Src oncogene at 42A


Protein Interactions

Physical binding of Src42A to Arm through kinase-domain/Arm-repeat interactions

To determine the molecular basis for genetic interactions between Src42A, shg and arm, whether or not protein products of these genes come together to form complexes within cells was examined using fractionated embryonic extracts. Membrane and cytosolic fractions were prepared from wild-type embryos and embryos with pnr-GAL4-dependent forced expression of either UAS-Src42A[WT], [KR] (dominant negative) or [YF] (activated). This section discusses endogenous interactions in wild-type embryos, whereas physical interactions in embryos with forced Src expression are described in the subsequent section (Takahashi, 2005).

Membrane and cytosolic fractions of wild-type embryos were treated with anti-Arm or anti-E-cad antibodies and the resultant immunoprecipitates were analyzed by SDS-PAGE and subsequent Western blotting. Any appreciable Src42A/Arm signals were detected in the E-cad immunoprecipitates of untransfected S2 cells, which express only a low level of E-cad (Takahashi, 2005).

Not only Arm but also Src42A and E-cad signals were detected in anti-Arm antibody immunoprecipitates obtained from the membrane fraction. Membranous Src42A signals were also coprecipitated by anti-E-cad antibody treatment. By contrast, there were no appreciable signals of E-cad on treating the cytosolic fraction with anti-Arm antibody. In the cytosolic fraction, Src42A signals co-precipitated with Arm appeared much less prominent than those in the membrane fraction. Accordingly, a significant fraction of membranous Arm, a core component of the putative adherens junction, may be considered to form a complex directly or indirectly with E-cad and Src42A as well (Takahashi, 2005).

Arm protein possesses 13 repeats referred to as Arm repeats, which provide binding sites for many Arm/ß-catenin-binding proteins. To determine whether Src42A binds to Arm directly and if so which part of Src42A is responsible for the Src-Arm interaction, a pull-down assay was carried out. Src42A was thus shown to bind to Arm through interaction of the 14 amino acid kinase domain peptide with Arm repeats (Takahashi, 2005).

Requirements of Src for Arm phosphorylation

In vertebrates, tyrosine phosphorylation has been shown to cause the binding of ß-catenin to E-cad to diminish significantly. Interactions between ß- and alpha-catenin may also be negatively regulated by tyrosine phosphorylation. Therefore studies were performed to see whether Arm phosphorylation requires Src activity. Arm was overexpressed in Drosophila S2 cells and RNAi was carried out to clarify any Src42A and/or Src64 involvement in Arm tyrosine-phosphorylation. The dsRNAs used specifically abolished protein expression of the corresponding target genes. Change in the degree of Arm tyrosine residue phosphorylation was monitored with Arm western blotting of anti-pTyr antibody immunoprecipitates of whole or E-cad-free cell extracts. The levels of Tyr-phosphorylated Arm were reduced by transfection with Src64 and/or Src42A dsRNAs, indicating that redundant Src function is required for Arm phosphorylation (Takahashi, 2005).

Consistent with the present findings on cytological accumulation of cytoplasmic E-cad and Arm signals in cells overexpressing wild type Src42A, increased signals of Arm and E-cad were noted in cytosolic fractions obtained from cells with forced expression of wild type or activated Src42A when using alpha-tubulin or Src42A as the control. Similar Src42A activity-dependent increase in cytosolic Arm signals was observed in anti-Arm precipitates and the supernatant fluid of anti-Src42A precipitates. Such increase may possibly be due to Arm tyrosine residue phosphorylation. However, it is considered that cytosolic Arm accumulation in cells with forced expression of wild type or activated Src42A may not necessarily arise from the tyrosine phosphorylation of Arm. Indeed, any Src42A-activity-dependent tyrosine phosphorylation appeared absent from Src42A-free Arm in the cytosolic fraction. Possibly, Src42A-dependent tyrosine phosphorylation activates some unknown factor responsible for cytosolic Arm stabilization, but not Arm itself (Takahashi, 2005).

Csk-deficient boundary cells are eliminated from normal Drosophila epithelia by exclusion, migration, and apoptosis; Loss of Csk functions leads enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion

The construction and maintenance of normal epithelia relies on local signals that guide cells into their proper niches and remove unwanted cells. Failure to execute this process properly may result in aberrant development or diseases, including cancer and associated metastasis. This study shows that local environment influences the behavior of dCsk-deficient cells. Broad loss of dCsk leads to enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion. Loss of dCsk in discrete patches leads to a different outcome: epithelial exclusion, invasive migration, and apoptotic death. These latter phenotypes required sharp differences in dCsk activity between neighbors; dE-cadherin, P120-catenin, Rho1, JNK, and MMP2 mediate this signal. Together, these data demonstrate how the cellular microenvironment plays a central role in determining the outcome of altered dCsk activity, and reveal a role for P120-catenin in a mechanism that protects epithelial integrity by removing abnormal cells (Vidal, 2006).

The mechanisms that regulate organ size and shape are not well understood, but recent studies have pointed to the importance of local interactions between neighboring cells. For example, in the process known as 'cell competition', cells with relatively higher proliferative rates actively eliminate their neighbors by programmed cell death. Conversely, apoptotic cells send proliferative signals to their neighbors to compensate for their loss. In this way, normal tissue size is achieved. The misregulation of such mechanisms may contribute to the development of cancer, since most solid tumors arise from intact epithelia and are resistant to size-control signals. Tumors are particularly dangerous when linked to metastasis, a process in which cells leave the primary tumor and invade distant tissues. These processes are best understood within the context of an intact epithelium, in which the full range of cell interactions is retained. Work in Drosophila has provided an important in situ view of the action of oncogenes within epithelia (Vidal, 2006).

Src family kinases (SFKs) are active in a broad range of cancer types, including tumors of the breast, colon, and hematopoietic systems. SFK activity typically increases as tumorigenesis progresses and is associated with metastatic behavior. The major inhibitor of SFK activity is C-terminal Src kinase (Csk) and its paralog Chk; these may act as tumor suppressors in, e.g., breast cancer, presumably through their ability to inhibit Src activity and perhaps other pathways. Drosophila Csk acts primarily or exclusively through Src pathway regulation, and the reduction of dCsk activity by itself led to increased organ size, organismal lethality, and increased cell proliferation due to a failure to exit the cell cycle. However, neither Csk loss nor Src activation has been clearly linked to early events in tumorigenesis, bringing into question the role of Csk/Src in proliferation in vivo. Instead, Src is currently thought to be a major player in the metastatic events that occur later in oncogenesis. How Csk or Src promotes the metastatic behavior of cells in situ remains largely unknown (Vidal, 2006).

