slipper: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - slipper

Synonyms - Mlk2

Cytological map position - 7D17

Function - signaling

Keywords - dorsal closure

Symbol - slpr

FlyBase ID: FBgn0030018

Genetic map position -

Classification - mixed lineage kinase

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene |
BIOLOGICAL OVERVIEW

The Jun kinase (JNK) pathway has been characterized for its role in stimulating AP-1 activity and for modulating the balance between cell growth and death during development, inflammation, and cancer. In mammals, six families of JNKKKs are known to serve as upstream regulators of JNK activity (MLK, LZK, TAK, ASK, MEKK, and TPL); however, the specificity of kinase utilization for transducing a distinct signal is poorly understood. In Drosophila, JNK signaling plays a central role in dorsal closure, controlling cell fate and cell sheet morphogenesis during embryogenesis. Notably, in the fly genome, there are single homologs of each of the mammalian JNKKK families. Mutations have been identified in one of those, a mixed lineage kinase, named slipper (slpr). slipper is required for basket/JNK activation during dorsal closure. Furthermore, other putative JNKKKs cannot compensate for the loss of slpr function and, thus, may regulate other JNK or MAPK-dependent processes (Stronach, 2002).

Eukaryotes transduce a variety of signals by use of a well-conserved kinase phosphorylation cascade culminating in activation of a class of proteins, generically referred to as mitogen-activated protein kinases (MAPKs). Typically, activated MAPKs phosphorylate transcription factor substrates, thus, ultimately regulating gene expression and cellular behavior. MAPKs are the most downstream kinases in a tripartite module of protein kinases. As such, MAPKs are phosphorylated and activated by dual specific MAPK kinases (MAPKKs/MKKs), which are themselves phosphorylated and activated by another family of upstream serine/threonine kinases, the MAPK kinase kinases (MAPKKKs, MKKKs) (Stronach, 2002).

Signals that stress cells during inflammation or changing environmental conditions induce a particular class of MAPKs called the stress-activated protein kinases (SAPKs). SAPKs are also referred to as JNKs because they phosphorylate the NH2 terminus of cJun, which together with cFos constitutes the AP-1 (activator protein-1) transcriptional complex that regulates primary response genes. As evidenced by the constitutive active nature of the viral v-jun and v-fos oncogenes described over a decade ago, proper regulation of AP-1 activity under varied conditions is critical for normal cellular behavior. In certain cell types, simply overexpressing proteins that compose AP-1 or positively regulate its activity can lead to transformation, whereas, in other contexts, JNK stimulation can induce apoptosis. Given the diverse and often conflicting roles ascribed to JNK signal transduction, it is important to define how distinct extracellular signals are specifically coupled to the JNK module and thus to the proper transcriptional output via JNK targets. Furthermore, characterization of a growing number of mammalian kinases with the ability to stimulate JNK activation in cell assays highlights the complexity and potential lack of specificity among JNKKKs. Many questions remain, such as why are there so many distinct kinase families at this level of the signaling hierarchy? Perhaps each kinase family interprets a distinct upstream signal or participates in unique cellular and developmental processes. Loss-of-function analysis will be a crucial step in answering these questions (Stronach, 2002).

Toward this end, a genetic approach has been taken to define regulators of the JNK pathway in vivo using Drosophila dorsal closure as a model system for JNK-dependent signal transduction. In the fly embryo, dorsal closure involves cell sheet morphogenesis, whereby the dorsal ectoderm moves from an initial lateral position toward the dorsal midline to enclose the mature embryo in a continuous protective epidermis. Mutations in the fly homologs of the vertebrate members of the JNK signal transduction pathway, hemipterous (hep), a JNKK related to vertebrate MKK7, basket (bsk), a JNK, dJun, and dFos, encoded by kayak (kay) result in dorsal closure failure, and thus, embryonic lethality. Despite significant progress in defining gene functions required for JNK signaling and dorsal closure in general, there are still several obvious missing players in the signaling machinery. slpr is an additional regulator of JNK activity. Cloning reveals that slpr encodes a member of the mixed lineage kinase (MLK) family of Jun kinase kinase kinases. These analyses provide the first demonstration of an absolute requirement for MLKs in the regulation of JNK signaling during morphogenesis in vivo and establish a specific genetic link between MLK, MKK7, and JNK (Stronach, 2002).

