Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity have not been identified. This study identifies ceramide as the activator of Drosophila MLK (Slipper) and ceramide and TNF-alpha are identified as agonists of mammalian MLK3. Slipper and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of Slipper and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-alpha also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of Slipper and MLK3, and MLK3 contributes to JNK activation induced by TNF-alpha (Sathyanarayana, 2002).
A MLK inhibitor, CEP-1347, prevents neuronal cell death induced by either the ectopic expression of MLKs or upon nerve growth factor (NGF) withdrawal (Maroney, 2001). Similarly, CEP-11004, an analog of CEP-1347, has also been reported to prevent neuronal cell death upon NGF withdrawal. However, the physiological agonist(s) of the MLKs has not yet been identified and the mechanisms involved in the regulation of MLK activity remain uncertain (Sathyanarayana, 2002).
Vertebrate JNKs are activated by proinflammatory cytokines such as TNF-alpha and IL-1ß, UV and gamma irradiation, heat shock, protein synthesis inhibitors such as anisomycin, and by other stress agents. Proinflammatory cytokines and cellular stress activate a number of MAPKKKs, which include MEKK1-4, members of MLK, ASK1, TAK1, and TPL2. These MAPKKKs in turn phosphorylate their downstream MEKs such as MKK4/SEK1 and MKK7 in the JNK pathway. The JNK pathway in vertebrates and Drosophila is quite conserved and regulates the process of embryonic dorsal closure in Drosophila. Slipper is a MAPKKK member that regulates dorsal closure in Drosophila by directly phosphorylating Hep and dMKK4. Endogenous ceramide is shown to be an activator of both Slipper and MLK3 and ceramide-induced activation of Slipper or MLK3 activates JNK without affecting the activation of p38 or ERK. These results demonstrate that TNF-alpha activates MLK3 and leads to JNK activation. These findings identify the agonists of MLK family members and have important implications for understanding the mechanism of TNF-alpha- and ceramide-mediated cell death (Sathyanarayana, 2002).
The dsRNAi-mediated gene silencing method has been used to identify an agonist that induces JNK activation through Slipper. After confirming by PCR analysis that Slipper transcripts are expressed in Drosophila S2 cells, a 672 base pair dsRNA corresponding to the 5' region of the Slipper gene was prepared. S2 cells were transfected with Slipper dsRNA and then treated with vanadate, arsenite, and a cell-permeable ceramide analog, C6-ceramide. The JNK activation was examined using phospho-JNK antibody. The dsRNAi significantly attenuates C6-ceramide-induced JNK activity but has no effect on arsenite- or vanadate-induced activation of JNK. The efficacy of dsRNAi in silencing slipper gene expression was confirmed by Northern hybridization using a Slipper probe made from a nonoverlapping sequence to dsRNA (Sathyanarayana, 2002).
To examine whether Slipper is activated by C6-ceramide in vivo, HEK293 cells were transfected with Myc-tagged Slipper and then treated with increasing concentrations of C6-ceramide for 45 min. The Slipper activation by ceramide was measured using bacterially expressed kinase-inactive SEK1/MKK4. The kinase activity of Slipper was increased by C6-ceramide in a dose-dependent fashion. The measurement of Slipper kinase activity at different time intervals after treatment with C6-ceramide (150 µM) shows that Slipper activation is time dependent. In order to exclude any contribution to changes in ceramide-induced Slipper activity by an associated kinase in the Slipper immune precipitates, the cells were transfected with a kinase-inactive mutant of Slipper (Slipper K156A); these immune precipitates did not phosphorylate SEK1 (Sathyanarayana, 2002).
Overexpression of Slipper in S2 cells and MLK3 in HEK293 cells stimulates ERK and p38 as well as JNK. The specificity of activation of MAPKs by endogenous Slipper in response to ceramide treatment was investigated. Endogenous Slipper transcripts in S2 cells were silenced posttranscriptionally by Slipper dsRNAi, and the activation of endogenous JNK, p38, and ERK by C6-ceramide was examined using appropriate phospho-specific antibodies. The Slipper dsRNAi specifically blocks JNK activation but does not affect activation of p38 or ERK by ceramide. In addition, a kinase-dead Slipper mutant (Slipper, K156A) substantially attenuates ceramide-induced JNK activation (Sathyanarayana, 2002).
