Gene name - Src oncogene at 64B
Cytological map position - 64B12--64B17
Function - signal transduction protein
Symbol - Src64B
FlyBase ID: FBgn0003501
Genetic map position - 3-
Classification - Src homolog
Cellular location - cytoplasmic
|Recent literature||Ho, D. M., Pallavi, S. K. and Artavanis-Tsakonas, S. (2015). The Notch-mediated hyperplasia circuitry in Drosophila reveals a Src-JNK signaling axis. Elife 4. PubMed ID: 26222204
Notch signaling controls a wide range of cell fate decisions during development and disease via synergistic interactions with other signaling pathways. Through a genome-wide genetic screen in Drosophila, this study uncovered a highly complex Notch-dependent genetic circuitry that profoundly affects proliferation and consequently hyperplasia. A novel synergistic relationship is reported between Notch and either of the non-receptor tyrosine kinases Src42A and Src64B to promote hyperplasia and tissue disorganization resulting in cell cycle perturbation, JAK/STAT signal activation, and differential regulation of Notch targets. Significantly, the JNK pathway is responsible for the majority of the phenotypes and transcriptional changes downstream of Notch-Src synergy. It has been reported that Notch-Mef2 also activates JNK, indicating that there are commonalities within the Notch-dependent proliferation circuitry; however, the current data indicate that Notch-Src accesses JNK in a significantly different fashion than Notch-Mef2.
When first identified (reviewed by Thomas, 1997), members of the Src family of protein tyrosine kinases (Src PTKs) were determined to be transforming proteins, encoded by oncogenic retroviruses. Drosophila Src oncogene at 64B has been shown to play a role in ring canal morphogenesis during oogenesis (Dobson, 1998). Before describing the biology of Drosophila Src oncogene at 64B, here termed Src64, a digression into the complex structural and functional world of Src biology is necessary; a brief overview of this information is provided to put Src developmental effects in Drosophila into a broader context.
At present, nine distinct Src PTKs have been identified in vertebrates and two in Drosophila. The Src PTKs share a common domain structure consisting of the following features from N- to C-terminal: an amino-terminal myristylation site, a region unique to Src PTKs, SH3 and SH2 domains, the protein tyrosine kinase catalytic domain and a carboxy-terminal negative regulatory region. The three domains that follow a region unique to all Src PTKs represent modular structures found in many classes of cellular proteins: the catalytic domain possesses tyrosine-specific protein kinase activity; the SH3 and SH2 domains are protein-binding domains present in lipid kinases, protein and lipid phosphatases, cytoskeletal proteins, adaptor molecules, transcription factors, and other proteins (reviewed by Thomas, 1997).
With regard to the SH3 domains of Src PTKs in particular, they are composed of 50 amino acids. The SH3 domain is important for intra- as well as intermolecular interactions that regulate Src catalytic activity, Src localization, and recruitment of substrates. SH3 domains bind short contiguous amino acid sequences rich in proline residues. All SH3 domain ligands contain a core consensus sequence of P-X-X-P; however, amino acids surrounding the prolines confer additional affinity and specificity for individual SH3 domains. SH3 ligands can bind in either one of two orientations: NH2 COOH (Class I) or COOH NH2 (Class II). The SH3 binding pocket has two hydrophobic grooves that contact the core X-P-X-X-P sequence. A second region contacts the residues N-terminal (class I) or C-terminal (Class II) to the proline core. Proteins that have been shown to interact with Src PTK SH3 domains, either in vitro or in vivo, include p68sam, p85 phosphatidylinositol-3' kinase (PI 3-K), and paxillin (reviewed by Thomas, 1997).
