Src oncogene at 42A: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Src oncogene at 42A

Synonyms -

Cytological map position - 42A6--7

Function - signaling

Keywords - JNK cascade, adherens junction, dorsal closure, eye, oncogene

Symbol - Src42A

FlyBase ID: FBgn0264959

Genetic map position - 2R

Classification - Src homology 3 (SH3) domain, SH2 motif, Tyrosine protein kinase,

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene | UniGene | HomoloGene

Recent literature
Ozturk-Colak, A., Moussian, B., Araujo, S. J. and Casanova, J. (2016). A feedback mechanism converts individual cell features into a supracellular ECM structure in trachea. Elife 5. PubMed ID: 26836303
The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. This study used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. An active feedback mechanism was demonstrated between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, it was revealed that cell-cell junctions are key players in this aECM patterning and organisation and that individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, it is proposed that Src42A phosphorylation levels provide a link for the extracellular matrix environment to ensure proper cytoskeletal organisation.
Vanderploeg, J. and Jacobs, J. R. (2017). Mapping heart development in flies: Src42A acts non-autonomously to promote heart tube formation in Drosophila. Vet Sci 4(2). PubMed ID: 29056682
Congenital heart defects, clinically identified in both small and large animals, are multifactorial and complex. Although heritable factors are known to have a role in cardiovascular disease, the full genetic aetiology remains unclear. Model organism research has proven valuable in providing a deeper understanding of the essential factors in heart development. For example, mouse knock-out studies reveal a role for the Integrin adhesion receptor in cardiac tissue. Recent research in Drosophila melanogaster (the fruit fly), a powerful experimental model, has demonstrated that the link between the extracellular matrix and the cell, mediated by Integrins, is required for multiple aspects of cardiogenesis. This study tested the hypothesis that Integrins signal to the heart cells through Src42A kinase. Using the powerful genetics and cell biology analysis possible in Drosophila, it was demonstrated that Src42A acts in early events of heart tube development. Careful examination of mutant heart tissue and genetic interaction data suggests that Src42A's role is independent of Integrin and the Integrin-related Focal Adhesion Kinase. Rather, Src42A acts non-autonomously by promoting programmed cell death of the amnioserosa, a transient tissue that neighbors the developing heart.
Roper, J. C., Mitrossilis, D., Stirnemann, G., Waharte, F., Brito, I., Fernandez-Sanchez, M. E., Baaden, M., Salamero, J. and Farge, E. (2018). The major beta-catenin/E-cadherin junctional binding site is a primary molecular mechano-transductor of differentiation in vivo. Elife 7. PubMed ID: 30024850
In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. This study finds the molecular stretching of the highly conserved Y654-beta-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the beta-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two alpha-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of beta-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the beta-catenin pathway process.
Hunter, M. V., Willoughby, P. M., Bruce, A. E. E. and Fernandez-Gonzalez, R. (2018). Oxidative stress orchestrates cell polarity to promote embryonic wound healing. Dev Cell 47(3): 377-387.e374. PubMed ID: 30399336
Embryos have a striking ability to heal wounds rapidly and without scarring. Embryonic wound repair is a conserved process, driven by polarization of cell-cell junctions and the actomyosin cytoskeleton in the cells around the wound. However, the upstream signals that trigger cell polarization around wounds are unknown. This study used quantitative in vivo microscopy in Drosophila and zebrafish embryos to identify reactive oxygen species (ROS) as a critical signal that orchestrates cell polarity around wounds. ROS promote trafficking of adherens junctions and accumulation of actin and myosin at the wound edge and are necessary for wound closure. In Drosophila, ROS drive wound healing in part through an ortholog of Src kinase, Src42A, which is identified as a redox sensor that promotes polarization of junctions and the cytoskeleton around wounds. It is proposed that ROS are a reparative signal that drives rapid embryonic wound healing in vertebrate and invertebrate species.
Olivares-Castineira, I. and Llimargas, M. (2018). Anisotropic Crb accumulation, modulated by Src42A, is coupled to polarised epithelial tube growth in Drosophila. PLoS Genet 14(11): e1007824. PubMed ID: 30475799
Tube size control and how tubular anisotropy is translated at the cellular level are still not fully understood. This study investigated these mechanisms using the Drosophila tracheal system. The apical polarity protein Crumbs transiently accumulates anisotropically at longitudinal cell junctions during tube elongation. Evidence is provided indicating that the accumulation of Crumbs in specific apical domains correlates with apical surface expansion, suggesting a link between the anisotropic accumulation of Crumbs at the cellular level and membrane expansion. This study finds that Src42A is required for the anisotropic accumulation of Crumbs, thereby identifying the first polarised cell behaviour downstream of Src42A. The results indicate that Src42A regulates a mechanism that increases the fraction of Crb protein at longitudinal junctions, and genetic interaction experiments are consistent with Crb acting downstream of Src42A in controlling tube size. Collectively, these results suggest a model in which Src42A would sense the inherent anisotropic mechanical tension of the tube and translate it into a polarised Crumbs accumulation, which may promote a bias towards longitudinal membrane expansion, orienting cell elongation and, as a consequence, longitudinal growth at the tissue level. This work provides new insights into the key question of how organ growth is controlled and polarised and unveils the function of two conserved proteins, Crumbs and Src42A, with important roles in development and homeostasis as well as in disease, in this biological process.