Jurkat T cells were treated with increasing concentrations of C6-ceramide. Then endogenous MLK3 was immunoprecipitated with a specific MLK3 antibody, and kinase activity was measured using SEK1 (K-R) protein as a substrate in vitro. C6-ceramide (10 µM) activates endogenous MLK3 about 4.5-fold. The effect of endogenously generated ceramides on MLK3 activities was examined by exposing Jurkat T cells to bacterial sphingomyelinase (bSMase) (300 mu/ml) for different time intervals (1-6 hr). Endogenous MLK3 was immunoprecipitated and assayed for its kinase activity. SMase treatment activates endogenous MLK3 in a time-dependent manner. These data demonstrate that release of endogenous ceramide activates MLK3 in a manner similar to C6-ceramide, a cell-permeable ceramide analog (Sathyanarayana, 2002).
To prove further that MLK3, rather than another coimmunoprecipitated kinase, is responsible for the ceramide-induced increased SEK1 kinase activity, CEP-11004, a specific inhibitor of MLKs, was used to block MLK3 kinase activity. Jurkat T cells were treated with CEP-11004 (500 nM) for 20 hr in serum-free media prior to stimulation with C6-ceramide, and MLK3 activity was estimated using SEK1 as the substrate. CEP-11004 was shown to block C6-ceramide-mediated MLK3 activity. In addition, CEP-11004 completely blocks JNK activation without affecting activation of p38 and ERK activities by ceramide. It is concluded that activation of MLK3 in response to ceramide leads to specific activation of JNK and does not mediate activation of ERK or p38 (Sathyanarayana, 2002).
Recombinant Slipper was immunoprecipitated from serum-starved HEK293 cells, and the kinase assay for Slipper was performed in the presence of either natural ceramide or C6-ceramide. Natural ceramide and C6-ceramide activate Slipper directly in an in vitro activation assay in a dose-dependent manner. The effect of increasing concentrations of either natural ceramide or C6-ceramide on the direct activation of MLK3 was examined. Endogenous MLK3 was immunoprecipitated from serum-deprived Jurkat T cells and assayed for in vitro kinase activity in the presence of either natural or C6-ceramides. The MLK3 kinase activity is directly activated in an in vitro kinase assay about 3-fold above basal activity by both natural and C6-ceramide treatments (Sathyanarayana, 2002).
Jurkat T cells were treated with two different doses of TNF-alpha, either in the presence or absence of CEP-11004. The activity of immunoprecipitated endogenous MLK3 was assayed using bacterially expressed SEK1 as the substrate. TNF-alpha activates the kinase activity of MLK3 in a dose-dependent manner, and the TNF-alpha-mediated MLK3 activity is brought back to basal level by CEP-11004 treatment. In addition, Jurkat T cells were treated with CEP-11004 for 20 hr prior to treatment with two different concentrations of TNF-alpha, and the activities of JNK, p38, and ERK MAPKs were measured by immunoblot using phosho-specific antibodies. While JNK activity is significantly attenuated, p38 and ERK activities are unaltered by inhibition of MLK3 (Sathyanarayana, 2002).
There are very few known kinases that are direct targets of ceramide. PKC-zeta, Raf-1, and CAPK (ceramide activated protein kinase) have been shown to be activated by ceramide. These results show that Slipper and MLK3 are targeted by ceramide. Reaper, a Drosophila protein known to cause ceramide generation, induces apoptosis during normal Drosophila development. In addition, overexpression of reaper induces apoptosis in S2 cells. It is therefore speculated that reaper may lead to apoptosis, at least in part, via ceramide-mediated activation of Slipper (Sathyanarayana, 2002).
TNF-alpha treatment is known to elevate endogenous ceramide in cells, and JNK is known to be activated in ceramide-induced apoptosis. It has been reported that members of MAPKKK, including MEKK1, ASK1, and TAK1, are part of the TNF-alpha-triggered JNK- and p38-activation pathway. Given the findings that TNF-alpha activates endogenous MLK3 kinase activity in a dose-dependent manner and the fact that the specific MLK inhibitor CEP-11004 blocks TNF-alpha-induced JNK activation specifically, it is reasonable to suggest that MLKs, and more specifically MLK3, is the downstream MAPKKK member mediating TNF-alpha signal to JNK in mammals. It is also speculated that TNF-mediated JNK activation in Jurkat T cells may be mediated via ceramides generated in response to TNF-alpha treatment (Sathyanarayana, 2002).