This study analyzed the phenotypes of dCsk in the context of developing epithelia. The outcome of a cell's loss of dCsk is linked to its cellular microenvironment. When dCsk activity is reduced broadly in the developing eye or wing, the result is overproliferation, inhibition of apoptosis, and decreased cell adhesion. Tissue integrity is retained, but dCsk cells become inappropriately mobile and fail to maintain their appropriate contacts. The outcome of these effects is an overgrown and mispatterned adult tissue. By contrast, loss of dCsk in discrete patches results in epithelial exclusion, invasive migration through the basal extracellular matrix, and eventual apoptotic death; these events occur exclusively at the boundary between dCsk and wild-type cells. Further emphasizing the unique nature of cells at this boundary, a specific requirement was found for a signal that includes Drosophila orthologs of E-cadherin, P120-catenin, RhoA, JNK, and the metalloprotease MMP2. Hence, this study explores the mechanisms by which the cellular microenvironment can direct different behaviors of cells, both in the regulation of apoptosis and epithelial integrity. It also uncovers a mechanism for the removal of abnormal cells from a normal epithelium (Vidal, 2006).

This study shows that reducing dCskactivity results in a blockade of apoptosis and downregulation of cellular adhesion. The work is consistent with the view that Csk is a tumor suppressor that acts at multiple steps. Mutations in the locus encoding the Csk paralog Chk have been described in breast tumors, and, in this study, it has been observed that human Chk can functionally replace dCsk. Therefore the experimental advantages of developing Drosophila imaginal epithelia were used to explore specific aspects of dCsk function that are relevant to the behavior of tumor cells (Vidal, 2006).

Visualization studies suggest that a reduction in dCsk activity leads to a failure of cells to stably retain associations with their neighbors, resulting in prolonged cell movement as cells slide across each other in a manner not observed in wild-type tissue. This may reflect a failure to establish stable junctions, excess cell motility, or both. Recent work has demonstrated a critical and dynamic role for the cadherin-based apical junctions in patterning the Drosophila retina. Misexpressing dE-cadherin prevents patterning defects in GMR>dCsk-IR retinas, suggesting that dCsk cells have reduced dE-cadherin function. Links between Csk, Src, cadherins, and junctional integrity have been reported in mammalian cell culture, and an association has been observed between Drosophila Src42A and dE-cadherin during embryonic development (Takahashi, 2005). The data are consistent with this view: misexpression of a kinase-dead form of Src42A leads to a disruption in the localization of the dE-cadherin-associated protein Armadillo; also, reduced Armadillo levels observed in dCsk retinas is rescued by dE-cadherin misexpression. Together, these data suggest that altering dCsk/Src activity affects cell movements by decreasing dE-cadherin adhesion (Vidal, 2006).

The mechanism by which dCsk alters dE-cadherin function is not clear, but it is relevant to note that Src activation can shift cadherin-based cell adhesion from a 'strong' to a 'weak' adhesive state in mammalian cultured cells. Phosphorylation of cadherins and catenins may mediate 'inside-out' signaling that can alter the adhesive strength of the homophilic bond between cells (reviewed in Gumbiner, 2005). Evidence for such a mechanism has been provided for integrin-mediated focal adhesions (reviewed in Hynes, 2002), and Src activity can alter focal adhesions (Yeatman, 2004). However, normal basal membrane architecture was observed in dCsk cells, as assessed both by anti-integrin staining and by transmission electron microscopy, indicating that at least the gross structure is not affected (Vidal, 2006).

The ability of dCsk to influence cell proliferation, apoptosis, and cell adhesion is consistent with its ability to direct tissue overgrowth: reducing dCsk activity throughout a tissue (or the entire organism) leads to significantly enlarged tissues. This ability demonstrates that dCsk can participate in the mechanisms that set tissue size. A small number of other proteins have been implicated in this process, including Salvador, Hippo, and Lats/Warts, which show phenotypes that are strikingly similar to dCsk. Furthermore, dCsk can directly phosphorylate Lats/Warts (Stewart, 2003) in vitro (Vidal, 2006).

However, reduction of dCsk activity shows some important differences. Mutations in salvador, hippo, or lats/warts lead to an increase in Diap1 levels, which, in turn, blocks apoptotic cell death. By contrast, reductions in dCsk does not significantly alter Diap1 protein levels. Furthermore, although both Hippo and dCsk are required to exit the cell cycle, the cell cycle profile from hippo mutant cells is normal, while dCsk cells contain a significant shift toward G2/M (Read, 2004; Stewart, 2003). Perhaps the most striking difference is the effects of these factors on discrete mutant patches. While broad loss of dCsk activity leads to expanded tissues, surprisingly discrete patches of dCsk tissue are eliminated by neighboring cells. Unlike salvador, hippo, or lats/warts, clonal patches of dCsk cells fail to survive to adulthood. The effects of dCsk reduction are more similar to those reported for the tumor suppressor gene scribble. The scribble locus encodes a component of the septate junction that regulates cell polarity and proliferation; mutant cells display neoplastic overgrowth in a homotypic environment, but are removed by JNK-dependent apoptosis in discrete clonal patches abutting wild-type tissue (Vidal, 2006).

This work provides evidence that neighboring wild-type tissue provides a locally nonautonomous signal that leads to the removal of dCsk mutant cells. For example, FRT-derived clones of dCsk cells were out-competed by neighbors with normal levels of dCsk: this was most easily seen by the clonally related 'twin spot' of wild-type tissue that was consistently larger than the few surviving dCsk clones. In contrast, FRT-mediated dCsk clones that encompassed the entire eye survived and overproliferated. In the developing wing, cells at the periphery of sd>dCsk-IR or ptc>dCsk-IR expression domains were preferentially removed by apoptosis. This death is dependent not on absolute dCsk activity, but on the juxtaposition of cells that are starkly different in their levels of dCsk. Small differences, for example across the ptc>dCsk-IR or omb>dCsk-IR graded expression domains, did not trigger cell death (Vidal, 2006).

This translocation and death of dCsk-IR cells at the patched/wild-type boundary requires at least two steps. At boundaries with wild-type tissue, dCsk cells initially lose their apical profile, shift downward, and eventually become basally excluded from the epithelium. Such excluded cells then migrate away from the boundaries in both directions and eventually die by apoptosis. These events are strikingly reminiscent of those described for tumor cells undergoing metastasis. Altered activity of both Csk and Src has been implicated in a broad variety of tumors. Typically, however, increased Src activity is associated with later events in tumorigenesis, particularly metastasis. Although the connections between high Src activity and metastases are not understood, they likely include Src's ability to break cell-cell junctions and increase cell motility. Another hallmark of metastatic behavior is the ability to degrade basal extracellular matrix: this study also demonstrate a functional requirement for MMP2 activity during the translocation of mutant cells out of the wing epithelium (Vidal, 2006).