Src42A is one of the two Src homologs in Drosophila. Src42A protein accumulates at sites of cell-cell or cell-matrix adhesion. Anti-Engrailed antibody staining of Src42A protein-null mutant embryos indicated that Src42A is essential for proper cell-cell matching during dorsal closure. Src42A, which is functionally redundant to Src64, was found to interact genetically with shotgun, a gene encoding E-cadherin, and armadillo, a Drosophila ß-catenin. Immunoprecipitation and a pull-down assay indicated that Src42A forms a ternary complex with E-cadherin and Armadillo, and that Src42A binds to Armadillo repeats via a 14 amino acid region, which contains the major autophosphorylation site. The leading edge of Src mutant embryos exhibiting the dorsal open phenotype is frequently kinked and associated with significant reduction in E-cadherin, Armadillo and F-actin accumulation. This phenotype suggests that not only Src signaling but also Src-dependent adherens-junction stabilization are essential for normal dorsal closure. Src42A and Src64 are required for Armadillo tyrosine residue phosphorylation but Src activity may not be directly involved in Armadillo tyrosine residue phosphorylation at the adherens junction (Takahashi, 2005).

The vertebrate Src family of non-receptor tyrosine kinases is comprised of nine members, three of which, Src, Yes and Fyn, are widely expressed in a variety of cells. These Src kinases are considered to have crucial roles in modulation of the actin cytoskeleton, a determinant of cell-shape change and cell migration. Transformation of fibroblasts with activated Src kinases gives rise not only to actin-cytoskeleton disruption but also increased tyrosine phosphorylation of many cytoskeleton-associated proteins involved in cell-substratum and cell-cell interactions. The importance of Src kinases as regulators of cell migration and cell-shape change is also underscored by studies using fibroblasts derived from mice deficient in Src, Yes and Fyn (Takahashi, 2005).

The major autophosphorylation site in focal adhesion kinase (FAK: see Drosophila FAK) serves as a binding site for Src homology 2 (SH2)-containing proteins. The FAK-Src complex mediates the phosphorylation of paxillin and p130-Crk-associated substrate, both of which are major scaffolding proteins capable of recruiting other molecules for integrin-based cell-substratum adhesions and which regulate cytoskeleton organization. The absence of FAK gives rise to increase in the number and extent of cell-substratum adhesions. A quantitative assay was conducted of the rate of incorporation of proteins into cell-substratum adhesion; departure of these proteins from this adhesion was measured (Webb, 2004). Src and FAK are crucial for adhesion turnover at the cell front. Thus, the rates of formation, disassembly and/or maturation of cell-substratum-adhesion appear controlled by FAK-Src activity (Takahashi, 2005 and references therein).