The SH2 domain also controls the repertoire of proteins interacting with Src PTKs. Binding interactions mediated by the SH2 domain function in regulating the catalytic activity of Src PTKs, as well as the localization of Src or its binding proteins. All SH2 domains bind to short contiguous amino acid sequences containing phosphotyrosine, and the specificity of individual SH2 domains lies in the 3-5 residues following the phosphotyrosine (+1, +2, +3, etc). Amino acids preceding phosphotyrosine may also be important for regulating binding affinity. Structural studies on Src family SH2 domains have shown that the ligand-binding surface of SH2 domains is composed of two pockets. One pocket contacts the phosphotyrosine; the other pocket contacts the +3 amino acid residue following the phosphotyrosine. Src family kinases show a preference for leucine at this position. Proteins that have been shown to interact with the Src SH2 domain in vivo include the focal adhesion protein FAK (focal adhesion kinase), p130cas (see CAS/CSE1 segregation protein), p85 PI 3-K, and p68sam (reviewed by Thomas, 1997).
Several of the vertebrate Src PTKs are expressed in specific hematopoietic lineages where their participation in receptor-mediated signaling is required for proper development and cellular function. The more broadly expressed Src PTKs (Src, Fyn and Yes) are activated in response to growth factors (such as PDGF, EGF, FGF and CSF-1) that signal through the activation of receptor tyrosine kinases (RTKs). Consistent with a role in RTK signaling, the inhibition of Src PTKs blocks mitogenesis in response to these growth factors. In addition to the involvement of Src PTKs in receptor-mediated signaling, the inhibition of Src PTKs blocks the G2-M transition of the cell cycle in fibroblasts (reviewed by Thomas, 1997).
There are multiple ways to activate Src family kinases. These include displacement of the intramolecular interactions of the SH2 or SH3 domains by high-affinity ligands or modification of certain residues, dephosphorylation of pY527 (phosphotyrosine 527 of Src) by a tyrosine phosphatase, or phosphorylation of Y416. The SH2 domain interacts with pTyr 527 of Src and adjacent residues in the negative regulatory tail. The primary sites of tyrosine phosphorylation in vivo are Y527 in c-Src, and the corresponding tyrosine in other Src PTKs. This residue is phosphorylated by the cytoplasmic tyrosine kinase Csk. Loss of Y527 phosphorylation leads to activation of Src catalytic activity. It is thought that Csk-mediated tyrosine phosphorylation of the C-terminal tail promotes an intramolecular interaction between the SH2 domain and the phosphorylated tail, keeping the kinase in a closed, inactive conformation. Biochemical and structural studies of Src suggest that the autophosphorylation site within the catalytic domain is also important for regulation of kinase activity. Phosphorylation of analogous residues within the catalytic domain of kinases induces a conformational change that allows the kinase to assume an active conformation. This site of phosphorylation corresponds to Y416 in c-Src, the endogenous cellular Src of mammals, which is not phosphorylated in inactive wild type Src, but is constitutively phosphorylated in activated oncogenic Src mutants. Mutation of Y416 diminishes the transforming potential of both v-src (the Src oncogene of viruses) and some oncogenic variants of c-Src, suggesting that phosphorylation of this residue may be important in vivo (reviewed by Thomas, 1997).
Numerous studies suggest that the Src PTKs also function to regulate the actin cytoskeleton. Transformation of fibroblasts with activated Src PTKs causes disruptions in the actin cytoskeleton. These changes are associated with increased tyrosine phosphorylation of many cytoskeletal associated proteins. These include proteins involved in cell substrate adhesion (tensin, vinculin, talin, paxillin, FAK, beta1 integrin, p130 cas, AFAP110); cell-cell adhesion (plakoglobin, beta-catenin, p120 cas), and other proteins thought to regulate the actin cytoskeleton (p190 rhoGAP and cortactin). Src PTK participation in cytoskeletal regulation is also supported by studies of src-deficient mice. These mice suffer from osteopetrosis, a bone remodeling disorder caused by a failure of osteoclast function. Examination of the src - osteoclasts shows that they are deficient in the formation of ruffled borders and have defects in the underlying actin cytoskeleton. Studies of fibroblasts derived from mice lacking Csk, a negative regulator of Src PTKs, have provided additional evidence for Src PTK involvement in actin cytoskeleton regulation. These cells have disrupted actin cytoskeletons and increased phosphorylation of p120, FAK, paxillin, tensin and cortactin. Furthermore, the removal of src activity suppresses the cytoskeletal defects of csk - cells and returns the phosphorylation of tensin and cortactin to normal levels (Dodson, 1998 and references).
