Gene name - HEM-protein/Kette
Cytological map position - 79D4
Function - signaling
Symbol - Hem
FlyBase ID: FBgn0011771
Genetic map position - 3-47
Classification - Hem family
Cellular location - surface transmembrane
|Recent literature||Hamp, J., Löwer, A., Dottermusch-Heidel, C., Beck, L., Moussian, B., Flötenmeyer, M. and Önel, S.F. (2016). Drosophila Kette/Nap1/Hem-2 coordinates myoblast junction dissolution and the Scar-WASp ratio during myoblast fusion. J Cell Sci [Epub ahead of print]. PubMed ID: 27521427
The fusion of founder (FCs) and fusion-competent myoblasts (FCMs) is crucial for muscle formation in Drosophila. Characteristic events of myoblast fusion are the recognition and adhesion of myoblasts and the formation of branched F-actin by the Arp2/3 complex at the site of cell-cell contact. At the ultrastructural level, these events are reflected by the appearance of finger-like protrusions and electron-dense plaques that appear prior to fusion. Severe defects in myoblast fusion are caused by the loss of Kette, a member of the regulatory Scar/WAVE complex. kette mutants form finger-like protrusions, but the electron-dense plaques are extended. This study shows that the electron-dense plaques in wild-type and kette mutant myoblasts resemble other electron-dense structures that are known to function as cellular junctions. Furthermore, analysis of double mutants and attempts to rescue the kette mutant phenotype with N-cadherin, wasp and genes of members of the regulatory Scar/WAVE complex reveals that Kette has two functions during myoblast fusion. First, Kette controls the dissolution of electron-dense plaques. Second, Kette controls the ratio of the Arp2/3 activators Scar/WAVE and WASp in FCMs.
In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. The gene kette (correctly termed Hem-protein, or simply Hem) is required for the normal projection of the VUM axons and interference with kette function disrupts glial migration. In spite of the fact that glial migration is disrupted in kette mutants, both the axon guidance and glial migration phenotypes have their origin in midline neuron expression and not in midline glial expression. Axonal projection defects are found for many moto- and interneurons in kette mutants. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The Hem/Kette protein is homologous to the transmembrane protein HEM-2/NAP1 (Nck-associated protein) evolutionary conserved from worms to vertebrates. In the CNS, the membrane protein Kette could be participating directly in the neuron-glial interaction at the midline, where it could act as a signal to direct glial migration. Alternatively, Kette could serve as a receptor of possibly glial-derived signals during VUM growth cone guidance. The experimental data suggest that Kette transduces information to the neuronal cytoskeleton, which is in agreement with a receptor function (Hummel, 2000).
In vitro analysis has shown a specific interaction of the vertebrate HEM-2/NAP1 with the SH2-SH3 adapter protein NCK and the small GTPase RAC1, which both have been implicated in regulating cytoskeleton organization and axonal growth. Hypomorphic kette mutations lead to axonal defects similar to mutations in the Drosophila NCK homolog dreadlocks. Furthermore, kette and dock mutants genetically interact. NCK is thought to interact with the small G proteins Rac1 and Cdc42, which play a role in axonal growth. In line with these observations, a kette phenocopy can be obtained following directed expression of mutant Cdc42 or RAC1 in the CNS midline. In addition, the kette mutant phenotype can be partially rescued by expression of an activated Rac1 transgene. These data suggest an important role of the HEM-2 protein in cytoskeletal organization during axonal pathfinding (Hummel, 2000).
Most of the Kette protein is found in the cytoplasm where it colocalizes with F-actin to which it can bind via its N-terminal domain. Some Kette protein is localized at the membrane and accumulates at focal contact sites. Loss of Kette protein results in the accumulation of cytosolic F-actin. Actin dynamics crucially depend on the ability of the protein to switch from a monomeric (G-actin) to a filamentous form (F-actin). Polymerization of F-actin starts with the de novo nucleation of an actin trimer, a process that occurs relatively slowly and requires the action of the Arp2/3 complex. Subsequent elongation is fast and cells have to prevent spontaneous actin polymerization by expressing a variety of actin-binding proteins such as profilin. The nucleation activity of the Arp2/3 complex in turn is regulated by a set of activators, such as the members of the Wasp (Wiskott-Aldrich syndrome protein) and Wave (Scar - FlyBase) families. Wave, which does not bind Cdc42, is trans-inhibited through its association with members of the Kette family: Sra1 (specifically Rac associated 1) and Abi (Abelson-interactor). Rac1 binding, presumably to Sra1, relieves the inhibitory function of this complex (Bogdan, 2003 and references therein).
