3 domains of c-Crk and CRKL. While KHS binds exclusively to Crk family proteins, HPK1 also interacts with both SH3 domains of Grb2 and weakly with Nck (Drosophila homolog: Dreadlocks), but not with more than 25 other SH3 domains tested. The interaction of HPK1 with c-Crk and CRKL was studied in more detail. HPK1-binding to the first SH3 domain of CRKL is direct and occurs via proline-rich motifs in the C-terminal, non-catalytic portion of HPK1. In vitro complexes are highly stable and in vivo complexes of c-Crk and CRKL with HPK1 are detectable by co-immunoprecipitation with transiently transfected cells but also with endogenous proteins. Furthermore, c-Crk II and, to a lesser extent, CRKL are substrates for HPK1. These results make it likely that HPK1 and KHS participate in the signal transduction of Crk family adapter proteins in certain cell types (Oehrl, 1998).

Ste20-related protein kinases have been implicated in the regulation of a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain; in transfected cells, it activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Candidate upstream regulators that might interact with HPK1 have been investigated. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 binds in vitro to specific proline-rich motifs in the HPK1 tail and functions synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation does not affect the binding of Grb2 to HPK1 but induces recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulate the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases (Anafi, 1997).

Transforming growth factor beta (TGF-beta)-activated kinase (TAK1) is known for its involvement in TGF-beta signaling and its ability to activate the p38-mitogen-activated protein kinase (MAPK) pathway. TAK1 is shown also to be a strong activator of c-Jun N-terminal kinase (JNK). Both the wild-type and a constitutively active mutant of TAK1 stimulate JNK in transient transfection assays. Mitogen-activated protein kinase kinase 4 (MKK4)/stress-activated protein kinase/extracellular signal-regulated kinase (SEK1), a dual-specificity kinase that phosphorylates and activates JNK, synergizes with TAK1 in activating JNK. Conversely, a dominant-negative (MKK4/SEK1 mutant inhibits TAK1-induced JNK activation. A kinase defective mutant of TAK1 effectively suppresses hematopoietic progenitor kinase-1 (HPK1)-induced JNK activity but has little effect on germinal center kinase activation of JNK. There are two additional MAPK kinase kinases, MEKK1 and mixed lineage kinase 3 (MLK3), that are also downstream of HPK1 and upstream of MKK4/SEK mutant. However, because the dominant-negative mutants of MEKK1 and MLK3 do not inhibit TAK1-induced JNK activity, it is concluded that activation of JNK1 by TAK1 is independent of MEKK1 and MLK3. In addition to TAK1, TGF-beta also stimulates JNK activity. Taken together, these results identify TAK1 as a regulator in the HPK1 --> TAK1 --> MKK4/SEK1 --> JNK kinase cascade and indicate the involvement of JNK in the TGF-beta signaling pathway. These results also suggest the potential roles of TAK1 not only in the TGF-beta pathway but also in the other HPK1/JNK1-mediated pathways (Wang, 1997).

The c-Jun amino-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) play a crucial role in stress responses in mammalian cells. The mechanism underlying this pathway in the hematopoietic system is unclear, but it is a key in understanding the molecular basis of blood cell differentiation. A novel protein kinase, termed hematopoietic progenitor kinase 1 (HPK1), has been cloned that is expressed predominantly in hematopoietic cells, including early progenitor cells. HPK1 is related distantly to the p21(Cdc42/Rac1)-activated kinase (PAK) and yeast STE20 implicated in the mitogen-activated protein kinase (MAPK) cascade. Expression of HPK1 activates JNK1 specifically, and it elevates strongly AP-1-mediated transcriptional activity in vivo. HPK1 binds and phosphorylates MEKK1 directly, whereas JNK1 activation by HPK1 is inhibited by a dominant-negative MEKK1 or MKK4/SEK mutant. Interestingly, unlike PAK65, HPK1 does not contain the small GTPase Rac1/Cdc42-binding domain and does not bind to either Rac1 or Cdc42, suggesting that HPK1 activation is Rac1/Cdc42-independent. These results indicate that HPK1 is a novel functional activator of the JNK/SAPK signaling pathway (Hu, 1996).