Considerable research has shown that dorsal closure is regulated by a canonical MAP kinase-signaling module remarkably similar to the mammalian stress-signaling pathway involving a phosphorylation cascade that culminates with activation of JNK and its substrate, Jun. During dorsal closure, JNK signaling mediates gene expression, accumulation of cortical cytoskeleton, and movement of the epidermis toward the dorsal midline. Loss of signaling results in defective cell shape changes, failed closure, and lethality. Precise regulation of signaling activity in leading edge cells is necessary for proper closure, however, the identities of signals and receptors that trigger JNK activation and link membrane components to the kinase cascade are still largely unknown. Genetic identification of mutants that fail to undergo dorsal closure may uncover such components. One such mutant, slpr, displays a severe dorsal open cuticle phenotype indicative of a complete failure of closure. Mutations in slpr phenocopy known JNK pathway mutants in Drosophila (Stronach, 2002).

The genetic analysis of slpr has uncovered the identity of a missing component in current understanding of JNK signal transduction during epithelial morphogenesis. Cloning has revealed that slpr encodes a mixed lineage kinase highly related to mammalian MLKs that have been shown to stimulate JNK activity when overexpressed in cell-based assays (Rana, 1996; Teramoto, 1996). Several lines of evidence indicate that slpr encodes Drosophila MLK. (1) A transgenic copy of the MLK gene is sufficient to rescue slpr mutants. (2) The MLK-coding sequence is mutated in genomic DNA and cDNA from slpr mutant embryos. Loss-of-function, dorsal open slpr mutants harboring molecular lesions in the kinase domain provides compelling genetic evidence that MLK proteins play a critical role in JNK signal transduction during morphogenesis, and that this role requires the kinase activity. (3) Genetic epistasis data support the biochemical data that MLK proteins serve to join upstream GTPase activity to downstream AP-1 transcriptional activity. This represents the first genetic evidence that MLKs are relevant physiological regulators of JNK activity in vivo (Stronach, 2002).

In Drosophila, signaling through JNK is required for a variety of processes including morphogenesis in the embryo and the adult, epithelial planar polarity, immunity, and apoptosis. The data indicate that slpr is absolutely required for dorsal closure and no essential role has been detected for slpr in either immunity or tissue polarity. Clonal analysis of slpr in the wing and notum does not indicate a role for slpr in planar polarity of hairs; however, lack of a polarity phenotype in clones has been noted for other members of the JNK pathway. Despite this, a role for members of the JNK cascade in the establishment of planar polarity has been proposed on the basis of the ability of loss-of-function mutations to suppress a polarity phenotype associated with gain-of-function Fz or dsh. Together, these observations raise the possibility that a redundant function may mask a requirement for slpr in planar polarity and additional experiments will be necessary to uncover this function (Stronach, 2002).

Studies on null Drosophila TGF-ß activated kinase 1 (Tak1) mutant flies have shown a requirement for dTAK in innate immunity to microbial infection. Further analysis of dTAK mutant flies indicates that dTAK does not play a major role, if any, in either tissue polarity or dorsal closure, because dTAK mutant flies are homozygous viable and fertile with no visible phenotype. Altogether, it is proposed that different JNKKKs are activated by different signals, and thus dedicated to different developmental processes. Further work will be needed to clarify this hypothesis. This includes the characterization of the mutant phenotypes of the other putative JNKKKs (LZK, ASK, MEKK4) and a clear demonstration that these kinases activate JNK activity in vivo. Tak1 has only been shown to activate JNK in overexpression assays, both in vitro and in vivo. Moreover, during an immune response, Tak1 activates NFkappaB. It has not yet been reported whether Tak1 can stimulate JNK activity during immune challenge. Possibly, the specificity of these JNKKKs will reside in their abilities to activate not only the JNK pathway but also additional pathways, such as NFkappaB in the case of Tak1 (Stronach, 2002).

