genghis khan

Protein Interactions

Genghis khan was isolated in a search for proteins that physically interact with the Drosophila small GTPases Rac1 and Cdc42. Gek does not bind to Cdc42N17, a dominant negative mutant that preferentially stays in the GDP-bound state. The ability of Gek to bind Cdc42 in its GTP-bound form (but not in its GDP-bound form) suggests that Gek is an effector of Cdc42. A mutation in the Cdc42 effector domain (A35), which is important for signaling to downstream targets, eliminates Gek binding. Gek does not bind to Drosophila Rac. Deletion of three residues in Gek, which correspond to three conserved residues of the Cdc42/Rac interactive binding (CRIB) domain, disrupt Gek's binding to Cdc42. Gek exhibits kinase activity using histone as a substrate (Luo, 1997).

Cdc42, a Rho family GTPase that acts through Gek

The small GTPases of the Rac/Rho/Cdc42 subfamily are implicated in actin cytoskeleton-membrane interaction in mammalian cells and budding yeast. The in vivo functions of these GTPases in multicellular organisms are not known. The Drosophila homologs of rac and CDC42 have been cloned: Drac1, and Dcdc42. They share 70% amino acid sequence identity with one another; both are highly expressed during neuronal and muscle differentiation in nervous system and mesoderm, respectively. Putative constitutively active and dominant-negative Drac1 proteins were expressed in these tissues. When expressed in neurons, Drac1 mutant proteins cause axon outgrowth defects in peripheral neurons without affecting dendrites. When expressed in muscle precursors, they cause either complete failure of myoblast fusion or abnormality of fusion. Expressions of analogous mutant Dcdc42 proteins cause qualitatively distinct morphological defects, suggesting that similar GTPases in the same subfamily have unique roles in morphogenesis (Luo, 1994).

The wing of Drosophila is covered by an array of distally pointing hairs. A hair begins as a single membrane outgrowth from each wing epithelial cell; its distal orientation is determined by the restriction of outgrowth to a single distal site on the cell circumference. The roles of Cdc42 and Rac1 have been examined in the formation of wing hairs. Cdc42 is required for localized actin polymerization in the extending hair. Interfering with Cdc42 activity by expression of a dominant negative protein abolishes both localized actin polymerization and hair outgrowth. In contrast, Rac1 is important for restricting the site at which hairs grow out. Cells expressing the dominant negative Rac1N17 fail to restrict outgrowth to a single site and give rise to multiple wing hairs. This polarity defect is associated with disturbances in the organization of junctional actin and also with disruption of an intricate microtubule network that is intimately associated with the junctional region. Apical junctions and microtubules are involved in structural aspects of hair outgrowth. The apical microtubules that point distally elongate during hair formation and fill the emerging wing hair. As the hair elongates, junctional proteins are reorganized on the proximal and distal edges of each cell (Eaton, 1996).

The Rho subfamily of GTPases has been shown to regulate cellular morphology. A new member of the Rho family, known as RhoL, has been identified. Rhol is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary causes nurse cells to collapse and fuse together. Mutant forms of Cdc42 mimic this effect. Expression of constitutively active RhoL leads to nurse cell subcortical actin breakdown and disruption of nurse cell-follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity is specifically required for migration of a subset of follicle cells called border cells. All three activities are necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that the three GTPases have cell type-specific effects on morphogenesis (Murphy, 1996).

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

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

Reverse genetic analysis in Drosophila has been greatly aided by a growing collection of lethal P transposable element insertions that provide molecular tags for the identification of essential genetic loci. However, because the screens performed to date have generated primarily autosomal P-element insertions, this collection has not been as useful for performing reverse genetic analysis of X-linked genes. A reverse genetic screen has been designed that takes advantage of the hemizygosity of the X chromosome in males together with a cosmid-based transgene, which serves as an autosomally linked duplication of a small region of the X chromosome. The efficacy and efficiency of this method is demonstrated by the isolation of mutations in Drosophila homologs of two well-studied genes: the human Neurofibromatosis 2 tumor suppressor and the yeast Cdc42 gene. The method described should be of general utility for the isolation of mutations in other X-linked genes, and should also provide an efficient method for the isolation of new alleles of existing X-linked or autosomal mutations in Drosophila (Fehon, 1997).

DEVELOPMENTAL BIOLOGY

Embryonic

Gek is present in precellular embryos, suggesting a maternal origin (Luo, 1997).

Effects of mutation or deletion

To study the in vivo function of gek, mutations in the Drosophila gek locus were generated. Homozygous gek mutants die as larvae. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. Although the number of eggs produced from gek mosaic females is comparable to that from control mosaic females, only 5% of the eggs can be fertilized. The F-actin rich ring canals between nurse cells and between the oocyte and the nurse cells are present in egg chambers mutant for gek. However, the cortical F-actin that surrounds the nurse cells appears abnormal, and ectopic F-actin blobs are frequently observed in the nurse cells and oocytes. Interestingly, ectopic F-actin blobs in nurse cells (but not in oocytes) also contain proteins normally restricted to ring canals, including Hu-li tai shao (Hts), kelch, and antigens recognized by antibodies to phosphotyrosine. Hts and Kelch are homologous to the actin binding proteins adducin and scruin, respectively. These findings suggest that loss of Gek function in germ-line cells alters the distribution of F-actin and actin binding proteins. In late-stage oocytes, ectopic actin polymerization is manifested as numerous F-actin spheres surrounding the yolk granules. These phenotypes can be rescued by a wild-type gek transgene. These results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42 (Luo, 1997).

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Fic, W., Bastock, R., Raimondi, F., Los, E., Inoue, Y., Gallop, J. L., Russell, R. B. and St Johnston, D. (2021). RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion. J Cell Biol 220(4). PubMed ID: 33646271

Harden, N., et al. (1996). A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures. Mol. Cell. Biol. 16(5): 1896-1908. PubMed Citation: 8628256

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Luo, L., et al. (1997). Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization. Proc. Natl. Acad. Sci. 94(24): 12963-12968. PubMed Citation: 9371783

Matsui, T., et al. (1996). Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 15(9): 2208-2216. PubMed Citation: 8641286

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Roberts, R., et al. (1997). Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice. Proc. Natl. Acad. Sci. 94(24): 13221-13226. PubMed Citation: 9371827

Wissmann, A., et al. (1997). Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape. Genes Dev. 11(4): 409-422. PubMed Citation: 9042856

date revised: 25 August 2021  

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