Src oncogene at 64B



Sequence analysis of a cDNA clone representing the Drosophila Src64 locus suggests that the gene encodes a 62 kd protein that is remarkably similar to the protein product of chicken c-src. The Drosophila Src64 locus is transcribed into three mRNAs that are each regulated independently during development. Drosophila SRC64 mRNA is abundant in embryos and pupae but rare in larvae and adults. In situ hybridization reveals that after the first 8 hr of development, SRC64 mRNA accumulates almost exclusively in neural tissues such as the brain, ventral nerve cord, and eye-antennal discs, and in differentiating smooth muscle. It is concluded that Drosophila Src64 may not be a mitotic signal but instead may play a role in the development of neural tissue and smooth muscle (Simon, 1985).

Differing Src signaling levels have distinct outcomes in Drosophila

High levels of Src activity are found in a broad spectrum of cancers. The roles of Src and its negative regulator Csk have been extensively studied, although results have often proved contradictory or the relevance to whole organisms is unclear. In Drosophila, overexpression of either Src orthologue resulted in apoptotic cell death, but paradoxically, reducing dCsk activity led to over-proliferation and tissue overgrowth. This study showed that in Drosophila epithelia in situ, the levels of Src signaling determine the cellular outcome of Src activation. Apoptotic cell death was triggered specifically at high Src signaling levels; lower levels directed antiapoptotic signals while promoting proliferation. Furthermore, the data indicate that expression of kinase-dead Src isoforms do not necessarily act as dominant-negative factors, but can instead increase Src pathway activity, most likely by titrating Csk activity away from endogenous Src. The importance of Src activity levels was emphasized when oncogenic cooperation between Src and Ras was examined: malignant overgrowth was observed specifically when high Src signaling levels were achieved. A model is proposed in which low levels of Src signaling promote survival and proliferation during early stages of tumorigenesis, whereas strong Src signaling, coupled with antiapoptotic signals, directs invasive migration and metastasis during advanced tumor stages (Vidal, 2007).

This study provides evidence that different levels of Src signaling lead to different biological outcomes. It is speculated that Src activity plays two important but separable roles during tumor maturation: early low levels of Src contribute to tumor overgrowth, whereas later high levels of Src, coupled with other oncogenes such as Ras, lead to invasive migration. Previous work emphasized the importance of Csk/Src-dependent signals present at tumor boundaries that can provoke metastatic-like behavior. In addition, the current data reconcile the contrasting phenotypes observed between partial reduction of Csk activity versus a strong increase in Src activity by demonstrating the importance of low versus high levels of Src pathway activation, respectively (Vidal, 2007).

It was also considered what precisely is being modeled with the use of altered Src isoforms, a common approach in the study of signaling pathways including Src. Unexpectedly, it was observed that kinase-dead versions of the two Drosophila SFKs did not behave as expected for dominant-negative isoforms. Furthermore, ectopic expression of dSrc42A, including as a kinase-dead isoform, led to the activation of its sole paralogue, dSrc64B. These data suggest an alternative explanation to paradoxical and controversial observations reported for vertebrate Src. The primary phenotype of src−/− knock-out mice, osteopetrosis caused by defective osteoclasts, was rescued by introducing a kinase-dead Src isoform (SrcKD). In addition, SrcKD rescued the reduced levels of phosphorylated tyrosine observed in src−/− osteoclasts. This led to the suggestion that the essential activities of Src are mediated exclusively through protein-protein interactions and not through kinase activity. The work suggests an alternative model in which endogenous SFKs are ectopically activated in SrcKD-rescued osteoclasts through the titration of Csk activity; this ectopic activation can then compensate for the loss of any individual SFK. In this view, the ability of SrcKD to rescue Src still invokes kinase activity, albeit indirectly (Vidal, 2007).

The results indicate that high levels of Src signaling can show oncogenic cooperation with Ras to direct tumoral growth and organismal lethality. Similarly to Ras/Scrib, Ras/dCsk cells displayed overgrowth, loss of epithelial polarity, migration, and invasion into the ECM. The data indicate that, although cells did not metastasize into distant tissues, they were capable of invading nearby tissues within the cephalic complex such as the brain. In mammals, the ability to invade a local blood or lymph vessel could provide a malignant cell the ability to reach and colonize most tissues of the organism. Therefore, current Drosophila models could model some (albeit not all) fundamental aspects of the metastatic process (Vidal, 2007).