To date it is still unclear how Kette could regulate the organization of the actin cytoskeleton in vivo. Since Kette has been predicted to be an integral membrane protein with six transmembrane domains, it might serve as a receptor recruiting Nck/Dock or Rho-GTPases to the membrane. Biochemical and genetic evidence reveals that Kette is found predominantly in the cytosol. Only a small amount of Kette is recruited to the plasma membrane. In vivo as well as in tissue culture models, Kette protein colocalizes with F-actin, and co-sedimentation assays reveal a direct interaction with F-actin. Within the membrane, Kette accumulates at the insertion sites of large F-actin bundles, suggesting that targeted localization of Kette may be required for its function. Loss of Kette protein leads to a Scar/Wave-dependent accumulation of F-actin within the cell. Ectopic expression of wild-type or different truncated Kette proteins in tissue culture cells or during Drosophila development does not affect F-actin formation or viability. However, expression of membrane-tethered Kette efficiently induces ectopic bundles of F-actin in a process depending on Wasp but not on Scar/Wave. These data indicate that Kette fulfils a novel role in regulating F-actin organization by antagonizing Wave and activating Wasp-dependent actin polymerization (Bogdan, 2003).
Thus Kette is able to modulate the activity of both Wasp and Scar/Wave and thus contribute to the regulation of F-actin dynamics. Wasp and Wave together with Cdc42 and Rac1 control different aspects of cortical actin dynamics (Takenawa, 2001). Cdc42 and Wasp are required for filopodia formation, whereas dominant-negative Wave disrupts the Rac1-dependent formation of branched network of F-actin bundles required to form lamellipodia (Fukuoka, 2001; Miki, 1998a). Wave localizes to membrane ruffles induced by activated Rac1 and Wasp accumulates in microspikes containing bundled F-actin (Miki, 1998b; Nakagawa, 2001; Bogdan, 2003 and references therein).
A large 500 kDa complex comprising Nap1/Kette, PIR121/Sra1, Abi, HSPC300 and Wave silences the otherwise constitutive activity of Wave in stimulating actin polymerization (Eden, 2002). The 500 kDa complex is stabilized by direct protein-protein interactions that have been demonstrated between Nap1/Kette and Abi, and Nap1/Kette and PIR121/Sra1 (also called Gex2) (Soto, 2002). The exact binding partners of Wave are presently unknown (Bogdan, 2003 and references therein).
Two modes of Wave activation have been demonstrated in vitro: (1) activated Rac1 is able to bind to PIR121/Sra1 (Kobayashi, 1998) and can relieve Wave inhibition by dissociating the Nap1/Kette, PIR121/Sra1 and Abi sub-complex (Eden, 2002); (2) PIR121/Sra1 is able to bind to the first SH3 domain of SH2SH3 adapter Nck (Kitamura, 1996; Kitamura, 1997), which is sufficient to activate the Wave complex (Eden, 2002). In vivo these two mechanisms may work at the same time to fully activate Wave (Bogdan, 2003 and references therein).
In agreement with the work of Eden (2002) disruption of Kette function leads to an excess formation of cytoplasmic F-actin. However, expression of even very high levels of wild-type Kette protein do not evoke any mutant phenotype. Thus, in wild type cells the inactive Wave complexes are already formed and Kette overexpression does not result in an additional sequestering of Wave into the silencing complexes and/or an incorporation of additional Kette into these complexes. In addition, since Kette requires Sra1 function to bind to membrane-associated adapters such as Nck, excess cytosolic Kette will not be able to reorganize subcellular Wave distribution (Bogdan, 2003).
Further support for the notion that Kette mediates repression of Scar/Wave activity stems from genetic analyses. Embryos lacking zygotic kette function display a characteristic CNS phenotype, whereas loss of zygotic Scar/Wave expression does not affect embryonic nervous system development (Zallen, 2002). The kette mutant phenotype, which is due to defects in neurite outgrowth (Hummel, 2000), is significantly suppressed by reducing the dose of Scar/Wave expression. This demonstrates that in wild-type embryos, Kette acts as a negative regulator of Scar/Wave. Similar results are obtained when Kette and or Scar/Wave expression is reduced in Drosophila S2 cells. These experiments also reveal that Scar/Wave is required for the normal subcellular distribution of Kette, which may, however, be an indirect effect caused by the disruption of the F-actin actin cytoskeleton (Bogdan, 2003).