In conclusion, ceramide is a potent agonist of Drosophila MLK and mammalian MLK3. The specificity of Slipper and MLK3 in mediating only ceramide-induced JNK activation without affecting ceramide-induced activation of ERK and p38 suggests an intriguing mechanism by which a specific MAPKKK can regulate different MAPK pathways in response to various physiological and pathological stimuli. These results also suggest that MLK3 plays a role in TNF-alpha-induced JNK activation. Studies showing that overexpression of MLK3 causes apoptosis and that neuronal cell death can be prevented by inhibition of the MLK family of kinases suggest a role for MLKs in apoptosis of neuronal cells. Since both ceramide and TNF are important triggers of cell death, these studies also indirectly suggest a role for MLK3 in modulating apoptosis. It is speculated that further elucidation of the role of MLKs, and specifically of MLK3, in apoptosis may ultimately facilitate the development of a targeted pharmacological intervention in neurodegenerative disorders such as idiopathic Parkinson's disease and Alzheimer's disease, both of which are associated with dysregulation of apoptosis (Sathyanarayana, 2002).
A highly diverse set of protein kinases function as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess fourteen MAPK kinase kinases (MAP3Ks) that activate Jun Kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, MAP3K signaling was assessed specificity in several JNK-dependent processes during development and stress response. The approach taken was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slipper and the TGF-beta activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure; then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. It was found that overexpression of the wildtype kinases stimulated JNK signaling in alternate contexts, so cells were capable to respond to both MAP3Ks, but with distinct outcomes. Relative to wildtype, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts including TNF-dependent cell death and innate immune signaling, however depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, these findings suggest that the selective deployment of a particular MAP3K can be attributed in part to their inherent sequence differences, cellular localization, and binding partner availability (Stronach, 2014).
Mixed lineage kinases (MLKs) belong to the family of mitogen activated protein kinase kinase kinase (MAPKKK) and cause neuronal cell death mediated through c-Jun, N-terminal kinase (JNK) pathway. Genetic studies in Drosophila have revealed the presence of an MLK termed slipper (slpr). However, its biochemical features like physiological substrate, role in different MAPK pathways and developmental and tissue-specific expression pattern have not been reported. Ectopic expression of dMLK either in Drosophila S2 or in mammalian HEK293 cells leads to activation of JNK, p38 and extracellular signal regulated kinase (ERK) pathways. Further, dMLK directly phosphorylates Hep, dMKK4 and also their mammalian counterparts, MKK7 and SEK1, in an in vitro kinase assay. Taken together, these results provide for the first time a comprehensive expression profile and new biochemical insight into dMLK/slipper (Sathyanarayana, 2003).
Slipper transcripts are expressed ubiquitously at low levels in embryos (Stronach, 2002).
In situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) analysis has revealed that dMLK is expressed in early embryonic stages, adult brain and thorax (Sathyanarayana, 2003).
Several large-scale genetic screens were undertaken in Drosophila to identify the maternal effects of zygotic lethal loci using the dominant female sterile technique. Among the collection of mutants, those mutations were sought that cause dorsal open embryonic cuticles, indicative of a failure in the process of dorsal closure. On the basis of that cuticle phenotype, several X-linked mutations were identified that produce consistent, severe, open cuticles when derived from females with mutant germ-line clones. Two mutations, 3P5 and 921, map to the same genetic region. Later, these mutations were found to define the same gene that has been called slpr, because the cuticle phenotype resembles an open shoe (Stronach, 2002).
The dorsal open cuticle phenotype of slipper mutant embryos resembles that caused by mutations in components of the JNK pathway known to regulate the progression of dorsal closure and expression of genes in the leading edge of the epidermis. To determine whether loss of slpr function affects JNK transcriptional targets, the expression of decapentaplegic (dpp) was characterized in slpr mutant embryos. It was found that dpp expression is absent from leading edge cells of approximately one-quarter of the observed embryos, consistent with the expected frequency of zygotically mutant slpr921/Y embryos. In the mutant embryos, the other tissue-specific patterns of dpp expression are unaffected. Furthermore, nearly half of all embryos derived from females with germ-line clones of the slpr3P5 allele show loss of leading edge dpp expression. Thus, the specific loss of dpp from leading edge cells of slpr mutant embryos indicates a significant reduction of JNK signaling and AP-1 activity (Stronach, 2002).