Increasing levels of Src activation correlate with advancing tumorigenic stages in a variety of human cancers, including lung, breast, pancreatic, ovarian, and colorectal cancer. Strikingly, this study observed a similar correlation with Src-driven oncogenesis in Drosophila: high (but not low) levels of Src signaling cooperated with oncogenic Ras to produce invasive overgrowth and organismal lethality. One question in the cancer field is whether ectopic expression studies truly model aspects of human tumors. The current results indicate that they can; however, attention must be paid to levels of activity and the specific aspects of tumorigenesis that each level emulates. Emerging models for tumorigenesis should consider not only the activation of multiple and specific pathways, but should also calibrate these levels to explore, e.g., benign overgrowth versus metastasis versus cell death (Vidal, 2007).

Although Src activity can affect cell proliferation and survival, a growing consensus in the field is that a major role for Src is to promote metastasis by stimulating migratory behavior in transformed cells. This study suggests that the proliferative and survival signals from low Src activation contributes to tumor growth in early stages. In line with this, a recent study shows that mice with keratinocyte-restricted deletion of Csk have mild SFK activation and develop epidermal hyperplasia, but without malignant transformation (Vidal, 2007).

In advanced stages, increased Src signaling, together with the acquisition of strong antiapoptotic signals that protect cells from Src-induced apoptosis such as oncogenic Ras, may reveal the ability of Src activation to drive invasive migration and, potentially, metastasis. For example, it is noted that pancreatic ductal adenocarcinoma involves the invasion of exocrine cells through intrapancreatic nerves, leading to severe damage and pain, promoting cancer spread, and precluding resection. Src, expressed at high levels in the majority of these tumors, is likely required for tumor progression, and cell culture studies suggest that invasion requires Ras signaling for pancreatic tumor targeting. Finally, Src-specific kinase inhibitors have entered clinical trials. The current results with dominant-negative Src isoforms suggest caution, however, as the potential of a Src:drug complex to titrate Csk/Chk activity may yield unpredictable results in the context of Src-overexpressing tumors (Vidal, 2007).

Effects of Mutation or Deletion

The cellular functions of the Drosophila Src64 gene product and of most vertebrate Src-family kinases, are unknown. The effects of over-expression of wild-type and mutated forms of Src64 were examined in transgenic Drosophila. Expression of both wild-type Src64 and a C-terminally truncated mutant at high levels during embryonic development induces extensive tyrosine phosphorylation of cellular proteins and caused considerable lethality, correlating with a block to germ-band retraction. Over-expression in the eye imaginal disc leads to the excess production of photoreceptor cells in the adult ommatidia. In contrast, expression of a kinase-inactive form of Src64 causes distinct nervous system abnormalities in embryos and decreases the numbers of photoreceptor cells in the adult eye ommatidia. This suggests that active forms of Src64 alter development by phosphorylation. Both the lethality and the eye roughening caused by activated Src64 are partially suppressed by mutations in the Drosophila Ras1 gene. These results suggest that over-expressed Src64 may function through Ras1 to stimulate differentiation in the embryonic nervous system and eye imaginal disc, and that kinase-active Src64 interferes with these processes (Kussick, 1993).

Vertebrate Src can be activated by specific mutations to become oncogenic. Analogous mutations in Drosophila Src64 induce abnormal differentiation of photoreceptor cells when expressed ectopically in the developing Drosophila adult eye. The roles played in this process by the adapter protein Downstream of receptor kinases (Drk), and the SH2 domain-containing tyrosine phosphatase Corkscrew (Csw), have been examined. Dominant-negative mutations in either the drk or csw genes ameliorate the developmental abnormalities induced by activated Src64. This suggests that Drk and Csw are required downstream of, or parallel to, Src64. Csw does not act solely as an upstream activator of Scr64. The results are discussed in relation to potential roles for the vertebrate homologs of Drk and Csw (Grb2 and SHP2, respectively) in the transformation of fibroblasts by vertebrate Src (Cooper, 1996).