Homophilic cadherin interaction is essential for cell-cell adhesion in vertebrates. The loss of E-cadherin (E-cad) expression has been shown to be related to invasive and metastatic cancers. ß-Catenin binds to alpha-catenin and the cytoplasmic domain of E-cad and is essential for linking E-cad to the actin cytoskeleton. Tyrosine-phosphorylation of ß-catenin or other adherens-junction-associated proteins is one means by which cadherin-mediated cell-cell adhesions may be altered. Enhanced tyrosine-phosphorylation of ß-catenin causes weakening of cadherin-actin interaction with consequent loss of cell adhesiveness. Src may be one of the tyrosine kinases responsible for this tyrosine-phosphorylation, because in cells transformed with Src, loss of epithelial cell differentiation and gain in invasiveness and cadherin-mediated adhesion detachment are all correlated with tyrosine-phosphorylation of the E-cad/ß-catenin complex (Takahashi, 2005 and references therein).

Nonetheless, precise determination of the functional roles of individual Src family kinases in vertebrates may be frought with considerable difficulty because compensatory interactions may occur among nine vertebrate Src kinase members. By contrast, Drosophila possesses only two Src kinases, Src64 and Src42A and accordingly, may provide a better and simpler system for clarifying Src functions in development (Takahashi, 2005).

Mutations in Src64 led to a reduction in female fertility, which is associated with nurse cell fusion and ring canal defects. Src64-mutant ring canals fail to undergo extensive tyrosine phosphorylation which normally occurs. Tec29 dominantly enhances the Src64 ring canal phenotype and loss of Tec29 results in a phenotype strikingly similar to that noted following loss of Src64 function. Tec29 kinase is localized in the ring canal, and this subcellular localization requires Src64 function, indicating that Tec29 is a downstream target of Src64 (Takahashi, 2005 and references therein).

Src42A is the closest relative of vertebrate Src in Drosophila. By localized expression of gain-of-function and dominant-negative forms of Src42A, it has been demonstrated that Src42A may be involved in the regulation of cytoskeleton organization and cell-cell contacts in developing ommatidia and that both dominant-negative and gain-of-function mutations of Src42A cause formation of supernumerary R7-type neurons, which is suppressible by one-dose reduction of various components involved in the Ras/MAPK pathway (Takahashi, 1996). A Src42A mutant has been isolated as an extragenic suppressor of Raf. With this and other mild Src42A mutants, it was found that Src42A may serve as a negative regulator of receptor tyrosine kinases in a Ras1-independent manner (Lu, 1999). Lu's genetic data for Src functions in ommatidium formation appear somewhat at variance with those of Takahashi (1996) using gain-of-function and dominant-negative types of Src42A transgenes (Takahashi, 2005).

As with Src64, Src42A may function in a synergistic manner with Tec29. A Tec29 mutation was noted to enhance the lethality of Src42A mutants dominantly (Tateno, 2000). Although no dorsal open phenotype was found in Src42A or Tec29 mutants, the double mutant embryos exhibit the dorsal open phenotype. Src42A has been shown to be functionally redundant to Src64 at least in the dorsal closure (Tateno, 2000). Both dorsal closure of the embryonic epidermis and thorax closure of the pupal epidermis require the Jun amino-terminal kinase (JNK) homolog Basket (Bsk). The severity of the epidermal closure defect in Src42A mutants depends on the degree of Bsk activity; Bsk activity depends on that of Src42A (Tateno, 2000), thus indicating that JNK-pathway activation is required downstream of Src42A (Takahashi, 2005).