While evidence supports the view that the activity of Csk -- and presumably Src and perhaps other effectors -- can regulate metastatic behavior, it alone is not sufficient. First, reducing dCsk activity by itself is not sufficient to allow migrating cells to survive; the data suggest that most or all eventually die. This is consistent with previous work highlighting the importance of a 'two-hit' model to allow for stable tumor overgrowth and metastasis. A second mutation that prevents apoptotic cell death would be minimally required. Second, all cells within a discrete dCsk patch are not equivalent: cells at the boundary of the clone that border cells of strongly differing dCsk levels are exclusively prone to release from the epithelium. This work predicts that cells at the borders of some human tumors are especially prone toward metastatic behavior. Metastasis is often the most serious aspect of a tumor, and approaches that address the metastatic behavior of cells may need to take into account the properties of cells at the periphery. Understanding whether and how these cells are unique may help to more effectively target therapeutic intervention (Vidal, 2006).

In addition to enabling a detailed examination of dCsk cells and their behavior within an epithelium, this model system permitted identification of signaling components that are necessary to execute the aberrant cell mobility and cell death. The results indicate important roles for dE-cadherin, dP120ctn, Rho1, dJnk, and MMP2 (Vidal, 2006).

JNK-dependent apoptosis is required for a broad palette of related mechanisms such as cell competition in developing tissues and the removal of scribble mutant cells. JNK signaling is also associated with the movement of cells within epithelia, including dorsal closure in Drosophila and in mammals. Interestingly, JNK activity is required for the synthesis of MMP2 by v-Src-transformed mammalian cells (Vidal, 2006).

JNK activity can be triggered by several upstream signaling factors, including the small GTPases of the Rho family, and genetic data provide a link between dCsk, dJnk, and Rho1. Rho family proteins are key regulators of cell shape and motility. They also promote the cytoskeletal rearrangements required for epithelial-to-mesenchymal transitions (EMTs), and it is noted that dCsk boundary cells show a number of features that are reminiscent of EMTs. In Drosophila, Rho1 was found to induce an 'invasive' phenotype in wing disc cells, but, in this study, it was demonstrated that, similar to dCsk boundary cells, ptc>Rho1 misexpressing cells also undergo apoptotic death. Most importantly, halving the genetic dose of Rho1 strongly suppresses discrete loss of dCsk, but does not appreciably affect broad loss. Thus, Rho1 activity is linked to dCsk, and activation of Rho1 is sufficient to phenocopy both the apoptotic and migratory phenotypes of dCsk cells located near wild-type tissue (Vidal, 2006).

Previous work in mammalian cell culture has provided direct links between Src and P120-catenin, between cadherins and P120-catenin, and between RhoA and P120-catenin; the latter two interactions have been reported in Drosophila tissue culture systems as well. This study further supports links between these factors in dCsk boundary cells. Interestingly, although normal levels of both dP120ctn and Rho1 were required for the efficient removal of dCsk boundary cells, they were not required for the phenotypes resulting from broad loss of dCsk. The requirement for p120ctn specifically in boundary cells may explain why, although it is the only ortholog present in Drosophila, dP120ctn (Drosophila p120-catenin) is not required for organism viability (Vidal, 2006).

Both Src and P120-catenins are known to directly interact with cadherins, and, in fact, a role was demonstrated for dE-cadherin/Shotgun in the removal of dCsk cells. A model is postulated in which the loss of dCsk results in the remodeling of the zonula adherens, presumably by the phosphorylation of catenins and dE-cadherin itself by Src. Src activation is known to switch cadherin from a strong adhesive state to a weak one, providing one potential explanation for why dCsk retinal cells displayed reduced cell adhesion in situ. One critical question regarding cadherins is whether they have signaling roles that are independent of their adhesive properties. Perhaps relevant to this point, it was surprising to find that reducing dE-cadherin function leads to a suppression of the effects of dCsk-IR at the boundary. A simple dCsk-IR-mediated reduction in dE-cadherin adhesion would be enhanced by further reducing dE-cadherin activity, suggesting that dE-cadherin may provide an active signal that promotes boundary cells' release from the epithelium. If such a signal does exist, neighboring wild-type cells must trigger it, either through their own endogenous dE-cadherin or through a separate, local signal. Why are multiple (3-4) rows affected? The results are consistent with the creation of a successive new boundary as the previous row of cells descends, although other longer-range signals cannot be ruled out (Vidal, 2006).

It is noted that reducing dCsk activity by itself is not sufficient to direct stable tumor overgrowth, supporting the importance of a 'two-hit' model in Drosophila. Loss of the junction protein Scribble showed similar phenotypes to dCsk, including apoptosis, but was found to confer survival and metastatic-like behavior to cells in the presence of an activated Ras isoform. Interestingly, coexpression of dE-cadherin prevents this metastatic behavior (Vidal, 2006).

Finally, how can dP120ctn and Rho1 promote release of dCsk near wild-type boundaries but not act similarly with other dCsk cells? One source of information is the cadherins themselves: the boundary creates an interface of cadherins that have been exposed to different levels of Csk and, presumably, Src activity. This unusual interface may generate the needed dE-cadherin signal. Importantly, recent work has noted a change in the subcellular localization of P120-catenin and E-cadherin specifically at the border of human tumor tissues. At the time that ptc>dCsk-IR boundary cells lose their apical profiles, this study found that dP120ctn is relocalized to the cytoplasm. These results again emphasize the possibility that cells at tumor boundaries pose a special risk of undergoing epithelial-to-mesenchymal-like transitions and metastatic behavior. Metastasis is often the most serious complication of progressing tumors. Targeting therapies to this aspect of cancer may benefit from considering boundary cells and their potentially distinctive properties (Vidal, 2006).

Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase signalling

The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. This study shows that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70 (Ferrante, 1995), binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. The Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. It is proposed that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade (Ziegenfuss, 2008).