It is thought that the 'Rpd3 mutation' described De Rubertis (1996) is actually in the adjacent gene Src64B. There is a small exon of Src64B between the P element insert described by De Rubertis and Rpd3. It therefore appears that the enhancement observed in that mutant is actually the result of disruption of Src64B and not the result of mutation of Rpd3. Mottus (2000) describes analysis of 3 point mutations all of which dominantly suppress PEV and 2 deletions in Rpd3 which do not display a dominant effect on PEV. At the time the Mottus paper was written, the genome project was not yet completed and the possible involvement of Src64B was not yet suspected (R. Mottus, 2006 personal communication to the editor of The Interactive Fly).

In Drosophila, the Jun amino-terminal kinase (JNK) homolog Basket (Bsk) is required for epidermal closure. Mutants for Src42A, a Drosophila c-src protooncogene homolog, are described. Src42A functions in epidermal closure during both embryogenesis and metamorphosis. The severity of the epidermal closure defect in the Src42A mutant depends on the amount of Bsk activity, and the amount of Bsk activity depends on the amount of Src42A. Thus, activation of the Bsk pathway is required downstream of Src42A in epidermal closure. This work confirms mammalian studies that demonstrate a physiological link between Src and JNK (Tateno, 2000).

Genes that regulate cell shape changes in Drosophila are required for dorsal closure of the embryonic epidermis and thorax closure of the pupal epidermis. Mutations in genes such as hemipterous (hep) and basket (bsk, also known as DJNK) result in abnormal embryos with a dorsal hole or abnormal adults with a dorsal midline cleft. Hep and Bsk are homologous to the mammalian MKK7 (MAPK kinase 7) and JNK, and they are components of a MAPK (mitogen-activated protein kinase) cascade. Although the role of the Hep-Bsk cascade during dorsal closure has been extensively studied, the upstream trigger of this cascade is poorly understood. To identify the trigger, a screen was carried out for mutants showing the dorsal midline cleft phenotype, like a mild hep mutant. The mutant for Src42A shows this phenotype and Src42A regulates Bsk during Drosophila development (Tateno, 2000).

Furthermore, the Tec29 Src42A double mutant shows complete embryonic lethality, and a certain fraction of the dead embryos show the dorsal open phenotype. Activated DJun, a transcription factor downstream of Bsk, partially rescues the dorsal open phenotype in the Tec29 Src42A double mutant. Thus, Src42A appears to regulate Bsk in the fusion of epithelial sheets during embryogenesis and metamorphosis, and Tec29 is involved in this regulation. The double mutant for Src64 and Src42A manifests a mild but clear dorsal open phenotype, which suggests a functional redundancy between Src64 and Src42A (Tateno, 2000).

Expression of puc is known to be induced by the Bsk signal. In the wing disc of the wild-type third-instar larva, puc is expressed in the dorsal midline of the adult notum. In the wing disc of the Src42AJp45 mutant, puc expression is reduced. In contrast, larvae with a constitutively activated form of Src42A (Src42ACA) shows ectopic expression of puc. Further, introduction of a hep null mutation reduces the amount of ectopic puc expression. It is known that Bsk induces expression of puc and decapentaplegic (dpp) during embryonic dorsal closure. The embryos of the Tec29 Src42A double mutant do not show any puc or dpp expression in the leading edge cells. These results indicate that Src42A, Tec29, Hep, and Bsk regulate dpp and puc expression during embryonic dorsal closure (Tateno, 2000).

During embryonic dorsal closure, the Hep-Bsk signal is required for elongation of the leading edge cells. In the absence of the Bsk signal, these cells do not fully elongate. The accumulation of F-actin and phosphotyrosine (P-Tyr) in leading edge cells is associated with the elongation of these cells. Accumulation of these substances is disturbed in the DJun and the puc mutants. In the double mutant for Tec29 and Src42A, the leading edge cells contain reduced quantities of F-actin and P-Tyr, and these cells are only partially elongated. Thus, the defect in embryonic dorsal closure in the Tec29 Src42A double mutant is caused by this failure in cell shape change, as is the case in the DJun mutant (Tateno, 2000).