Dynamic changes in cellular and subcellular localization of Src42A have been found and phenotypes of a Src42A protein-null and Scr42A Src64 mutants have been described. Genetic and biochemical analyses indicate that E-cad and Armadillo (Arm) form a complex with Src in the membrane and the resultant putative adherens junction complex is required for proper regulation of F-actin accumulation and actin cytoskeleton dynamics in leading edge cells during dorsal closure (Takahashi, 2005).

The results of this study clearly demonstrate the redundant function of Src42A and Src64 to be indispensable in numerous aspects of Drosophila development. Though Src42A is distributed over the entire plasma membrane of all cells, its signal distribution is not uniform. Two major types of Src42A deposition in the membrane could be clearly recognized (Takahashi, 2005).

In ectodermal cells, strong Src42A signals in apical or apicolateral regions were always associated with strong E-cad signals. E-cad is a core component of the adherens junction that is responsible for cell-cell adhesion and, hence, most, if not all, E-cad-associated membranous Src42A is probably related to adherens junction-dependent cell-cell adhesion (Takahashi, 2005).

A considerable fraction of ectodermal cells were also found associated with the second type of basal Src42A free of E-cad. E-cad-free Src42A is localized on the ectoderm/mesoderm interface and eliminated from ectodermal cells that have evaginated or invaginated without mesoderm association. The extracellular matrix (ECM) comprises several groups of secreted proteins such as integrin ligands. During embryogenesis, different cell layers become properly connected, most probably via cell adhesion to ECM. E-cad-free Src42A may thus be related to integrin-mediated cell-matrix adhesion. Cell-ECM adhesion may not be restricted to the interface between ectodermal and mesodermal cell layers. Strong Src42A signals have actually been found present on the interface between mesodermal and endodermal cell layers (Takahashi, 2005).

The current study shows that, as with JNK signaling genes, Src is required not only for thick F-actin accumulation at the leading edge but proper cell-cell matching along the midline seam as well. JNK signaling, which includes hemipterous (hep) and basket (bsk), is essential for dorsal closure of the embryonic epidermis in Drosophila. Based on examination of Tec29 Src42A mutant phenotypes, it has been suggested (Tateno, 2000) that Src42A acts upstream of bsk (Takahashi, 2005).

In vertebrates, JNK is considered to be situated downstream of Src in integrin signaling (Oktay, 1999; Schlaepfer, 1999). Genetic experiments indicate that interactions between Src and arm/shg, genes encoding the core components of the adherens junction are essential for JNK signaling regulation required for dorsal closure. A pull-down assay also shows that Src protein is capable of directly binding to Arm. Both putative adherens-junction Src and integrin-associated Src thus would appear involved in the regulation of JNK signaling (Takahashi, 2005).

The adherens junction is necessary for cell-cell adhesion and thick F-actin accumulation occurs at the level of the adherens junction at the leading edge. Since E-cad and Arm signals along with actin signals are reduced significantly at the leading edge in Src42A26-1;Src64P1/+ embryos and the leading edge of the mutants is significantly kinked, the absence of Src protein from the adherens junction may possibly result in destruction of structural integrity, implying that the adherens junction is also involved in dorsal closure regulation in a structural way (Takahashi, 2005).

Dorsal closure and CNS defects similar to those in Src mutants have been observed in abl mutants. In vertebrates, Abl is tyrosine-phosphorylated with Src (Plattner, 1999) and is capable of interacting with delta-catenin, an E-cad-binding protein. Abl may thus function as well downstream of Src signaling in Drosophila. Germ-band retraction and possibly too, head involution, both of which require Src activity, may be regulated by the two above distinct Src functions. alpha1,2-laminin and alphaPS3ßPS integrin have clearly been shown to be essential for spreading a small group of amnioserosa epithelium cells over the tail end of the germ band during germ-band retraction. shg activity has also been shown to be essential for normal germ-band retraction and head involution (Takahashi, 2005).