Developing tissues produce excessive numbers of cells and selectively destroy a subpopulation through programmed cell death to regulate growth. Rapid clearance of cell corpses is essential for maintaining tissue homeostasis and preventing the release of potentially cytotoxic or antigenic molecules from dying cells, and defects in cell corpse clearance are closely associated with autoimmune and inflammatory diseases. In C. elegans the CED-1 receptor is expressed in engulfing cells, where it acts to recognize cell corpses and drive their phagocytosis. CED-1 promotes engulfment through an intracellular NPXY motif, a binding site for proteins containing a phosphotyrosine-binding (PTB) domain, and a YXXL motif, a potential interaction site for proteins containing SH2 domains. The PTB domain adaptor protein CED-6 can bind the NPXY motif of CED-1, is required for cell corpse engulfment and acts in the same genetic pathway as CED-1. CED-1 ultimately mediates actin-dependent cytoskeletal reorganization through the Rac1 GTPase, and Dynamin modulates vesicle dynamics downstream of CED-1 during engulfment, but the molecular signalling cascade that allows CED-1 to execute phagocytic events remains poorly defined (Ziegenfuss, 2008).

Glia are the primary phagocytic cell type in the developing and mature brain. Glia rapidly engulf neuronal cell corpses produced during development, as well as neuronal debris generated during axon pruning or during Wallerian degeneration in the adult brain. In Drosophila, glial phagocytosis of these engulfment targets requires Draper, the fly orthologue of CED-1. Draper, like CED-1, contains 15 extracellular atypical epidermal growth factor (EGF) repeats, a single transmembrane domain, and NPXY and YXXL motifs in its intracellular domain. Drosophila Ced-6 is also required for the clearance of pruned axons, indicating possible conservation of the interaction between CED-1 and CED-6 in flies, but additional signalling molecules acting downstream of Draper have not been identified (Ziegenfuss, 2008).

Draper was identified in a yeast two-hybrid screen for molecules interacting with the regulatory region of Shark. When LexA-Shark, constitutively active Src kinase and AD-Draper are present, Shark and Draper interact physically. In the absence of Src kinase, Shark and Draper fail to interact, indicating that phosphorylation of Draper by Src may be essential for Shark-Draper interactions. The Draper intracellular domain contains an ITAM (YXXI/L-X6-12-YXXL), a key domain found in many mammalian immunoreceptors including Fc, T-cell and B-cell receptors. SFKs phosphorylate the tyrosines in ITAM domains, thereby allowing ITAM association with SH2-domain-containing signal transduction proteins including Syk and Zap-70. Y-->F substitutions of the tyrosine residues were generated within or near the Draper ITAM, and it was found that Tyr 949 and Tyr 934 are critical for robust Draper-Shark binding. These correspond to the consensus tyrosine residues in the predicted Draper ITAM. Plasmids were transfected with carboxy-terminally haemagglutinin-tagged Draper (Draper-HA) or with Draper-HA and Shark with an amino-terminal Myc tag (Myc-Shark) into Drosophila S2 cells, immunoprecipitated with anti-HA antibodies, and western blots were performed with anti-phosphotyrosine, anti-Myc and anti-HA antibodies. Myc-Shark was found to co-immunoprecipitate with Draper-HA, and that anti-phosphotyrosine antibodies label a band corresponding to the position of Draper-HA that is absent in empty vector controls. Further, it was found that a Y949F substitution markedly reduces Draper-Shark association. Taken together, these data indicate Draper and Shark can associate physically through the Draper ITAM domain (Ziegenfuss, 2008).

Attempts were made to determine whether Shark is required for glial phagocytic activity in vivo. Severing adult Drosophila olfactory receptor neurons (ORNs) initiates Wallerian degeneration of ORN axons. Antennal lobe glia surrounding these severed axons respond to this injury by extending membranes towards severed axons and engulfing degenerating axonal debris. These glia express high levels of Draper, and in draperδ5 null mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the central nervous system (CNS). Thus, both the extension of glial membranes to severed axons and the phagocytosis of degenerating axonal debris require Draper signalling (Ziegenfuss, 2008).

Whether Shark function in glia is essential for glial responses to axon injury was explored by driving a UAS-regulated double-stranded RNAi construct designed to target shark (sharkRNAi) with the glial-specific repo-Gal4 driver, severing ORN axons, and assaying the recruitment of Draper and green fluorescent protein (GFP)-labelled glial membranes to severed axons. Maxillary palp-derived ORN axons project to 6 of the roughly 50 glomeruli in the antennal lobe. Within hours after maxillary palps have been ablated in control animals, Draper immunoreactivity decorates severed axons projecting to and within maxillary palp ORN-innervated glomeruli, and GFP-labelled glial membranes are recruited to these severed axons. Strikingly, knocking down Shark in glia completely suppresses these events. Next, antennal ORN axons were severed; these axons project to about 44 of the 50 antennal lobe glomeruli. Antennal ablation therefore injures nearly all glomeruli in the antennal lobe and results in the majority of antennal lobe glia in control animals upregulating Draper and undergoing hypertrophy. It was found that knocking down Shark in glia also blocks this glial response to axon injury. Thus, Shark is essential for all axon-injury-induced changes in glial morphology and Draper expression (Ziegenfuss, 2008).

To determine whether Shark is required for glial phagocytosis of severed axons, a subset of maxillary palp ORN axons were labelled with mCD8::GFP, Shark function was knocked down in glia, and the clearance of severed axons was assayed. In control animals severed GFP-labelled ORN axonal debris was cleared from the CNS within 5 days. In contrast, glial-specific sharkRNAi potently suppresses the clearance of degenerating axons, with severed axons lingering in the CNS for at least 5 days. Then whether mutations in the shark gene affected the glial clearance of degenerating axons was examined. The null allele of shark, shark1, is pupal lethal (Fernandez, 2000). Therefore glial responses to axon injury were assayed in shark1 heterozygous mutants, and dominant genetic interactions between draperδ5 and shark1 were tested. It was found that both draperδ5/+ and shark1/+ animals showed defects in glial phagocytic function: 5 days after injury, significant amounts of axonal debris remained within OR85e-innervated glomeruli. Moreover, shark1/+; draperδ5/+ animals showed a striking suppression of glial clearance of severed axons almost equivalent to that of draperδ5 mutants. Thus, shark mutations dominantly suppress the glial clearance of degenerating ORN axons, and this phenotype is strongly enhanced by removing one copy of draper. These data, taken together with sharkRNAi data, show that Shark is essential for the clearance of degenerating axons by glia (Ziegenfuss, 2008).

Is Shark required for the glial clearance of neuronal cell corpses? In embryonic stage 14-15 control animals, 24.4 cell corpses were found per hemisegment. In contrast, it was found that shark1 null mutants showed a marked increase in CNS cell corpses, with null mutants containing almost twice as many corpses per hemisegment. shark1/Df(2R)6063 mutants accumulate cell corpses at levels similar to those in shark1, indicating that this phenotype maps to shark. These cell corpse engulfment phenotypes are indistinguishable from that of draperδ5 mutants. It is concluded that Shark, like Draper, is also essential for the efficient clearance of embryonic neuronal cell corpses by glia (Ziegenfuss, 2008).