A model is proposed in which Src42A, upon receiving an unidentified signal, activates the Hep-Bsk pathway to regulate cell shape change and epidermal layer movement. This is consistent with the observation in mammals that c-Src regulates the cell morphogenetic and migratory processes and is known to activate JNK. As in Drosophila, c-Src definitely affects F-actin organization and P-Tyr localization during cell morphogenesis. Therefore, Src regulation of JNK activity toward a change in cell shape may be conserved (Tateno, 2000).

It can be also interpreted that Src42A acts upstream of DFos, a dimerization partner of DJun. Although the Src42A, Tec29, and Src64 single mutants do not show a dorsal open phenotype, the DFos mutant clearly exhibits it. This relationship is also analogous to that in mammals. Both c-src and c-fos knockout mice have a similar defect, osteopetrosis caused by reduced osteoclast function. But the phenotypic severity is milder in c-src than in c-fos knockouts; this can be explained by the functional overlap in multiple Src-family tyrosine kinases. Accordingly, in both Drosophila and mammals, multiple nonreceptor tyrosine kinases may cooperate to regulate the function of the Jun/Fos complex (Tateno, 2000).

Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Csw functions throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unambiguously elucidated, current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question. The isolation and characterization of a new, spontaneously arising, viable allele of csw, cswlf, has allowed a genetic approach to identify loci required for Csw function. The rough eye and wing vein gap phenotypes exhibited by adult flies homo- or hemi-zygous for cswlf has provided a sensitized background from which a collection of second and third chromosome deficiencies have been screened to identify 33 intervals that enhance and 21 intervals that suppress these phenotypes. Intervals encoding known positive mediators of RTK signaling, e.g., drk, dos, Egfr, E(Egfr)B56, pnt, Ras1, rolled/MAPK, sina, spen, Src64B, Star, Su(Raf)3C, and vein, as well as known negative mediators of RTK signaling, e.g., aos, ed, net, Src42A, sty, and su(ve), have been identified. Of particular interest are the 5 lethal enhancing intervals and 14 suppressing intervals for which no candidate genes have been identified (Firth, 2000).

Interestingly, enhancing and suppressing genetic interactions were detected with genomic intervals containing the nonreceptor tyrosine kinases Src64B and Src42A, respectively. With regard to Src64B, a small deletion that encompasses the Src64B gene, Df(3L)10H as well as the adult viable hypomorphic mutation Src64BDelta 17 were tested, both of which enhance the wing phenotype of cswlf; however, unlike the deficiency Df(3L)10H, no obvious enhancement of the cswlf rough eye phenotype was observed in combination with Src64BDelta 17. While the role of Src64B in RTK signaling in Drosophila has not been broadly explored, ectopic expression studies have suggested that Src64B plays a positive role during photoreceptor differentiation. These observations have been extended to include a putative role for this gene in Egfr-mediated specification of wing veins. With regard to Src42A, there are conflicting reports with respect to Ras pathway regulation. Src42A maps to 41A (Src41A) and plays a positive role in Ras signaling. The same gene has been mapped to 42A and a negative role for this kinase in Egfr signaling has been demonstrated. When a genetic interaction between cswlf and a P-element insertion allelic to Src42A was tested, a suppression of the cswlf phenotypes was observed, consistent with a negative regulatory role for this gene (Firth, 2000 and references therein).

Raf is an essential downstream effector of activated Ras in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified 'Raf activator.' In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, a genetic screen was conducted to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). A collection of chromosomal deficiencies representing ~70% of the autosomal euchromatic genomic regions was examinied for their abilities to enhance the lethality associated with a hypomorphic viable allele of Draf, DrafSu2. Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of DrafSu2, causing lethality in DrafSu2 hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3epsilon, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. Src64B interacts genetically with Draf and an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, a genetic interaction was discovered between DrafSu2 and Df(3L)vin5 that reveals a novel role for Draf in limb development. Loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs (Li, 2000).