The underlying defects that contribute to aborted dorsal closure in JNK pathway mutants are not only the failure to maintain leading edge specific gene expression but also the failure to maintain a stretched epithelial cell morphology. Using a marker for epidermal cell boundaries, it was found that slpr mutant embryos also do not maintain the dramatic cell shape changes within the dorsal epithelium. In particular, the concerted elongation of leading edge cells initiates properly but fails shortly afterward. Eventually, these cells round up, the dorsal epidermis slackens laterally, and the internal organs herniate. Because the cuticle is secreted from epidermal cells, and the dorsal epidermis has failed to close dorsally, the resulting cuticle has a large hole on the dorsal side (Stronach, 2002).
The phenotypic similarities between slipper and genes encoding the JNK signaling cascade, hep, bsk, and dJun, suggest that slpr may regulate JNK signaling. To further test whether slpr mutants diminish signaling through the JNK pathway, genetic epistasis tests were performed. Activation of positive components functioning downstream of slpr may be expected to alleviate the defect caused by slpr loss-of-function. Inducible expression of a constitutive active form of the Jun transcription factor that normally serves as a substrate for phosphorylation by Bsk significantly rescues the slpr mutant phenotype. Similarly, loss-of-function mutations in downstream negative components may augment residual signaling activity to functional levels. Consistent with this line of reasoning, slpr is dominantly suppressed by reducing the dosage of a negative regulator of JNK signaling, puc, encoding a JNK phosphatase. Heterozygosity at the puc locus significantly suppresses the severe cuticle phenotype of the strong slpr921 allele, indicated by the clear reduction in size of dorsal cuticle holes. Moreover, loss of one copy of puc rescues embryos mutant for the weaker slpr3P5 allele such that they develop to adulthood. Mutant male flies emerge but are weakly viable and show no gross morphological defects. Taken together, these data support a role for slpr in JNK signal transduction, upstream of bsk (Stronach, 2002).
Total RNA transcripts were prepared from handpicked embryos mutant for each slpr allele and subjected to RT-PCR amplification with cDNA-derived primers. Sequence analysis of cDNA derived from slpr mutant embryos reveals molecular lesions associated with this locus. In fact, cDNA from both slpr alleles harbors a single-point mutation within the MLK-coding region. These lesions were verified to be present in slpr mutant genomic DNA, indicating that slpr encodes Drosophila MLK. The lesions are located in the kinase domain. The slpr921 allele produces an amino acid substitution in kinase subdomain IX that changes a highly conserved glutamate to lysine. The weaker allele, 3P5, produces a premature stop codon predicted to truncate the protein within the last kinase subdomain. In addition to finding mutations in the MLK gene in slpr mutant DNA and transcripts, the slpr mutant phenotype was rescued with a transgene encompassing 14 kb of genomic DNA, including CG2272 and the closely neighboring CG15339 gene. Rescue with this genomic fragment, coupled with the presence of mutations in the MLK-coding region in slpr mutant DNA, confirms that MLK is encoded by the slpr gene (Stronach, 2002).
In the process of dorsal closure, Rac1 appears to be a primary upstream activator of JNK signaling. To position slipper function in the JNK pathway relative to the GTPase, additional genetic epistasis tests were performed. Due to the difficulty in following all relevant chromosomes in the embryo, the adult Drosophila eye was used to evaluate a possible genetic interaction. Expression of wild-type dRac1 in the eye, under regulation by the glass promotor, causes a rough, glazed appearance. This phenotype is dominantly suppressed by 50% reduction in the levels of JNK signal transducers, msn, slpr, hep, and bsk. Heterozygosity at the puc locus, encoding a negative regulator of JNK signaling acting in opposition to bsk, dominantly enhances the Rac1-induced rough eye. To assess whether other putative JNKKKs in Drosophila can interact in this assay, Drosophila TGF-ß activated kinase 1 (Tak1) was included in this analysis. Unlike slpr, removal of one copy of Tak1 has no effect on the Rac1-induced eye phenotype. These data suggest that there is a dosage-sensitive interaction between the JNK pathway and Rac1 function in this tissue whereby increased Rac1 activity can be suppressed by reduction in downstream components, including slpr. Although it is not known whether Tak1 is expressed in the eye or at what level, Tak1 shows no genetic interaction with GMR-dRac1. Thus, slpr appears to be a relevant JNKKK in this assay. Taken together, these epistasis tests are consistent with slpr function being required downstream of Rac1 and upstream of bsk (Stronach, 2002).