Src-dependent dynamical regulation of E-cad-dependent cell-cell adhesion may also be necessary for visual system formation. E-cad overexpression or elimination of EGFR activity have been shown to render optic placode cells incapable of invaginating and prevent the separation of Bolwig's organ precursors from the optic lobe. Virtually identical phenotypes were induced by loss of Src activity, suggesting involvement of at least the adherens junction Src in larval visual system formation and that Src should function either upstream or downstream of EGFR signaling (Takahashi, 2005).

Duox, Flotillin-2, and Src42A are required to activate or delimit the spread of the transcriptional response to epidermal wounds in Drosophila

The epidermis is the largest organ of the body for most animals, and the first line of defense against invading pathogens. A breach in the epidermal cell layer triggers a variety of localized responses that in favorable circumstances result in the repair of the wound. Many cellular and genetic responses must be limited to epidermal cells that are close to wounds, but how this is regulated is still poorly understood. The order and hierarchy of epidermal wound signaling factors are also still obscure. The Drosophila embryonic epidermis provides an excellent system to study genes that regulate wound healing processes. A variety of fluorescent reporters were developed that provide a visible readout of wound-dependent transcriptional activation near epidermal wound sites. A large screen for mutants that alter the activity of these wound reporters has identified seven new genes required to activate or delimit wound-induced transcriptional responses to a narrow zone of cells surrounding wound sites. Among the genes required to delimit the spread of wound responses are Drosophila Flotillin-2 and Src42A, both of which are transcriptionally activated around wound sites. Flotillin-2 and constitutively active Src42A are also sufficient, when overexpressed at high levels, to inhibit wound-induced transcription in epidermal cells. One gene required to activate epidermal wound reporters encodes Dual oxidase, an enzyme that produces hydrogen peroxide. Four biochemical treatments (a serine protease, a Src kinase inhibitor, methyl-β-cyclodextrin, and hydrogen peroxide) were found to be sufficient to globally activate epidermal wound response genes in Drosophila embryos. The epistatic relationships among the factors that induce or delimit the spread of epidermal wound signals were examined. The results define new genetic functions that interact to instruct only a limited number of cells around puncture wounds to mount a transcriptional response, mediating local repair and regeneration (Juarez, 2011).

Drosophila wound healing is an example of a regenerative process, which requires localized epidermal cytoskeletal changes, and localized wound-induced changes in epidermal transcriptional activity. This genetic screen with wound-dependent reporters has allowed identification of novel components that regulate the localized transcriptional response to wounding in epidermal cells. This research identifies seven genes that are required to either activate (Duox and ghost/stenosis) or localize (Flo-2, Src42A, wurst, varicose, and Drosophila homolog of yeast Mak3) the expression patterns of epidermal wound reporters. The number of new functions involved in the delimitation of epidermal wound response near wound sites was unexpected, but indicates that considerable genetic effort is devoted to localizing the activity of transcriptional wound responses during regeneration (Juarez, 2011).

One of the genes that limits the spread of epidermal wound reporters after clean epidermal punctures is Flo-2, as mutants of this gene show a broad expansion of epidermal wound gene activation. Drosophila Flo-2 is itself transcriptionally activated around epidermal wound sites, consistent with an evolutionarily conserved role in regeneration after wounding. In vertebrates, reggie-1/Flo-2 gene expression is activated in wounded fish optic neurons, and reggie-1/Flo-2 and reggie-2/Flo-1 morpholino knockdowns in wounded zebrafish retinal explants reduced axon outgrowth compared to controls. Flo-2 transcriptional activation around Drosophila epidermal wound sites is dependent on the grh genetic function, which is required to activate at least a few other epidermal wound response genes. Flo-2 thus appears to act in the same pathway as grh, although it may act both downstream and upstream of grh, since overexpression of Flo-2 can inhibit the activation of other grh-dependent wound response genes. In this respect, Flo-2 resembles stit receptor tyrosine kinase gene (Wang, 2009), which is both transcriptionally activated by Grh, as well as required for grh-dependent activation of other downstream wound genes. Amazingly, overexpression of Flo-2 can even inhibit the global activation of the Ddc and ple-WE1 wound reporters that are induced by the serine protease trypsin, or by hydrogen peroxide. The inhibitory function of overexpression of Flo-2 on wound induced transcription is cell non-autonomous, at least over the range of a few cell diameters, as shown by the ability of striped overexpression of Flo-2 to silence puncture or trypsin-induced gene activation throughout the epidermis (Juarez, 2011).