Src64B and Tec29A are removed by two deficiencies that each dominantly enhance the lethality of DrafSu2. They were selected as candidate genes for these two deficiencies because a survey of FlyBase for genes in the regions removed by the deficiencies did not yield other genes more likely to be involved in Draf function. The Src64BDelta17 allele in homozygotes enhances DrafSu2, confirming that Src64B genetically interacts with DrafSu2. Overexpression of an activated form of Src64B in early embryos can cause activation of the Tor target gene tll and cuticular defects similar to those caused by gain-of-function mutations in tor. These results are consistent with a role for Src64B in Tor signaling and/or Draf activation. It was not possible to demonstrate that Tec29A could enhance Draf using an available mutant allele of Tec29A. However, indirect evidence has been obtained suggesting a requirement of Tec29A in Tor signaling. (1) Tec29A206 homozygous mutant embryos exhibit defects in the terminal structures that are specified by the Tor pathway. Specifically, they show defective mouth parts and shortened Filzkörper, phenotypes consistent with disruption of Tor signaling. (2) Reducing the activity of Tec29A suppresses a gain-of-function tor allele. Most strikingly, embryos zygotically homozygous for Tec29A206 that are derived from torY9 mothers exhibit mouth parts and Filzkörper indistinguishable from those of Tec29A206 embryos. (3) Mutation of Tec29A restores most of the ventral denticle bands that would have been deleted due to torY9, suggesting that Tec29A is genetically epistatic to tor. However, many of the embryos still exhibit minor disruptions in the ventral denticle bands, a defect reminiscent of weak tor gain-of-function mutations. This suggests that homozygosity for Tec29A206 cannot completely suppress torY9. (4) Possibly, while Tec29A may be required for Tor signaling, Tec29A206 may not be a null allele and therefore cannot completely suppress torY9. This would be consistent with the inability of this allele to enhance DrafSu2. Alternatively, the maternally contributed Tec29A may be able to partially mediate signaling by the mutant TorY9 protein. (5) Tec29A may not be an absolute requirement for Tor signaling, but rather it may function in a separate pathway that in conjunction with Tor is required for the differentiation of terminal structures (Li, 2000).

The likelihood that Src64B and Tec29A are involved in Draf activation is based upon data from in vitro studies of mammalian c-Src function. Src kinases can phosphorylate and activate Raf-1 in vitro, and the tyrosine residues phosphorylated by Src are important for Raf-1 activation. Tec kinases are very similar to Src kinases in the kinase domain, but lack the C-terminal regulatory tyrosine and the N-terminal myristylation site that are specific for Src family members. Tec kinases interact with and are activated by Src through phosphorylation. It has been shown in Drosophila that Tec29A is regulated by Src64B and that both are required for the growth of ring canals of the egg chamber. Although it has not been documented that Tec can phosphorylate Raf in vivo, given the similarities in the kinase domain, it is not unreasonable to propose that Tec could do so. Finally, consistent with these results, the two genomic regions containing Src64B and Tec29A have also been identified as required for the function of Corkscrew (Csw) in a similar screen for modifiers of a partial loss-of-function csw allele (Li, 2000).

Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference has identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. RhoGAP inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by RhoGAP. Local regulation of RhoGAP could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of RhoGAP by integrin and Src, both implicated in neural plasticity (Billuart, 2001).

Since rat p190 can rescue the Drosophila RhoGAP (p190) loss-of-function phenotypes, tests were performed to see if upstream regulators of mammalian p190 could interact with Drosophila p190 to regulate MB axon morphogenesis. The Src family of tyrosine kinases phosphorylate mammalian p190. Tests were performed to see if the Drosophila Src homolog, Src64, regulates p190 activity. Heterozygosity for two Src64 alleles significantly suppresses the p190 RNAi phenotype, with the strength of suppression correlating with the strength of the alleles used. This result is consistent with the notion that Src64 negatively regulates p190 (Billuart, 2001).