The acquisition of polarity within the plane of an epithelium is important for the function of epithelial layers in addition to the polarity associated with the apical-basal axis. Previous studies of epithelial planar polarity have implicated a noncanonical Frizzled pathway. The current model is that Fz1 activates JNK activity through a pathway that includes Dishevelled (Dsh) and the RhoA GTPase. Tissue polarity mutants show disruptions in the polarized orientation of hairs and bristles. To determine whether Slpr links RhoA activity to the JNK pathway, the adult cuticle phenotype of slpr mutant clones was examined in both the wing and notum, in which the tissue polarity phenotype is clearly visible. Loss of slpr activity in mutant clones is not associated with any obvious polarity defects, suggesting that another JNKKK may mediate the activation of JNK activity during the establishment of planar polarity. It is unlikely that Tak1 is this JNKKK, because dTAK mutant animals are homozygous viable with no apparent tissue polarity defects (Stronach, 2002).
Loss-of-function slpr mutant clones in adult tissues have normal epithelial planar polarity. Adult wings of wild-type flies show a uniform polarized orientation of hairs, pointing distally downward. slpr clones marked by the gene yellow (y) are only visualized in bristles at the margin, yet large y;slpr clones show normal orientation of hairs neighboring the margin. This is in comparison with the disrupted orientation of hairs in the dsh1 wing. Planar polarity is also evident in hairs of the adult notum. Smaller hairs in the region of the clone show normal polarized orientation, pointing posteriorly downward. In contrast, the hairs of dsh1 mutant nota are randomly oriented, indicating a defect in planar polarity (Stronach, 2002).
Mixed Lineage Kinases (MLKs) function as Jun-N-terminal kinase (JNK) kinase kinases to transduce extracellular signals during development and homeostasis in adults. slipper (slpr), which encodes the Drosophila homolog of mammalian MLKs is implicated in activation of the JNK pathway during embryonic dorsal epidermal closure. To further define the specific functions of Slpr, the phenotypic consequences of slpr loss- and gain-of function was studied throughout development, using a semi-viable maternal effect allele and wildtype or dominant negative transgenes. From these analyses it was confirmed that failure of dorsal closure is the null phenotype in slpr germline clones. In addition, there is a functional maternal contribution, which can suffice for embryogenesis in the zygotic null mutant, but rarely suffices for pupal metamorphosis, revealing later functions for slpr as the maternal contribution is depleted. Zygotic null mutants that eclose as adults display an array of morphological defects, many of which are shared by hep mutant animals, deficient in the JNK kinase (JNKK/MKK7) substrate for Slpr, suggesting that the defects observed in slpr mutants primarily reflect loss of hep-dependent JNK activation. Consistent with this, the maternal slpr contribution is sensitive to the dosage of positive and negative JNK pathway regulators, which attenuate or potentiate Slpr-dependent signaling in development. Though Slpr and TAK1, another JNKKK family member, are differentially used in dorsal closure and TNF/Eiger-stimulated apoptosis, respectively, a Tak1 mutant shows dominant genetic interactions with slpr suggesting potential redundant or combinatorial functions. Finally, it was demonstrated that SLPR overexpression can induce ectopic JNK signaling and that the Slpr protein is enriched at the epithelial cell cortex (Polaski, 2006).
Previous genetic studies have established a role for SLPR/MLK in JNK pathway activation during embryonic tissue closure. This study characterizes the phenotype of an allele affecting postembryonic development as well as protein products encoded by wildtype and mutant alleles. slprBS06 is a newly isolated null allele that encodes an early nonsense mutation and consequently, no protein product is detected in mutant tissue clones or by Western immunoblot. Phenotypic comparison between the null allele and existing alleles confirms the role for SLPR in dorsal closure, clarifies that slpr has a maternal contribution and that the prior two alleles encode dominant negative proteins, and uncovers additional roles for Slpr in metamorphosis of the adult (Polaski, 2006).