The only animal where Flo-2 null mutants have so far been characterized is Drosophila, where Flo-2 has been shown to regulate the spread of Wingless (Wg) and Hedgehog (Hh) signals in the wing imaginal discs. In the wing discs, both the secretion rate and the diffusion rate of these two lipid-modified morphogens were increased when Flo-2 was overexpressed, and decreased when Flo-2 and Flo-1 proteins were not expressed. Despite the reduced spread of Wg and Hh morphogen proteins in Flo-2 mutant imaginal discs, adult morphology of mutants was normal, presumably because of compensatory mechanisms that occur later in development. Whereas a reduced range of activation of wg and hh long range transcription target genes was observed in Flo-2 mutant imaginal discs, a greatly increased range of wound-induced gene activation was observed in Flo-2 mutant embryos. This apparent discrepancy could be explained if one invokes of a long-range wound-induced inhibitory signal that in wild type embryos diffuses faster and farther than a wound activating signal, and thereby functions to limit the wound response to nearby epidermal cells, and that in Flo-2 mutants this potential inhibitory signal has reduced secretion, concentration, and/or diffusion range. This notion is consistent with the cell non-autonomous effect of overexpressed Flo-2 on inhibiting wound- or trypsin-induced gene activation. A similar scheme of controlling signal spreading has been seen in the way that Mmp2 acts cell non-autonomously to limit FGF signaling during Drosophila tracheal development and branch morphogenesis. It's also possible that Flo-2 normally is required to set a global threshold that wound-induced signals must overcome in order to activate wound transcription, for example via Flo-2-dependent endocytosis/degradation of a diffusible wound signal and its receptor (perhaps the Stit RTK), and that signal strength normally surpasses the Flo-2 threshold only in the vicinity of a wound. In this model, loss of Flo-2 would result in all epidermal cells being able to exceed the wound signal threshold, and overexpression of Flo-2 would prevent any cells from exceeding the wound signal threshold. The cell non-autonomous effects of Flo-2 overexpression under this model might be explained by an increase in Flo-2-dependent endocytosis/degradation that rapidly depletes an activating signal from the extracellular space (Juarez, 2011).

Many previous studies have documented biochemical, molecular biological, and cell biological interactions between Src family kinases and Flotillins. In Drosophila, lack of Src42A and Flo-2 leads to expanded spread of wound gene activation, and overexpression of Flo-2 or activated Src42A can inhibit wound gene activation, which is consistent with an interaction between the two functions during the process of wound gene regulation. In cultured mammalian cells, Flo-2 can be phosphorylated by Src family kinases in an extracellular signal-dependent fashion. This phosphorylation is associated with changes in the normal intracellular trafficking of Flotillin-containing membrane microdomains and vesicles. Since overexpressed Flo-2 in Drosophila can act in a cell non-autonomous fashion to inhibit wound gene activation, and overexpressed Src42A acts in a cell autonomous fashion to inhibit wound gene activation, one interpretation is that Flo-2 lies genetically upstream of Src42A in the epidermal wound response. This hypothesis appears to be inconsistent with the vertebrate biochemical data indicating that Src kinases phosphorylate Flotillins to activate their diverse functions. However, an observation that is consistent with Src42A activating Flo-2 protein function, is that even when Flo-2 is overexpressed, addition of chemical inhibitors of Src family kinases to wounded embryos, results in widespread Ddc .47 or ple-WE1 wound reporter activation. One interpretation of this suggests Flo-2 protein, no matter the level of expression, is inactive in the absence of Src42A function. Complex feedback loops involving signaling proteins being regulated by a transcription factor, while the activity of the same transcription factors is regulated by the same signaling pathway, have been observed in the control of Drosophila epidermal wound gene expression and reepithelialization, so there may be similar dynamic cross-regulatory interactions between Flo-2 and Src42A in the localization of the epidermal wound response, interactions not easily captured in linear genetic pathway diagrams (Juarez, 2011).