Kette protein localizes to the plasma membrane where it accumulates in focal adhesion contacts. A prime candidate that may mediate recruitment of Kette to the membrane is the SH2 SH3 adapter protein Nck which, besides binding of the Sra1/Kette/Abi complex, also recruits numerous other proteins to focal contact sites. Among these is Wasp, which binds to the third SH3 domain of Nck (Bogdan, 2003 and references therein).
kette interacts with dock, which encodes the Drosophila Nck homolog (Hummel, 2000). Membrane recruitment of Kette is sufficient to activate actin polymerization in the cell cortex mediated by Wasp. How is this brought about? One explanation might be that recruitment of Kette to the membrane disintegrates the inhibitory Wave complex, independent of the Nck/Sra1 association. This would then lead to an excess of Wave activity and subsequently to an excess of actin polymerization. However, the genetic data clearly show that membrane bound Kette functions independent of Scar/Wave but depends on Wasp (Bogdan, 2003).
Wasp usually adopts an auto-inhibited conformation and is activated after Cdc42, Nck binding or phosphorylation. A structure-function analysis of the Drosophila Wasp has demonstrated that the Cdc42-binding domain is not necessary for function, suggesting that alternative pathways, such as phosphorylation can activate Wasp. Kette might be a part of such an alternative pathway; a genetic interaction between kette and wasp in the regulation of actin dynamics has been demonstrated. Regulation of Wasp by Kette is not mediated by direct protein-protein interaction, but probably involves Abi that is able to link Kette and Wasp. The Abl interactor (Abi) protein localizes to sites of actin polymerization at the tips of lamellipodia and filopodia and has been implicated in the cytoskeletal reorganization in response to growth factor stimulation. As a positive regulator of the non-receptor tyrosine-kinase Abelson (Abl) Abi may bring Abl into position to phosphorylate and thus activate Wasp. Abl is known to phosphorylate many proteins regulating focal adhesion and F-actin dynamics and overexpression of activated Abl induces F-Actin formation in Cdc42-independent manner. Some tyrosine kinases are activate by phosphorylation of Wasp; however, direct phosphorylation of Wasp by Abl remains to be demonstrated (Bogdan, 2003 and references therein).
Further support of the model that suggests in vivo Kette activates Wasp but suppresses Wave comes from the phenotypic analyses of Drosophila kette, wasp and scar/wave mutants. Mutations in kette have been isolated due to defects in commissure formation in the embryonic CNS (Hummel, 2000). If Kette acts via activating Wasp, similar phenotypes are expected following disruption of either gene. This is indeed the case and loss of zygotic and maternal Wasp function results in a kette-like embryonic CNS phenotype (Hummel, 2000; Zallen, 2002; Bogdan, 2003 and references therein).
In agreement with the proposed function of Kette in regulating both, Wasp and Wave, is its subcellular distribution. Whereas the majority of Kette is present in the cytoplasm to keep Wave in its inactive state (Eden, 2002) some is present in the leading edge of lamellipodia-like structures. However, the highest amounts of Kette are present at the insertion points of large F-actin bundles where N-Wasp is also present. Kette might be recruited to these focal adhesion sites via Sra1/Nck; via Wasp, it could enhance the formation of F-actin bundles (Bogdan, 2003 and references therein).
It is presently unclear just how Wasp activity results in straight F-actin bundles, whereas Wave stimulates the formation of a meshed F-actin network (Takenawa, 2001). In the cytosol, Kette may act as a scaffold protein that keeps Wave close to F-actin and recruits additional factors to F-actin such as Profilin, which not only binds to Kette but also enhances actin nucleation (Tsuboi, 2002). Thus, Kette could promote the formation of a meshed F-actin network characteristic for lamellipodia. At the membrane other proteins may interact with Kette and in this respect it is interesting to note that the F-actin crosslinking protein Filamin, which plays an important role in filopodia formation, also binds to Kette. This suggests that Kette, in addition to regulating Wasp and Wave, may also contribute to the decision whether filopodia or lamellipodia are formed (Bogdan, 2003).
A new family of proteins, termed the HEM family, has been identified which show distinct expression patterns in blood cells and the central nervous system. Through the isolation and characterization of the corresponding brain-specific Drosophila (Hem-protein) and rat orthologs (Hem-2), and through the detection of the Caenorhabditis elegans Hem-2 ortholog in the database, it has been shown that this family is conserved throughout evolution. HEM proteins show a conserved length ranging from 1118 to 1126 amino acid residues. Moreover, they are at least 35% identical with one another and harbour several conserved membrane-spanning domains, indicative for their location on the cell surface (Baumgartner, 1995).
date revised: 18 August 2003
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