Concluding this digression into Src biological structure and function is a description of Tec29 (formerly, and more properly termed Btk family kinase at 29A), a second Drosophila non-receptor tyrosine kinase, originally cloned because of sequence homology to the kinase domain of v-src. As will be described in the protein interaction section, Tec29 interacts with Src64 in ring canal development in Drosophila oocytes (Roulier, 1998 and Guarnieri, 1998). In vertebrates (Roulier, 1998 and references), Tec family kinases interact with a variety of membrane associated and cytoplasmic proteins. For example, Tec family members can act in a cytokine-stimulated signaling pathway: Tec and Btk act downstrem of janus kinase (JAK) to link interleukin 6 signaling to PI-3 kinase. Tec family members may also link signals to the cytoskeleton, since they bind via their Src homology 3 (SH3) domains to WASP and Vav, both of which are associated with the actin cytoskeleton. Tec kinases also interact with Src family kinases. In vitro binding data indicate direct interactions between proline-rich regions of Tec family kinases and the SH3 domains of Src family members. Thus mammalian Src family kinases are directly upstream of Tec family kinases in signaling pathways. This heirarchy appears to be conserved in Drosophila (Roulier, 1998 and Guarnieri, 1998).
Drosophila Tec29 shows a dynamic expression pattern throughout development ( Gregory, 1987; Vincent, 1989; Katzen, 1990 and reviewed in Roulier, 1998). Tec29 mRNA is contributed maternally to the embryo and is first localized uniformly with refinement during gastrulation, followed by expression in segmental stripes. Subsequently, it is expressed strongly in the developing salivary primordia and invaginating salivary glands, as well as in other epithelial tissues that undergo defined cell movements, including regions of the head. During early embryogenesis, Tec29 is expressed as a 66 kDa protein isoform, p66, which is membrane-localized, while the expression in the central nervous system (CNS) beginning during germband retraction is of a smaller 55 kDA isoform, p55 (Vincent, 1989 and Wadsworth, 1990). Expression continues during larval stages in the CNS as well as in the lymph glands, the site of hemocyte production. All third instar imaginal discs express Tec29 at moderate levels, except the eye disc, in which expression is lower. Pupal expression is strongest in the lamellocytes, the differentiated hemocytes responsible for the removal of larval tissues during metamorphosis. The immune-associate expression of Tec29 in Drosophila is particularly interesting in light of its roles in immune system development in the vertebrate Tec family members. Although the cloning and expression pattern of Tec29 have been reported previously, until recently no mutations have been available for the functional analysis of this gene.
Drosophila Src64 plays a role in ring canal morphogenesis during oogenesis. The biology of Drosophila ring canals (reviewed by Dodson, 1998) will be presented briefly, before specific discussion of Src64. Drosophila oocytes develop from one of 16 germline cells (which in turn have developed from a single germline stem cell). These 16 cells are interconnected by a network of 15 cytoplasmic bridges, termed ring canals. Of the 16 cells, the one cell destined to become the oocyte is connected to four ring canals. Therefore, the developing oocyte is connected directly (via 4 ring canals) to four of the 15 germline cells, and indirectly (via the ring canal network) to the remaining germline cells. These 15 remaining cells will differentiate as nurse cells. Early in oogenesis, the developing oocyte becomes transcriptionally inactive. Thus, most of the maternal products required for early embryogenesis are synthesized in the nurse cells and transported to the oocyte through the network of ring canals. Throughout most of oogenesis, the cytoplasmic transport from the nurse cells to the oocyte is gradual. At late stages (beginning at stage 11) of oogenesis, the nurse cells contract and rapidly transfer the remainder of their cytoplasmic contents to the oocyte. Following the completion of cytoplasmic transfer, the nurse cells degenerate.