To date, the increasing number of mammalian protein kinases implicated as JNKKKs on the basis of sequence information and ability to activate or block JNK signaling in cell transfection or other in vitro assays underscores the potential for stunning complexity at the upper tiers of the pathway. This notion serves to reinforce and emphasize the need for thorough genetic analysis of these proteins. To date, targeted gene disruptions in the mouse have been reported only for the MEKK family, yet these studies suggest that individual MEKKs regulate unique cell behaviors including morphogenesis and apoptosis. By taking advantage of the reduced gene redundancy in Drosophila relative to mammalian systems and the various defined processes in which JNK signaling is required, questions of specificity at the level of cellular and developmental responses as well as at the level of upstream and downstream partners are open to investigation (Stronach, 2002).


GENE STRUCTURE

cDNA clone length - 5129

Bases in 5' UTR - 724

Exons - 9

Bases in 3' UTR - 919


PROTEIN STRUCTURE

Amino Acids - 1161

Structural Domains

CG2272/slipper is predicted to encode a protein with sequence homology to mammalian members of the MLK family. These kinases have been implicated in JNK signal transduction at the level of the JNKKK. MLK proteins consist of an N-terminal src-homology 3 (SH3) domain, followed by a kinase domain with key amino acids resembling the known signatures of both tyrosine and serine/threonine kinases; hence the name 'mixed lineage kinase' (Dorow, 1993; Katoh, 1995). Just C-terminal to the kinase domain are two leucine zipper (LZ) motifs thought to mediate dimerization (Leung, 1998; Vacratsis, 2000). Following the LZ region, there is a short stretch of amino acids with homology to Cdc42/Rac interacting binding (CRIB) motifs that may serve as a site of interaction with those small GTPases. The remainder of the Slpr protein displays no known motifs and has no significant homology to its mammalian counterparts. Slpr is most related to the human MLK family of three proteins with up to 56% amino acid identity within the N-terminal region, consisting of ~510 amino acids, including the kinase domain (Dorow, 1993, 1995; Gallo, 1994; Ing, 1994; Katoh, 1995). The structural features of MLK proteins have provided clues to their function and support their role as important signal transducers for the JNK pathway, conveying signals from upstream GTPases to downstream JNKKs, (Rana, 1996, Teramoto, 1996, Tibbles, 1996, Hirai, 1997, and Merritt, 1999 as cited by Stronach, 2002).

Phosphorylation sites have been identified that act in a kinase activation loop that is key to MLK-3 activation via both autophosphorylation and hematopoietic progenitor kinase 1 phosphorylation (Leung, 2001). Interestingly, sequence comparisons of the kinase activation loop among MLK family members reveal that Slpr shares two critical target phosphorylation sites: Thr 287, analogous to Thr 277 in human MLK3 required for kinase autophosphorylation, and Ser 291, analogous to Ser 281, targeted by members of the GCK-IV family of MKKKKs related to the fly Misshapen (Msn) protein kinase. Thus, as suggested by results from mammalian studies, it is possible that Msn, Slpr, and dRac1 participate in a ternary signaling complex to stimulate JNK signal transduction (Stronach, 2002).


EVOLUTIONARY HOMOLOGS

The MAPK cascades regulate a wide variety of cellular functions, including cell proliferation, differentiation, and stress responses. A novel MAP kinase kinase kinase (MAPKKK), termed MLTK (for MLK-like mitogen-activated protein triple kinase), has been identified whose expression is increased by activation of the ERK/MAPK pathway. There are two alternatively spliced forms of MLTK, MLTKalpha and MLTKbeta. When overexpressed in cells, both MLTKalpha and MLTKbeta are able to activate the ERK, JNK/SAPK, p38, and ERK5 pathways. Moreover, both MLTKalpha and MLTKbeta are activated in response to osmotic shock with hyperosmolar media through autophosphorylation. Remarkably, expression of MLTKalpha, but not MLTKbeta, in Swiss 3T3 cells results in the disruption of actin stress fibers and dramatic morphological changes. A kinase-dead form of MLTKalpha does not cause these phenomena. Inhibition of the p38 pathway significantly blocks MLTKalpha-induced stress fiber disruption and morphological changes. These results suggest that MLTK is a stress-activated MAPKKK that may be involved in the regulation of actin organization (Gotoh, 2001).

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocks JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response is selective because CEP-1347 does not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro is competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 leads to death in Chinese hamster ovary cells, and CEP-1347 blocks this death at doses comparable to those that inhibit MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death (Maroney, 2001).


slipper: Developmental Biology | Regulation | Effects of Mutation | References

date revised: 7 March 2002

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.