The inhibitory effect of Src42A on wound gene activation suggests that it might antagonize a signaling cascade that leads to the epidermal wound response. A good candidate for such a signaling cascade is the RTK pathway involving the Stit kinase. Stit is a RET-family RTK that is required for robust activation of the Ddc and stit wound reporter genes in wounded embryos (Wang, 2009). Other evidence consistent with RTK pathway importance in wound gene activation is that phosphotyrosine accumulates persistently around wound sites, and that ERK kinase function is required for robust activation of the Ddc wound reporter gene. Interestingly, Src42A has been shown to act as an inhibitor of some Drosophila RTK proteins (those encoded by the torso, Egfr, and sevenless genes) in a few different tissues during Drosophila development. The Flo-2 and Src42A functions in epidermal wound localization after clean wounding are reminiscent of the role of Drosophila WntD during infectious wounding. WntD mutants show higher levels of some antimicrobial peptide genes after septic injury of adults (Juarez, 2011).

Previous evidence suggested that H2O2 and Duox could provide wound-induced inflammatory signals and antimicrobial activities. The current studies show that Duox is required to activate wound reporter genes after epidermal wounding, and that injected exogenous H2O2 is sufficient to activate widespread epidermal wound gene expression. Overexpression of either Flo-2 or Src42A.CA can inhibit the H2O2 -dependent wound reporter expression, suggesting that all of these components are in a common pathway controlling the activation of epidermal wound reporters. However, the ability of trypsin injection to activate the Ddc .47 and ple-WE1 wound reporters in Duox mutants suggests that a serine protease might act downstream of, or in parallel to, H2O2-dependent wound signals. A recent report showed that in cultured mammalian cells, a Src kinase phosphorylates and inhibits a Flo-2-associated enzyme, peroxiredoxin-1, which results in increased stability of H2O2. This is consistent with the results placing Flo-2, Src42A, and H2O2 in a common wound signaling pathway (Juarez, 2011).

Like H2O2, the injection of methyl-β-cyclodextrin (MβCD) into wounded embryos triggers a global wound response in the epidermis. MβCD strongly depletes cholesterol and other sterols from membranes and disrupt lipid rafts, but was also shown to remove sphingolipid-associated proteins such as Src-Family Kinases. The effects of MβCD, in combination with the effects of loss of Flo-2, suggests that the integrity of lipid rafts and associated proteins are required to inhibit epidermal wound signals. In cultured cells, MβCD treatments trigger a release of EGF receptors from membrane microdomains, which increases EGFR, and perhaps other RTK, signaling in a ligand-independent manner. Interestingly, in cultured keratinocytes, MβCD treatment can induce the expression of involucrin, which encodes a protein, analogous to Drosophila Ple/tyrosine hydroxylase, which is required for the formation of an epidermal barrier. Similarly, MβCD injections into Drosophila embryos might also cause an increase the levels of a wound signal produced or released from cells adjacent to the wound site, allowing more widespread transcriptional activation of wound reporter genes. The observations that overexpression of Src42A or Flo-2 can inhibit the MβCD -triggered activation of epidermal wound reporter genes suggest that high levels of these proteins might overcome lipid raft-inhibitory effects on wound signaling pathways (Juarez, 2011).