Other SPS1 family members

A human protein kinase (termed MST1) has been cloned and characterized. The MST1 catalytic domain is most homologous to Ste20 and other Ste20-like kinases (62-65% similar). MST1 is expressed ubiquitously, and the MST1 protein is present in all human cell lines examined. Biochemical characterization of MST1 catalytic activity demonstrates that it is a serine/threonine kinase, and that it can phosphorylate an exogenous substrate as well as itself in an in vitro kinase assay. Further characterization of the protein indicates MST1 activity increases approximately three- to four-fold upon treatment with PP2A, suggesting that MST1 is negatively regulated by phosphorylation. MST1 activity decreases approximately 2-fold upon treatment with epidermal growth factor; however, overexpression of MST1 does not affect extracellular signal-regulated kinase-1 and -2 activation. MST1 is unaffected by heat shock or high osmolarity, indicating that it is not involved in the stress-activated or high osmolarity glycerol signal transduction pathways. Thus MST1, although homologous to a member of a yeast MAPK cascade, is not involved in the regulation of a known mammalian MAPK pathway and potentially regulates a novel signaling cascade (Creasy, 1995).

A novel human member of the STE20 serine/threonine protein kinase family named mst-3 has been cloned and characterized. Based on its domain structure, mst-3 belongs to the SPS1 subgroup of STE20-like proteins, which includes germinal center (GC) kinase, hematopoietic progenitor kinase (HPK), kinase homologous to STE20/SPS-1 (KHS), kinases responsive to stress (KRS1/2), the mammalian STE20-like kinases (mst1/2), and the recently published STE20/oxidant stress response kinase SOK-1. mst-3 is most closely related to SOK-1, with 88% amino acid similarity in the kinase domain. The similarity of the mst-3 kinase domain to STE20 is 42%. The mst-3 transcript is ubiquitously expressed, and the protein has been found in all human, mouse, and monkey cell lines tested. An in vitro kinase assay shows that mst-3 can phosphorylate basic exogenous substrates as well as itself. Interestingly, mst-3 prefers Mn2+ to Mg2+ as a divalent cation and can use both GTP and ATP as phosphate donors. Like SOK-1, mst-3 is activated by autophosphorylation. However, a physiological stimulus of mst-3 activity has not been identified. mst-3 activity does not change when exposed to several mitogenic and stress stimuli. Overexpression of mst-3 wild-type or kinase dead protein affects neither the extracellular signal-regulated kinases (ERK1/2 or ERK6), c-Jun N-terminal kinase (JNK), p38, nor pp70S6 kinase, suggesting that mst-3 is part of a novel signaling pathway (Schinkmann, 1997).

The human serine/threonine protein kinases, Mst1 and Mst2, share considerable homology to Ste20 and p21-activated kinase (Pak) throughout their catalytic domains. However, outside the catalytic domains there are no significant homologies to previously described Ste20-like kinases or other proteins. To understand the role of the nonhomologous regions, a structure/function analysis of Mst1 was performed. A series of COOH-terminal and internal deletions indicates that there is an element within a central 63-amino acid region of the molecule that inhibits kinase activity. Removal of this domain increases kinase activity approximately 9-fold. Coimmunoprecipitation assays, the yeast two-hybrid procedure, and in vitro cross-linking analysis indicate that Mst1 homodimerizes and that the extreme COOH-terminal 57 amino acids are required for self-association. Size exclusion chromatography indicates that Mst1 is associated with a high molecular weight complex in cells, suggesting that other proteins may also oligomerize with this kinase. While loss of dimerization alone does not affect kinase activity, a molecule lacking both the dimerization and inhibitory domains is not as active as one that lacks only the inhibitory domain. Comparison of Mst1 and Mst2 indicates that both functional domains lie in regions conserved between the two molecules (Creasy, 1996).

A novel cDNA encoding a protein kinase (termed PASK) was isolated from rat brain. The PASK catalytic domain is most similar to Ste20-related protein kinases, showing 45.5% and 39.2% amino acid identity with human SOK1 and yeast Sps1, respectively. The amino-terminal noncatalytic domain of 71 amino acids is rich in alanine and proline and contains several proline-alanine repeats. PASK is widely expressed in rat tissues but negligible in liver and skeletal muscle. Immunohistochemical analysis reveals that PASK is localized to a distinct set of cells including neurons, adrenal glomerulosa cells, and transporting epithelia such as the epithelial cells of brain choroid plexus, the distal tubule and collecting duct of kidney, the duct of the salivary gland, and parietal cells of the stomach. Subcellular fractionation shows that PASK is present in both the cytosol and the Triton X-100-insoluble cytoskeletal fraction in brain (Ushiro, 1998).