The severe dorsal open phenotype of slprBS06 germline clones, maternally and zygotically mutant, indicates that dorsal closure is the earliest requirement for Slpr in embryogenesis and that a failure of dorsal closure is the null phenotype, consistent with the phenotype of the previously characterized 921 and 3P5 slpr alleles. In contrast though, most slprBS06 zygotic mutants survive embryogenesis and adult mutant males are recovered at a low frequency. These males display several visible morphological phenotypes of variable penetrance, presumably as a consequence of the eventual depletion of functional maternal product. These observations indicate that the maternal slpr gene product is nearly sufficient for embryogenesis in the absence of zygotic product, but rarely provides enough function for metamorphosis, revealing additional roles for Slpr in postembryonic processes (Polaski, 2006).
Defects observed in mutant adults implicate Slpr function for proper metamorphosis of the genital discs, dorsal abdomen and notum, maxillary palps, and wing. Females show somatic defects during oogenesis demonstrating that Slpr is required for proper morphology of the chorionic dorsal appendages. Many of the defects, including those affecting the thorax, genitals and dorsal appendages, have been documented previously to result from loss of JNK signaling. Thus, the data reported here implicate Slpr as the upstream JNKKK family member required for JNK activation in these processes. The current study also suggests that slpr function is mediated primarily, if not entirely, via HEP/MKK7 and the JNK pathway, as evidenced by the fact that hep mutants share in common all of the defects observed in slpr mutants and that reducing the dosage of two known negative regulators of JNK signaling, puc and raw, suppresses the slpr phenotypes. In light of these results, it will be informative to systematically test whether, in vivo, the mammalian MLK proteins activate alternative substrates and pathways as has been suggested from tissue culture studies (Polaski, 2006).
Given that the slpr921 and slpr3P5 alleles are phenotypically more severe in zygotic mutants than the null allele indicates that the encoded products have dominant negative activity, which interferes with the functional pool of maternal slpr gene product. This is consistent with the molecular nature of the alleles, which predict that full length (921) or partial (3P5) protein product would be expressed in the mutants. Indeed, clonal analysis and immunofluoresence staining confirm the expression of mutant protein in slpr921 animals. Protein levels in slpr3P5 mutant tissue appear reduced relative to wildtype, but the encoded fragment retains the SH3 domain and most of the kinase domain, each of which, if folded properly, could engage in protein-protein interactions. Similarly, the catalytically inactive, full length protein encoded by slpr921 retains several functional protein interaction motifs, which could account for the antimorphic properties of the protein (Polaski, 2006).
What candidates are known to interact with the various regions of the Slpr protein to account for dominant negative activity? By analogy with the mammalian MLK proteins, at least three recognized domains have potential protein binding activity. The leucine zipper mediates homodimerization, which is requisite for autophosphorylation and substrate activation. Mutant Slpr protein in slpr921 cells might trap wildtype protein in unproductive dimers. The CRIB domain binds to the activated form of the small GTPases, Cdc42 and Rac1, both implicated in dorsal closure. Titration of these GTPases by non-catalytic Slpr921 protein could also contribute to dominant interference of the wildtype Slpr protein. Finally, the N-terminal SH3 domain, retained in the proteins encoded by both slpr3P5 and slpr921, has the potential to engage in both intra- and intermolecular interactions. The SH3 domain of mammalian MLK3 can bind to a region between the LZ and CRIB domains through a critical proline residue that is conserved in Drosophila Slpr. The postulated intramolecular binding is thought to negatively regulate MLK activation by locking the protein in a closed conformation (Polaski, 2006).
This type of autoinhibition has been demonstrated for other modular kinases, such as Src tyrosine kinase. Also, the SH3 domain may serve as a docking site for upstream activating kinases of the Ste20 family, for which titration by an SH3-containing protein fragment could impair signal relay to the JNK pathway. Therefore, the modular domain organization of the Slpr protein with the potential for multiple regulatory protein interactions is likely to explain why residual mutant protein is more detrimental than complete loss of protein in the null mutant (Polaski, 2006).
Why then does overexpression of an engineered kinase dead Slpr transgene that is functionally equivalent to the protein encoded by slpr921 have such mild consequences in the embryo? Evidence suggests that it is due to a substantial functional maternal component, in addition to the zygotic contribution, because reducing the maternal pool in embryos derived from slpr-/+ heterozygous mothers exacerbates the effect of dominant negative Slpr transgene expression. Further support for the function of the maternal gene product is demonstrated by the sensitivity of the maternal contribution to the dosage of additional positive and negative JNK pathway regulators, which has been monitored as the extent of recovery of slprBS06 mutant adult males (Polaski, 2006).