The actin cytoskeletal rearrangements that occur during ring canal morphogenesis have been extensively studied. Shortly after the arrest of the mitotic cleavage furrow, one or more of the unidentified phosphotyrosine-containing proteins localizes to the outer rim of the presumptive ring canal (Robinson, 1994). After the final mitotic divisions give rise to the 16 germ-cell cluster, an inner rim forms at the ring canals. Initially, several proteins become localized to the inner rim of the ring canal. These include a ring-canal-specific product (HTS-RC) of the hu-li tai shao (hts) gene; F-actin, and additional phosphotyrosine-containing protein(s) (Robinson, 1994). The accumulation of F-actin is dependent on hts function since, in hts mutant egg chambers, the inner rim does not form at the majority of cytoplasmic bridges and only phosphotyrosine can be detected at most ring canals (Yue, 1992; Robinson, 1994).
Subsequent to the addition of F-actin, HTS-RC and phosphotyrosine protein(s), the Kelch protein (Kelch) also becomes localized to the inner rim of the ring canal (Xue, 1993). The function of Kelch is to maintain the compaction of the ring canal rim. In late stage kelch mutant egg chambers, F-actin diffuses into the inner lumen of the ring canals and partially blocks the transfer of nurse cell cytoplasm to the oocyte. In addition to these known components of the ring canal, the product of the cheerio gene is also required for proper ring canal formation. cheerio ring canals are small and lack F-actin, HTS-RC and Kelch. Furthermore, fusions between the nurse cell and the oocyte are frequently observed in cheerio egg chambers indicating that the integrity of the plasma membrane has been compromised (Robinson, 1997). To date, the cheerio gene has not been cloned, so it is not known whether its product is a ring canal component. Once the ring canals are established, they do not remain static. The rims of newly formed ring canals have diameters of 0.5-1 mm. By stage 11, at the onset of rapid cytoplasmic transfer from the nurse cells to the oocyte, the ring canals have attained their maximum size, with a diameter of roughly 10 mm. EM studies have shown that the early phase of growth (prior to stage 5) is accompanied by the addition of new actin filaments to the ring canal. After stage 5, there is an increase in total F-actin at the ring canal, but it is unclear as to whether this increase results from the addition of new filaments or the lengthening of existing filaments. However, during this developmental period, the filaments become organized into large bundles (Tilney, 1996).
Drosophila Src64 function has been shown to be required during oogenesis for ring canal morphogenesis. Females homozygous for any of three Src64 alleles have reduced fertility. Eggs laid by Src64 females hatch at reduced frequency, when compared to wild type. In addition, Src64 females lay fewer eggs than wild type, which may suggest that the defective eggs are resorbed as has been previously observed for other female-sterile mutations. In contrast, mutant Src64 males are fully fertile. The loss of female fertility is associated with a defect in cytoplasmic transfer from the nurse cells to the developing oocyte. Unlike wild-type egg chambers, 55% of late stage Src64 egg chambers have nurse cell cytoplasm remaining at the anterior end of the oocyte. As a result, eggs from Src64 females range from 50%-100% of the length of eggs oviposited by wild-type females. Both the reduction of female fertility and the defect in cytoplasmic transfer can be reverted by excision of the downstream P-element present in the Src64 PI allele. This indicates that these phenotypes are due to disruption of the Src64 gene (Dodson, 1998).
