Gene name - misshapen
Cytological map position - 62E6--62E7
Function - protein kinase
Symbol - msn
Genetic map position -
Classification - SPS1 family kinases
Cellular location - presumably cytoplasmic
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).
Msn shows higher homology to those kinases that phosphorylate serine and threonine than to those that phosphorylate tyrosine residues. The kinase domain is at the N-terminus of the protein and is followed by a long C-terminal domain. The last 333 amino acids of the protein are 79% identical to a sequence present in C. elegans and identified by the genome project (Treisman, 1997).
The Drosophila Msn and C. elegans Mig-15 proteins are highly homologous to mammalian NIK, an activator of the JNK module. These three proteins share the same overall structure, containing an amino-terminal kinase domain and a carboxy-terminal putative regulatory domain. Moreover, these three proteins are highly conserved within both the kinase domain and the carboxy-terminal regulatory domain (Su, 1998).
date revised: 7 November 98
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