Because it was found that Shark binds Draper only in the presence of an active Src kinase in two-hybrid assays, Drosophila Src kinases were screened for roles in glial phagocytic activity. Interestingly, it was found that glia-specific knockdown of Src42A (src42ARNAi) potently suppress glial phagocytic activity: in src42ARNAi animals, Draper is not recruited to severed maxillary palp axons; glial hypertrophy and upregulation of Draper after antennal ablation was blocked; and GFP-labelled severed axons lingered in the CNS for 5 days. Knockdown of two other Drosophila Src kinases, Btk29A and Src64B, had no effect on the glial phagocytosis of severed axons. Thus, Src42A seems to be essential for all morphological responses of glia to axon injury and for the efficient clearance of degenerating axonal debris from the CNS (Ziegenfuss, 2008).

It was predicted that Draper phosphorylation status should be sensitive to the SFK inhibitor PP2. Indeed, addition of PP2 to S2 cultures led to a decrease in the phosphorylation of Draper and Draper-Shark association. Strikingly, co-transfection of Draper and Src42A led to a marked increase in Draper phosphorylation, which was PP2-sensitive and Draper-specific. Draper-Shark interactions are not dependent on Shark kinase activity because kinase-dead Shark (Shark K698R) associates with Draper. These data indicate that Src42A may phosphorylate the Draper intracellular domain, thereby increasing the association of Shark with Draper and the activation of downstream glial phagocytic signalling (Ziegenfuss, 2008).

This study has identified Shark and Src42A as novel components of the Draper pathway. One potential model for Draper-Shark-Src42A interactions is that Shark and Src42A drive the recruitment of Draper to engulfment targets. However, CED-1 has been shown to cluster around cell corpses even in the absence of its intracellular domain. Moreover, Zap-70 and Syk bind phosphorylated ITAM domains in mammalian immunoreceptors when ITAM domains are phosphorylated by Src after ligand-dependent receptor. Therefore a model is favoured in which the engagement of Draper with its ligand (presumably presented by engulfment targets) promotes receptor clustering, tyrosine phosphorylation of Draper by Src42A, association of Shark, and activation of downstream phagocytic signalling events. This work suggests that Draper is an ancient immunoreceptor in which the extracellular domain is tuned to recognize modified self and the intracellular domain signals through ITAM-Src-Syk-mediated mechanisms. This is the first identification of ITAM-Src-Syk signalling in invertebrates, and it suggests that a pathway similar to Draper-Ced-1 may ultimately have given rise to ITAM-based signalling cascades in mammalian myeloid and lymphoid cells, including those regulated by Fc, B-cell and T-cell receptors (Ziegenfuss, 2008).

Tissue deformation modulates twist expression to determine anterior midgut differentiation in Drosophila embryos

Mechanical deformations associated with embryonic morphogenetic movements have been suggested to actively participate in the signaling cascades regulating developmental gene expression. This paper develops an appropriate experimental approach to ascertain the existence and the physiological relevance of this phenomenon. By combining the use of magnetic tweezers with in vivo laser ablation, physiologically relevant deformations were locally control in wild-type Drosophila embryonic tissues. The deformations caused by germ band extension upregulate Twist expression in the stomodeal primordium. Stomodeal compression triggers Src42A-dependent nuclear translocation of Armadillo/beta-catenin, which is required for Twist mechanical induction in the stomodeum. Finally, stomodeal-specific RNAi-mediated silencing of Twist during compression impairs the differentiation of midgut cells, resulting in larval lethality. These experiments show that mechanically induced Twist upregulation in stomodeal cells is necessary for subsequent midgut differentiation (Desprat, 2008).

Demonstrating the role of mechanical deformations in the regulation of developmental gene expression requires an ability to reproduce endogenous deformations by locally controlling tissue deformations within the living embryo. Although tools for measuring and applying global forces had been previously reported for studying Xenopus embryo tissue explants, approaches for locally manipulating tissues within developing embryos were still lacking. In this study, magnetized cells were remotely manipulated to produce a 60 ± 20 nN force necessary to generate deformations similar to those produced endogenously. The magnitude of this force is smaller by a factor of ~20 than the 1 μN force associated with the convergent extension movements in Xenopus explants measured using the deflection of an optical fiber. This is consistent with the fact that the Xenopus embryo is 10 times larger that the Drosophila embryo. This value is also consistent with the 13 nN force developed by a 20 MDCK cell assembly on a soft micropillar surface, noting that the cell colony is five times smaller than the Drosophila embryo length. Importantly, both magnetic and external uncontrolled forces rescued mechano-sensitive Twist expression in the stomodeum. This indicates that Twist expression might not be highly sensitive to the intensity or symmetry of tissue deformations (Desprat, 2008).

The remote manipulation of magnetized cells in the Drosophila embryo enabled demonstration that mechanical compression of stomodeal cells comparable to those induced by endogenous morphogenetic movements upregulates Twist expression in the stomodeal primordium. Arm nuclear translocation is a major instructive step in the mechanical-to-genetic transduction pathway, coupling the macroscopic events of morphogenetic shape changes to the molecular processes regulating developmental gene expression. Moreover, previous studies showed that Src family kinases are involved in mechano-transduction through two distinct modes: either though direct mechanical activation resulting in phosphorylation of Src (Wang, 2005), or through a permissive mode where a mechanically induced conformational change in a Src substrate makes its phosphorylation site accessible to the already activated p-Src (Sawada, 2006). This study found that Src42A acts in the permissive mode in the mechano-transduction pathway upstream of Arm. Because β-catenin is a substrate of Src in mammalian cells, one might speculate that the mechano-sensitive substrate of p-Src42A in Drosophila embryos may be junctional Arm. Further study will be necessary to determine whether this is the case, or if an unknown mechano-sensitive Src42A substrate controls Arm activation (Desprat, 2008).

At later stages of development during organogenesis, mechanical cues generated by organ functions were also suggested to shape the physiological function of specialized organs. For instance, embryonic muscle activity is involved in mouse bone development through β-catenin activation. This study has found that endogenous morphogenetic movements at early stages of development are able to control gene expression, thus identifying a feedback loop of the embryo morphological development onto the genome. Such mechanical cues may mediate long-range effects that coordinate and synchronize differentiation events throughout the whole embryo. Such effects may be especially important under conditions in which dynamical and complex topology prevents the establishment of the long-range morphogen gradients that are efficient at earlier stages, when cells are arranged in simpler, static geometrical patterns (Desprat, 2008).