Incomplete cytoplasmic transfer is often indicative of defects in the actin cytoskeleton of the nurse cells. Two unique cytoskeletal features are particularly important for efficient transfer. The first is the formation prior to the rapid transfer phase (stage 10B) of actin cables, which hold the nurse cell nuclei in place. In the absence of these actin cables, the nurse cell nuclei become lodged in the ring canals and block transfer. Staining of Src64 egg chambers with fluorescein-conjugated phalloidin to visualize filamentous actin at stage 10B shows that the actin cables are present. The second important cytoskeletal structure of the nurse cells are the ring canals. In order to assess the role of SRC64 in ring canal morphogenesis, early to mid-stage mutant egg chambers were stained with fluorescein phalloidin and with antibodies directed against phosphotyrosine, HTS-RC or Kelch. These experiments show that the normal complement of 15 ring canals is present and that F-actin, HTS-RC and Kelch all localize properly at the ring canals. The intensity of the staining for these components does not differ appreciably from that observed for wild-type ring canals, but the ring canal morphology appears abnormal. In contrast, there is a significantly reduced level of anti-phosphotyrosine staining in Src64 egg chambers. This reduction is particularly dramatic at the ring canals, where only faint staining can generally be observed. However, mutant ring canals that maintain elevated anti-phosphotyrosine are observed occasionally. Reduced anti-phosphotyrosine staining is also observed in the cortical regions of the mutant nurse cells. These results indicate that Src64 function is required for the majority of phosphotyrosine accumulation at ring canals, but that the formation of the ring canals and the localization of known ring canal components does not depend on this accumulation (Dodson, 1998).
During the analysis of ring canal morphogenesis, it was observed that Src64 ring canals are smaller than their wild- type counterparts. This phenotype is particularly obvious in the later stages of oogenesis. This effect was quantitated by measuring the outer ring canal diameters during both mid and late stages (stages 5 and 10A) of oogenesis. In stage 5 wild-type egg chambers, the ring canals vary in size between 2.0 and 4.5 mm, with an average size of 3.1 mm. In contrast, Src64 PI ring canals vary in size from 1.0-3.5 mm with an average size 2.6 mm.The difference in ring canal size is more apparent at stage 10A. At this stage, wild-type ring canals vary between 6 and 14 mm with an average diameter of 9.5 mm. This represents a 3.1-fold increase in the average outer diameter of wild-type ring canals between stage 5 and stage 10A. In Src64 PI egg chambers, there is only a 2.3-fold increase in ring canal diameter between stages 5 and 10A. Src64 PI ring canals range from 3-10 mm with an average of 5.9 mm. These measurements indicate that ring canal growth is defective during both early and late phases of ring canal morphogenesis. In order to assess whether these smaller ring canals have other morphological abnormalities, F-actin-, HTS-RC- and Kelch-stained ring canals were examined at high magnification. The small ring canals appear normal except for the presence of a slightly concave inner rim that is reminiscent of the inner rims of earlier stage wild-type ring canals (Dodson, 1998).
Many (45%) stage 10A Src64 PI egg chambers contain fewer than 15 ring canals. This reduction in ring canal number is only observed in stage 9-10 egg chambers, indicating that some ring canals must degenerate during these later stages of oogenesis. One possible consequence of ring canal degeneration would be fusion between cells. Evidence for such fusions was sought by staining mutant egg chambers with fluorescein phalloidin to visualize the filamentous cortical actin at the nurse cell boundaries, and propidium iodide to visualize the nurse cell nuclei. At stages 9-10 approximately 70% of Src64 PI mutant egg chambers have fusions between nurse cells (Dodson, 1998).
The altered morphogenesis and stability of Src64 ring canals and their reduced phosphotyrosine content suggested that Src64 might be a ring canal component. To test this possibility, wild-type egg chambers were stained with anti-Src64 antibodies. Specific staining is detected in the cortex of the nurse cells and appears enriched at the ring canals in wild type egg chambers. Co-staining for F-actin and Src64 shows that Src64 localization overlaps F-actin at the ring canal. These results are consistent with Src64 being localized to the nurse cell and oocyte plasma membranes, as well as to the inner rim of the ring canals (Dodson, 1998).