Given that the maternal product is nearly sufficient for mutant males to survive to adulthood, it is curious that immunodetection of endogenous Slpr protein in the embryo has been difficult relative to the ease with which transgenic protein can be detected. This may suggest that the maternal pool of slpr gene product is largely mRNA rather than protein, that an active mechanism exists to maintain low levels of embryonic protein, or that protein complexes mask the ability to detect the epitope on the Slpr protein, either of which could be overcome by the abundant expression of exogenous transgenic protein. Though the mechanism is not clear, the genetic loss-of-function and overexpression data together indicate that certain cell types or developmental contexts are sensitive to the levels of Slpr protein in modulating JNK signaling. For example, while exogenous Slpr can induce JNK signaling in embryonic dorsal ectoderm cells, normally limited for JNK activity, not all cells are equally inducible, suggesting there may be other limiting components or brakes that modulate the precise levels of JNK activity in cells. Inferring function from overexpression experiments in the absence of loss-of-function data can be misleading however, because wildtype transgene expression can stimulate JNK signaling promiscuously, or at least where the endogenous protein appears not to be required. For example, transgenic expression of either Slpr or TAK1 can induce JNK signaling ectopically in the embryonic dorsal ectoderm under the control of pnr-GAL4, even though endogenous levels of TAK1 cannot provide enough JNK signaling activity in slpr null embryos to rescue dorsal closure. Moreover, Tak1 mutants are viable providing corroborating evidence that Tak1 is not required for dorsal closure. In sum, JNK signaling activity may be at a threshold level in most cells, easily overactivated by expression of many different upstream regulators, but whose selective use in physiological circumstances is only revealed through analysis of loss-of-function (Polaski, 2006).
The combined gain- and loss- of function analysis for Slpr described here supports two proposed mechanisms of signaling specificity among JNKKK proteins; first, that individual family members are used selectively in particular contexts and second, that potential combinatorial or redundant functions may exist among members with common substrates. With respect to TNF/Eiger signaling, both loss-of-function analysis and dominant negative constructs consistently implicate TAK1 rather than Slpr in this JNK-dependent response. In addition, JNK-dependent developmental morphogenetic events, in particular dorsal closure in the embryo, selectively require Slpr. It was consistently observed that the slprBS06, Tak1 double mutant is more severe than either single mutant alone, suggesting that there are likely to be additional, perhaps redundant, functions of Slpr and TAK1 that are only revealed in the double mutant. These functions have yet to be investigated in detail (Polaski, 2006).
At face value, genetic analysis has allowed the assignment of Slpr to mediate many JNK-dependent morphogenetic events and TAK1 to mediate JNK-dependent homeostatic responses including apoptosis and immunity. However, it is still unclear whether the selective functions reflect differential transcriptional responses or whether cell and developmental context shapes what appear to be quite different cellular behaviors. In other words, though the developmental defects that arise as a consequence of loss of Slpr function may suggest a common failure in cell shape change or cytoskeletal functions, similar to the defects that underlie the failure of embryonic dorsal closure in the mutants, that assumption may be too simplistic. It is a formal possibility that Slpr could regulate additional or alternative JNK-dependent cell responses in distinct contexts. For example, though Slpr appears not to mediate TNF-induced apoptosis in the Drosophila eye under conditions where Eiger is overexpressed, the male genital misrotation phenotype observed in slpr mutants may be linked to JNK-dependent developmental programmed cell death. Defective genital rotation is observed in certain viable alleles of hid, encoding a protein with proapoptotic function. The basis of the rotation defect may be due to an excess of genital disc cells, similar to the embryonic defects in head involution, the namesake phenotype of hid (Polaski, 2006).
Thus, JNK signaling and HID function are both required for proper genital rotation and interestingly, there is precedent for hid being a transcriptional target of the JNK pathway downstream of Eiger. Thus, it will be important to determine specifically whether Slpr mediates JNK-dependent HID expression or even apoptosis in imaginal discs, or whether the requirement for Slpr in male genital rotation is unrelated to apoptosis. More generally, a full understanding of Slpr function will require systematic definition of the molecular and cellular mechanisms that underlie the morphological defects. If Slpr functions to regulate different outputs in different contexts, determining what regulates a selective response will be of considerable interest for future studies (Polaski, 2006).
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date revised: 5 June 2007
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