The dASPP-dRASSF8 complex regulates cell-cell adhesion during Drosophila retinal morphogenesis

Adherens junctions (AJs) provide structure to epithelial tissues by connecting adjacent cells through homophilic E-cadherin interactions and are linked to the actin cytoskeleton via the intermediate binding proteins beta-catenin and alpha-catenin. Rather than being static structures, AJs are extensively remodeled during development, allowing the cell rearrangements required for morphogenesis. Several 'noncore' AJ components have been identified that modulate AJs to promote this plasticity but are not absolutely required for cell-cell adhesion. dASPP has been identified as a positive regulator of dCsk (Drosophila C-terminal Src kinase) (Langton, 2007). This study shows that dRASSF8, the Drosophila RASSF8 homolog, binds to dASPP and that this interaction is required for normal dASPP levels. genetic and biochemical data suggest that dRASSF8 acts in concert with dASPP to promote dCsk activity. Both proteins specifically localize to AJs and are mutually required for each other's localization. Furthermore, abnormal E-cadherin localization is observed in mutant pupal retinas, correlating with aberrant cellular arrangements. Loss of dCsk or overexpression of Src elicited similar AJ defects. Because Src is known to regulate AJs in both Drosophila and mammals, it is proposed that dASPP and dRASSF8 fine tune cell-cell adhesion during development by directing dCsk and Src activity. The dASPP-dRASSF8 interaction is conserved in humans, suggesting that mammalian ASPP1/2 and RASSF8, which are candidate tumor-suppressor genes, restrict the activity of the Src proto-oncogene (Langton, 2009).

Cell-cell contacts are essential for development and adult life of multicellular organisms. The best-characterized form of cell-cell contact is the adherens junction (AJ), which links neighboring cells via homotypic E-cadherin (E-Cad) interactions. The highly conserved intracellular domain of E-Cad binds to β-catenin, which itself binds to α-catenin. Transient interactions between α-catenin and actin filaments link AJs to the cytoskeleton, though the exact nature of this connection remains controversial. AJs are particularly important for the integrity of epithelial tissues. In addition to establishing and maintaining cell-cell adhesion, AJs regulate several aspects of cellular behavior, including cytoskeletal rearrangement and transcription. Inappropriate disruption of cell-cell contacts can lead to excess proliferation and is a hallmark of the metastatic process (Langton, 2009).

Dynamic remodeling of AJs occurs during all major morphogenetic events involving movement and rearrangement of epithelial cells, including convergent extension and gastrulation. AJ remodeling is necessary for the generation of epithelial structures with extremely precise patterns, such as the hexagonal array of ommatidia in the Drosophila compound eye (Langton, 2009).

SRC signaling is a major cellular pathway known to promote AJ remodeling in development and metastasis. Cellular SRC (c-SRC) is a member of the SRC family kinases (SFKs), which include c-SRC, FYN, and YES. Activated c-SRC is known to regulate AJs by several mechanisms. For example, c-SRC can induce the ubiquitylation of E-Cad by an E3 ubiquitin ligase called Hakai, promoting E-Cad internalization or degradation. In Drosophila, Src42A (one of two c-Src homologs) genetically interacts with E-Cad (encoded by shotgun [shg] in Drosophila), localizes to AJs, and forms a ternary complex with E-Cad and Armadillo (Drosophila β-catenin). Furthermore, Src42A activation leads to decreased E-Cad protein levels and concurrent stimulation of E-Cad transcription by Armadillo and TCF, which is thought to be important for AJ turnover during morphogenesis (Langton, 2009).

The C-terminal region of c-SRC and other SFKs is targeted by C-terminal SRC kinase (CSK), which negatively regulates c-SRC by phosphorylating a conserved tyrosine residue (Tyr527 in avian c-SRC). Drosophila CSK appears to function analogously to mammalian CSK as a negative regulator of SFKs. dCsk is a negative regulator of tissue growth; mutants die as giant pupae and imaginal discs are enlarged as a result of increased proliferation. These observations are seemingly at odds with studies showing that Src activation in Drosophila tissues stimulates proliferation but also leads to considerable apoptosis. A recent report attempted to reconcile this discrepancy, suggesting that lower levels of Src activation induce proliferation and protection from apoptosis, whereas high levels lead to apoptosis and invasive migration (Langton, 2009 and references therein).

It has been shown that dCsk activity is modulated by dASPP, the Drosophila homolog of mammalian ASPP1 and ASPP2, which physically interacts with dCsk and enhances its capacity to phosphorylate Src42A (Langton, 2007). Accordingly, dASPP phenotypes are enhanced by reducing dCsk gene dosage and are rescued by complete removal of Src64B, which functions redundantly with Src42A. This study identifies dRASSF8 as a new dASPP regulator. dRASSF8 is the homolog of mammalian RASSF7/8 (Ras association domain family 7/8). Ras association (RA) domain-containing proteins are putative Ras effectors; they specifically bind the activated (GTP-bound) form of Ras family GTPases, which function in numerous signal transduction pathways regulating proliferation, apoptosis, and differentiation. Mammalian RASSF family members 1–6 are characterized by their domain structure, with a C-terminal RA domain, a C1-like zinc finger, and a SARAH (Salvador-RASSF-Hippo) domain. Mammalian RASSF7–10 are atypical RASSF proteins because they contain an N-terminal RA domain and lack a C1-like or SARAH domain. Recently, Xenopus RASSF7 was shown to be required for completing mitosis. Human RASSF8 is a putative tumor-suppressor gene; when expressed in lung cancer cells, RASSF8 inhibits anchorage-independent growth. Importantly, the molecular function of RASSF8 has not been elucidated (Langton, 2009).

Two RASSF family proteins are encoded by the Drosophila genome. dRASSF is similar to human RASSF1–6 and has been linked to the Hippo pathway. dRASSF8 is similar to human RASSF7 and RASSF8, having an N-terminal RA domain. Published genome-wide yeast two-hybrid data suggested that dRASSF8 interacts with dASPP, prompting an investigation of the relationship between these proteins. Based on genetic and biochemical data, it is suggested that the dASPP-dRASSF8 complex regulates AJs by directing the activity of dCsk and Src (Langton, 2009).

dRASSF8 is the sole Drosophila homolog of mammalian RASSF7 and RASSF8, which are so-called N-terminal RASSF proteins and the least-studied members of the RASSF family. This study demonstrates that dRASSF8 binds to dASPP in Drosophila cells and that RASSF8 binds to ASPP1 and ASPP2 in human cells, indicating that an evolutionarily conserved relationship between these proteins has been uncovered. The function of RASSF8 is currently unknown, and this study thus provides new insights into the function of N-terminal RASSF proteins (Langton, 2009).

