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
Summary:
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.
BIOLOGICAL OVERVIEW

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).


GENE STRUCTURE

cDNA clone length - 2588bp

Bases in 5' UTR - 538

Exons - 10 (Src42A-RA)

Bases in 3' UTR - 496

PROTEIN STRUCTURE

Amino Acids - 517

Structural Domains

See NCBI Conserved Domain Summary for information on Src42A structure.


EVOLUTIONARY HOMOLOGS

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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