Src64 regulates the localization of Tec29, a Tec-family kinase required for ring canal growth. A genetic screen was carried out to search for downstream components of the Src64 signaling pathway. Mutations affecting Tec29 dominantly enhance the Src64 ring canal phenotype. Loss of Tec29 function in the female germ line results in a phenotype strikingly similar to that caused by loss of Src64 function. In each case, the ring canals are reduced in size and phosphotyrosine content. Although Src64 and Tec29 mutations have similar effects on ring canal growth, there are differences in their phenotypes that suggest that the two proteins may have nonoverlapping functions. Neither nurse cell fusion nor ring canal detachment is observed in Tec29 mutant egg chambers. This phenotypic difference suggests that Src64 may activate biochemical pathways in which Tec29 does not participate (Guarnieri, 1998). In addition, while Src64 mutations have no effect on viability, animals lacking Tec29 function do not survive to adulthood, indicating a different requirement for Tec29 and Src64 in zygotic development. The phenotype associated with Tec29 lethality is a defect in the process of head involution in which several distinct morphogenetic movements during mid to late embryogenesis normally result in the positioning of the head primordia inside the embryo. The head skeletal defects in Tec29 mutants can be explained by the idea that the correct juxtaposition of tissues in the head during and after head involution is necessary for proper head skeleton sclerotization (Roulier, 1998). The embryonic phenotype correlates with the expression of Tec29 in regions of the head, including the labial and maxillary segments and the cleft between the acron and labrum (Katzen, 1990 and Erica M. Roulier, unpublished experiments, 1998 cited in Roulier, 1998).
Previous studies have suggested that Src64 may function at other times during Drosophila development. If so, what other roles might it play? SRC64 mRNA is broadly expressed throughout development with elevated levels detectable in the central nervous system (CNS) and developing visceral mesoderm of embryos and early pupae. High levels of Src64 are also detected in the photoreceptors of third instar eye imaginal discs (Simon, 1985). Since no Src64 alleles were available prior to Dodson's 1998 report, previous attempts to examine the role of Src64 have relied on ectopic expression of a kinase inactive Src64 protein. In some cases, kinase inactive forms of Src PTKs have been shown to mimic the effects of disrupting the corresponding gene. In the case of Src64, ubiquitous overexpression of the kinase inactive protein during embryogenesis leads to defects in CNS development. During eye development, expression under sevenless transcriptional control of the kinase inactive Src64 leads to the loss of photoreceptors (Kussick et al., 1993). These results suggested that Src64 might have an important role in CNS and photoreceptor development. However, no CNS or photoreceptor defects have been detected in Src64 animals. One possible explanation for this discrepancy is that the amount of Src64 required during CNS and photoreceptor development is much lower than that required during oogenesis. Thus, the very low level of Src64 present in the mutant animals might be sufficient for these processes. Alternatively, expression of the kinase inactive Src64 may interfere with biochemical pathways in which Src64 either does not normally participate or in which Src64 function can be replaced by Src41, the second Drosophila Src PTKs (Takahashi et al., 1996). During embryonic and larval development, the expression of Src41 largely overlaps Src64, including high levels of expression in the CNS and visceral mesoderm (Takahashi, 1996). Thus Src41 may be able to compensate for a lack of Src64 in these tissues. Interestingly, the lack of maternally contributed Src41 in preblastoderm embryos suggests that the female germline is a tissue where Src41 expression may not overlap that of Src64. This difference in expression patterns may explain why the Src64 mutant phenotype is limited to defects in oogenesis. Ultimately, understanding the full range of Src64 and SrcPTK function during Drosophila development will require the identification of mutations in Src41 and any as yet unidentified Drosophila SrcPTKs (Dodson, 1998).
In the region of the polypeptide responsible for kinase and transforming activity, the Drosophila amino acid sequence is 54% homologous with that from v-src; beginning with the tryosine-416 codon, it is 62% homologous with Drosophila Abl (Hoffman, 1983)
date revised: 14 August 98
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