To clarify the upstream regulatory mechanism of mitogen-activated protein kinase (MAPK), the reverse transcriptase-based polymerase chain reaction (RT-PCR) was performed with degenerate primers synthesized based on sequences conserved among the kinase domains of yeast MAPK kinase kinases (MAPKKKs), Stell, Bck1, and Byr2. Several mammalian cDNA fragments were isolated that encode kinase subdomains sharing significant sequence homology with yeast MAPKKKs. Subsequent screening of a HeLa cell cDNA library using one of these cDNA fragments as a probe resulted in the isolation of a full-length cDNA that encodes a novel protein kinase. The catalytic domain sequence of this gene product is closely related to those of budding yeast Sps1 and Ste20 protein kinases. Thus, this protein has been called YSK1 (Yeast Sps1/Ste20-related Kinase 1). The transcript of YSK1 is detected in a wide range of tissues and cells. Immunoprecipitated YSK1 shows protein kinase activity. Although YSK1 is significantly similar in its kinase domain to kinases of the yeast and mammalian MAPK pathways, the overexpression of YSK1 does not lead to the activation of the ERK (extracellular signal-regulated kinase) pathway, JNK (c-Jun NH2-terminal kinase)/SAPK (stress-activated protein kinase) pathway, or p38/Mpk2 pathway. These results suggest that YSK1 may be involved in the regulation of a novel intracellular signaling pathway (Osada, 1997).

STE20-homologous proteins have been implicated in mammalian MAP kinase pathways as important transducers of signals from p21 family GTPases. A novel STE20 family member has been cloned and called KHS, for kinase homologous to SPS1/STE20. KHS encodes a kinase of 95 kD, which is expressed in a variety of tissues. Transiently expressed fusion protein GST-KHS exhibits phosphotransferase activity toward a panel of test substrates, including myelin basic protein (MBP), which is phosphorylated by all known STE20 homologs. KHS is most closely related to another human STE20, GC kinase (74% similar in the catalytic domain), which has recently been placed upstream of the stress-activated MAP kinases (SAPKs/JNKs). KHS also activates JNK in transient coexpression experiments, suggesting a role for KHS in the stress response of fibroblasts. Characterization and comparison of the regulation of these two kinases will be important in elucidating MAP kinase signaling cascades (Tung, 1997).

The c-Jun N-terminal kinase (JNK), or stress-activated protein kinase plays a crucial role in cellular responses stimulated by environmental stress and proinflammatory cytokines. However, the mechanisms that lead to the activation of the JNK pathway have not been elucidated. A cDNA has been isolated encoding a novel protein kinase that has significant sequence similarities to human germinal center kinase (GCK) and human hematopoietic progenitor kinase 1. The novel GCK-like kinase (GLK) has a nucleotide sequence that encodes an ORF of 885 amino acids with 11 kinase subdomains. Endogenous GLK can be activated by UV radiation and proinflammatory cytokine tumor necrosis factor alpha. When transiently expressed in 293 cells, GLK specifically activates the JNK, but not the p42/44(MAPK)/extracellular signal-regulated kinase or p38 kinase signaling pathways. Interestingly, deletion of amino acids 353-835 in the putative C-terminal regulatory region, or mutation of Lys-35 in the putative ATP-binding domain, markedly reduces the ability of GLK to activate JNK. This result indicates that both kinase activity and the C-terminal region of GLK are required for maximal activation of JNK. Furthermore, GLK-induced JNK activation can be inhibited by a dominant-negative mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1) or mitogen-activated protein kinase kinase 4/SAPK/ERK kinase 1 (SEK1), suggesting that GLK may function upstream of MEKK1 in the JNK signaling pathway (Diener, 1997).

Eukaryotic cells respond to different extracellular stimuli by recruiting homologous signaling pathways that use members of the MEKK, MEK and ERK families of protein kinases. The MEKK-->MEK-->ERK core pathways of Saccharomyces cerevisiae may themselves be regulated by members of the STE20 family of protein kinases. Specific activation of the mammalian stress-activated protein kinase (SAPK) pathway by germinal center kinase (GCK), a human STE20 homolog is reported. SAPKs, members of the ERK family, are activated in situ by inflammatory stimuli, including tumour-necrosis factor (TNF) and interleukin-1, and phosphorylate and probably stimulate the transactivation function of c-Jun. Although GCK is found in many tissues, its expression in lymphoid follicles is restricted to the cells of the germinal center, where it may participate in B-cell differentiation. Activation of the SAPK pathway by GCK illustrates further the striking conservation of eukaryotic signaling mechanisms and defines the first physiological function of a mammalian Ste20 (Pombo, 1995).

REFERENCES

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