Other genes (wurst and varicose) identified in the screen have phenotypes similar to Flo-2 and Src42A mutants. wurst encodes an evolutionarily conserved trans-membrane protein, containing a heat shock cognate protein 70 binding domain and a clathrin binding motif. wurst is ubiquitously expressed in embryonic epithelial cells, strongly up-regulated during endocytosis-dependent luminal clearance, and mislocalized in mutants with endocytosis defects. wurst mutant embryos have tortuous tracheal tubes, due to a failure to properly endocytose matrix material from the tracheal lumen. varicose encodes an evolutionarily-conserved septate junction scaffolding protein, in the Membrane Associated GUanylate Kinase (MAGUK) family. varicose is expressed in epidermally-derived cells (including the hindgut and trachea) and co-localizes with the septate junction proteins, Coracle and Neurexin4. varicose mutant embryos develop permeable tracheal tubes and paracellular barrier defects in epithelia. Like wurst mutants, varicose mutants also have abnormal matrix composition in the tracheal lumen, and may also have abnormal extracellular matrix composition produced by other epidermal cells (Juarez, 2011).

Another gene (ghost), also known as stenosis) identified in this screen is required for wound reporter activation like Duox or grh. ghost encodes the Drosophila Sec24CD homolog, a coat protein of COPII vesicles in the ER/Golgi trafficking pathway. Transport of cargo from the ER to the Golgi via COPII vesicles is required to achieve normal amounts of secretion of extracellular matrix proteins into the developing Drosophila tracheae and normal apical-basal localization of membrane proteins. Presumably, similar secretion and membrane localization defects occur in non-tracheal epidermal cells, which account for the severe cuticle deposition defects in ghost (Sec24CD) mutants. It is fascinating to note that the finding that ghost (Sec24CD) is required for transcriptional activation of epidermal wound reporter genes is consistent with the finding that RNAi knockdowns of Sec24C in a planaria (Schmidtea mediterranea) interfered with normal regeneration after amputation wounds. It is possible that the ghost mutants do not secrete enough wound signals, or the protein matrix necessary for the propagation of a wound signal (Juarez, 2011).

Another gene required for the activation of wound reporters is shroud (sro). It is believed sro to be an allele in the Drosophila Fos-D isoform, and it was hypothesized that one of the Drosophila kayak/Fos transcription factors was required for the activation of some epidermal wound gene reporters. However, has been recently discovered that sro[1] and other sro point mutant alleles do not map in the kayak/Fos gene, but in an immediately adjacent transcription unit (Nm-g/sro) that encodes an enzyme in the sterol metabolic pathway that is necessary for production of ecdysone hormone. At first glance, the requirement of sro to activate some wound reporters suggested that these reporters rely on ecdysone signaling. This is possible, although deletions were tested that eliminate zygotic functions of the ecdysone receptor gene, as well as of the phantom gene (which encodes another enzyme in the ecdysone synthesis pathway), and embryos that are zygotic mutants in either gene show normal activation of the ple-WE1 wound reporter after puncture wounding (Juarez, 2011).

In summary, though this large unbiased screen, several genes were identified that add to the understanding of the complex pathways that control the signals that activate wound response transcription near puncture wounds. At the cellular level, there appears to be a correlation between genetic functions required to localize wound-induced gene activation, and cellular functions required for endocytosis and/or apical-basal polarity. For example, one function of Flo-2 is in signal-dependent endocytosis, although Flo-2 also plays other roles in vesicular trafficking. There have been many studies showing that endocytosis can regulate extracellular signaling strength and duration. For example, one study found that tagged-FGF8 showed increased accumulation, spread, and target gene activation when Rab-5-mediated endocytosis was reduced in zebrafish embryos. It is believed that further studies on wound response signaling may provide new insights into how membrane microdomains, endocytosis of membrane receptors, and the composition and organization of the extracellular matrix, regulates the transmission of wound signals (Juarez, 2011).


cDNA clone length - 2588bp

Bases in 5' UTR - 538

Exons - 10 (Src42A-RA)

Bases in 3' UTR - 496


Amino Acids - 517

Structural Domains

See NCBI Conserved Domain Summary for information on Src42A structure.


See Src oncogene at 64B for information on Src family evolutionary homologs.

Src oncogene at 42A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 14 October 2005

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