Future experiments will determine whether RASSF7 also binds ASPP family proteins or whether this function is specific to RASSF8. RASSF7 has been studied in Xenopus and was found to associate with centrosomes and to be required for completing mitosis. In contrast, the current data suggest that dRASSF8 is not required for cell-cycle progression because null mutants for dRASSF8 are viable. These findings are suggestive of divergent functions for RASSF7 and RASSF8 in vertebrates, with dRASSF8 being functionally analogous to RASSF8 rather than RASSF7. Indeed, GFP-tagged RASSF7 localizes to the nucleus and centrosomes in Xenopus embryos, whereas this study never observed nuclear localization of dRASSF8. Further studies of N-terminal RASSF proteins in vertebrates should clarify whether RASSF7 and RASSF8 have overlapping or independent functions (Langton, 2009).

In vivo data point at a close relationship between dRASSF8 and dASPP, which colocalize and are required for each other's presence at AJs in epithelial cells. dRASSF8 posttranscriptionally regulates the levels of dASPP protein in epithelia. Thus, it seems likely that binding to dRASSF8 stabilizes dASPP and prevents its degradation, which can be observed for many protein complexes. Overall, these data provide compelling evidence for a functional link between dRASSF8 and dASPP, which is likely to be conserved through to their closest mammalian counterparts, RASSF8 and ASPP1/2 (Langton, 2009).

The data suggest that dRASSF8 has some dASPP-independent roles. For example, dRASSF8 mutant wings are large and broadened, whereas dASPP mutant wings are large but of normal shape. In addition, the dRASSF8 adult eye phenotype is more marked than that of dASPP mutants. Accordingly, it was found that dRASSF8, but not dASPP, is required for apoptosis of excess IOCs in the developing pupal retina. It therefore appears that the dRASSF8 eye phenotype results from both reduced apoptosis of IOCs and cell-cell adhesion defects. The subtle differences between the dASPP and dRASSF8 phenotypes indicate unknown functions for dRASSF8, which are not due to its effects on dASPP. Future efforts will be aimed at elucidating these functions (Langton, 2009).

These data are consistent with a model in which dRASSF8 binds to and positively regulates dASPP and, in this way, promotes dCsk activity indirectly. Coimmunoprecipitation experiments support this idea, showing that dRASSF8 and dASPP associate and that dASPP and dCsk associate. However, no detect interaction was detected between dRASSF8 and dCsk, indicating that dRASSF8 does not directly associate with dCsk. The proposed model is also supported by genetic data; the dRASSF8-dCsk genetic interaction is weaker than the dASPP-dCsk interaction, suggesting that dASPP is the primary regulator of dCsk. The weaker genetic relationship between dRASSF8 and dCsk can be explained by the observation that some dASPP protein persists in dRASSF8 mutant tissue. These observations suggest that dRASSF8 regulates dCsk via dASPP (Langton, 2009).

Retinal morphogenesis involves dynamic changes in cell-cell contacts to create the final ordered array of photoreceptors and accessory cells. dASPP and dRASSF8 are required for normal E-Cad localization in 26-27 hr APF retinas, providing an explanation for the patterning defects in mutant eyes. It is proposed that the abnormal E-Cad localization in dASPP mutant eyes results from increased Src activity based on several lines of evidence. dASPP binds to and positively regulates dCsk, leading to Src inhibition; therefore, loss of dASPP increases Src activity, which is known to reduce cell-cell adhesion by promoting the internalization and degradation of E-Cad. In agreement with this, it was shown that loss of dCsk or overexpression of either Drosophila Src leads to loss of AJ material in 26-27 hr APF retinas. This claim is further supported by the fact that the dASPP eye phenotype is suppressed by loss of Src64B. Thus, the presence of the dASPP-dRASSF8 complex at AJs may be required to locally prevent inappropriate Src activation and dissolution of AJs (Langton, 2009).

The fact that dASPP and dRASSF8 mutants are homozygous viable implies that these genes are dispensable for the majority of morphogenetic processes occurring during development. Therefore, the regulation of AJs by dASPP and dRASSF8 may be restricted to the eye. However, as they are expressed in other epithelial tissues, a closer examination of dASPP and dRASSF8 mutants may reveal subtle defects in other morphogenetic processes (Langton, 2009).

It is suggested that dASPP and dRASSF8 are new noncore AJ components and part of the machinery that ensures the fine regulation of AJs by Src during development. This regulation is crucial to provide precisely the right amount of junctional plasticity to allow cell-cell rearrangements and patterning to take place while limiting this plasticity to maintain epithelial coherence and prevent cell delamination. Because the interaction between these proteins is conserved in mammals, this finding is likely to be relevant to mammalian development and to the metastatic process, which is associated with downregulation of E-Cad and loss of cell-cell adhesions. Indeed, ASPP1 knockout mice present defects in the assembly of lymphatic vessels consistent with a potential adhesion defect. This suggests that regulation of cell-cell adhesion may underlie the function of ASPP1/2 and RASSF8 as mammalian tumor suppressors (Langton, 2009).

Src kinases mediate the interaction of the apical determinant Bazooka/PAR3 with STAT92E and increase signalling efficiency in Drosophila ectodermal cells

Intercellular communication depends on the correct organization of the signal transduction complexes. In many signalling pathways, the mechanisms controlling the overall cell polarity also localize components of these pathways to different domains of the plasma membrane. In the Drosophila ectoderm, the JAK/STAT pathway components are highly polarized with apical localization of the receptor, the associated kinase and the STAT92E protein itself. The apical localization of STAT92E is independent of the receptor complex and is due to its direct association with the apical determining protein Bazooka (Baz). This study found that Baz-STAT92E interaction depends on the presence of the Drosophila Src kinases. In the absence of Src, STAT92E cannot bind to Baz in cells or in whole embryos, and this correlates with an impairment of JAK/STAT signalling function. It is believed that the requirement of Src proteins for STAT92E apical localization is mediated through Baz, since can Src can be co-precipitated with Baz but not with STAT92E. This is the first time that a functional link between cell polarity, the JAK/STAT signalling pathway and the Src kinases has been established in a whole organism (Sotillos, 2013).

In vertebrates, there is strong evidence showing that, besides JAK, the Src kinases can activate STAT signalling. First, STAT3 and STAT5 are crucial downstream factors in Src-induced transformation. Second, Src kinase activation has been shown to result in STAT tyrosine phosphorylation. By contrast, in Drosophila the available data suggest that the involvement of Src in STAT92E activation is probably marginal. This is shown by the fact that in Drosophila the phenotypes of null stat92E alleles are very similar to those of mutants in which the function of the JAK kinase, the receptor or of all ligands, is abolished, indicating that STAT92E activation via alternative kinases occurs in a minority of tissues. One of the few cases reported of JAK-independent STAT92E activation occurs during the proliferation and migration of the embryo pole cells where STAT92E is activated by the Ras/Raf pathway downstream of the Torso tyrosine kinase receptor. The study of mutations in the C-terminal Src kinase (Csk), a negative regulator of Src signalling, offers indirect evidence to suggest that Src may activate STAT92E in the Drosophila eye. Eyes that lack Csk are larger than normal, a phenotype also observed when the canonical JAK/STAT pathway is ectopically activated during eye development. Moreover, Csk clones exhibit higher levels of STAT92E expression, which has been interpreted as being due to the induction of a STAT92E positive-feedback loop. However, these results do not clarify whether Src-induced STAT92E activation is due to direct STAT92E phosphorylation or caused by indirect regulation (Sotillos, 2013).

In the ectoderm, where the JAK/STAT pathway is highly polarized, efficient signalling requires STAT92E apical localization achieved through interaction with the membrane-associated apical polarity protein Baz (Sotillos, 2008). This study obtained several pieces of evidence showing that Src is also required for the correct STAT92E membrane localization and signalling. First, although heterozygous stat92E or Src mutant embryos are normal, double heterozygous stat92E and Src mutant embryos display phenotypes that resemble a partial JAK/STAT signalling failure. Second, the expression of a direct target of STAT92E is downregulated in a Src mutant background. Third, decrease of Src gene activity affects STAT92E localization to the membrane of epithelial cells or to the membrane of mesodermal cells expressing Baz. Fourth, in S2R+ cultured cells STAT92E co-localizes with Baz only when co-expressed with Src42A or Src64B. The data also show that tyrosine 711 is not required for membrane localization, demonstrating that this function of Src is independent of STAT92E activation (Sotillos, 2013).

Although it was possible to co-precipitate Baz with STAT92E and Baz with Src42A, co-precipitation of Src42A with STAT92E was not achieved. This suggests that the interaction between Src42A and STAT92E is either too labile to be detected or that Src modifies Baz and that this allows the recruitment of STAT92E to Baz. The data also suggest that the kinase activity of Src42A is dispensable for STAT92E-Baz interactions, as a kinase-dead isoform of Src42A is able to rescue membrane localization in Src mutant backgrounds to the same level that a constitutively activated form can. Although a direct activation of STAT92E in response to Src and growth factors was possible in the minority of cell types, the genetic interactions do not reveal any phenotype apart from those of the canonical JAK/STAT pathway (Sotillos, 2013).

In addition to the parallel dimers formed by SH2 interaction with phosphor-Tyrosine in activated STATs (Mohr, 2012), vertebrate STAT proteins have been shown to form 'inactive' antiparallel dimers mediated by the region that includes the N-terminal and the coiled-coil domains (Mao, 2005; Neculai, 2005; Ota, 2004). In Drosophila, active phosphorylated STAT92E also forms parallel homodimers through an SH2-phosphotyrosine interaction. This study suggests that STAT92E may also form inactive homodimers mediated through the N-terminal coiled-coil domain. Thus, formation of STAT92E dimers prior to pathway activation could be an ancestral STAT characteristic, reinforcing Drosophila as a model for studying vertebrate STAT signalling (Sotillos, 2013).

Previous papers have described the requirement of a STAT92E-Baz interaction for efficient JAK/STAT signalling (Sotillos, 2008). This study has uncovered the domains in both molecules involved in this physical interaction. In the case of STAT92E the region was narrowed down to the transactivation (TA) domain of the protein: only the most C-terminal part of the molecule is able to localize to the apical membrane cortex on its own and to co-precipitate with Baz. Moreover, when the TA domain is removed, the C-terminal domain is unable to localize to the cell membrane where Baz is located and reduces its ability to co-precipitate with Baz, probably owing to the loss of binding to the N-terminal part of Baz. However, this construct is still able to bind to the C-terminal part of Baz, indicating that another domain in this fragment is also involved in this interaction. In vertebrates, the TA domain has been shown to be crucial for the regulation of the activity of STAT through the interaction with several proteins. This study has added a new function to this domain as a mediator of the interaction of STAT92E with Ba (Sotillos, 2013).

Baz is a scaffolding protein that is able to interact with various proteins and lipids through different regions. Interaction with STAT92E requires both the Baz N-terminal region (1-317) that includes the oligomerization domain and the C-terminal region (1048-1464) that includes the phosphatidylinositol-binding site. Given that both Baz N- and C-terminal domains are conserved, and that STAT92E TA domain is conserved in vertebrate STATs, it is possible that the PAR3-STAT interaction is a conserved feature of JAK/STAT signalling. S2 cell and embryo experiments show a requirement of Src in the STAT92E-Baz interaction. Paradoxically, it is possible to precipitate STAT92E in the absence of Src using the N- and the C-terminal fragments of Baz in vitro. Since in vitro Baz fragments are being used that most probably have a different conformation from the full-length protein, this paradox can be explained if, in vivo, Src function was required to change the conformation of Baz or to displace another protein that interferes with the binding, events that would not take place in the in vitro binding (Sotillos, 2013).

In summary, these results show that Src42A and Src64B are required redundantly in the ectoderm to allow STAT92E to bind to Baz. This interaction leads to the priming of JAK/STAT signalling by concentrating inactive STAT92E dimers apically near the polarized receptor kinase complex, contributing in this way to the efficient canonical JAK/STAT signalling. Although it cannot be completely discarded that future studies in Drosophila may find some specific cell type in which direct STAT92E activation by Src kinases exists, the data indicate that, in general, there is no direct STAT92E activation by Src in Drosophila. It is speculated that the existence of complexes where STAT, PAR3 and Src interact might have allowed the evolution of STAT-activation shortcuts that in vertebrates would have led to Src directly phosphorylating STAT in tyrosine 700 without the intervention of JAK or the canonical receptors. Considering the relevance of these proteins in development and disease, future studies should address whether apical STAT localization through PAR3-Src activity is also functioning in the vertebrate lineage (Sotillos, 2013).

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

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