Protein kinase A (PKA) holoenzyme is anchored to specific subcellular regions by interactions between regulatory subunits (Pka-R) and A-kinase anchoring proteins (AKAPs). The functional importance of PKA anchoring during Drosophila oogenesis has been examined by analyzing membrane integrity and actin structures in mutants with disruptions in A kinase anchor protein 200 (Akap200). In wild-type ovaries, cAMP-dependent protein kinase R2 (Pka-RII) the regulatory subunit of cAMP-dependent protein kinase 1 (Pka-C1, also known as Protein kinase A) and Akap200 localize to membranes and to the outer rim of ring canals (actin-rich structures that connect germline cells). In Akap200 mutant ovaries, Pka-RII membrane localization decreases, leading to a destabilization of membrane structures and the formation of binucleate nurse cells. Defects in membrane integrity could be mimicked by expressing a constitutively active PKA catalytic subunit (Pka-C) throughout germline cells. Unexpectedly, nurse cells in Akap200 mutant ovaries also have enlarged, thin ring canals. In contrast, overexpressing Akap200 in the germline results in thicker, smaller ring canals. To investigate the role of Akap200 in regulating ring canal growth, genetic interactions with other genes that are known to regulate ring canal morphology were examined. Akap200 mutations suppress the small ring canal phenotype produced by Src64B mutants, linking Akap200 with the non-receptor tyrosine kinase pathway. Together, these results provide the first evidence that PKA localization is required for morphogenesis of actin structures in an intact organism (Jackson, 2002).

Although many genes have been identified that alter ring canal function, altered ring canal size has only been observed in mutants of the non-receptor tyrosine kinases Src64B and Tec29. In these mutants, ring canals are smaller, a phenotype opposite that of the Akap200 mutants. Ring canals were examined in Src64B mutant ovaries when Akap200 gene dose is reduced, to determine whether Akap200 could antagonize Src64B in regulating ring canal size. The hypomorphic Src64BPI allele is homozygous viable and produces ring canals that are smaller than wild type. Akap200k07118 acted as a dominant suppressor of the small ring canal size phenotype produced by these mutants. Females of genotype Akap200k07118/+; Src64BPI/Src64BPI produce ring canals that are almost wild type in size and have near normal amounts of phosphotyrosine. Reducing Src64B gene dose by half, however, fails to affect the size of the ring canals produced by Akap200k07118 mutants. Ring canals produced by Akap200k07118/Akap200k07118; Src64BPI/+ are slightly smaller than those produced by Akap200k07118 mutants. These results suggest that Src64B and Akap200 act antagonistically to regulate ring canal growth. Akap200 protein localization is not altered detectably in Src64BPI mutant egg chambers, nor is Src64B localization changed visibly in Akap200 mutant ovaries. These observations suggest that post-translational mechanisms may be responsible for the genetic interaction (Jackson, 2002).

Cooperation of the BTB-Zinc finger protein, Abrupt, with cytoskeletal regulators in Drosophila epithelial tumorigenesis

The deregulation of cell polarity or cytoskeletal regulators is a common occurrence in human epithelial cancers. Moreover, there is accumulating evidence in human epithelial cancer that BTB-ZF genes, such as Bcl6 and ZBTB7A, are oncogenic. Previous studies on Drosophila melanogaster have identified a cooperative interaction between a mutation in the apico-basal cell polarity regulator Scribble (Scrib) and overexpression of the BTB-ZF protein Abrupt (Ab). This study shows that co-expression of ab with actin cytoskeletal regulators, RhoGEF2 or Src64B, in the developing eye-antennal epithelial tissue results in the formation of overgrown amorphous tumours, whereas ab and DRac1 co-expression leads to non-cell autonomous overgrowth. Together with ab, these genes affect the expression of differentiation genes, resulting in tumours locked in a progenitor cell fate. Finally, the study shows that the expression of two mammalian genes related to ab, Bcl6 and ZBTB7A, which are oncogenes in mammalian epithelial cancers, significantly correlate with the upregulation of cytoskeletal genes or downregulation of apico-basal cell polarity neoplastic tumour suppressor genes in colorectal, lung and other human epithelial cancers. Altogether, this analysis reveals that upregulation of cytoskeletal regulators cooperate with Abrupt in Drosophila epithelial tumorigenesis, and that high expression of human BTB-ZF genes, Bcl6 and ZBTB7A, shows significant correlations with cytoskeletal and cell polarity gene expression in specific epithelial tumour types. This highlights the need for further investigation of the cooperation between these genes in mammalian systems (Turkel, 2015).

This study has shown that over-expression of the Ab BTB-ZF protein cooperates with upregulation of RhoGEF2 or Src64B in tumorigenesis, whereas Ab and DRac1 do not cooperate. Furthermore, expression of Ab with each of these cytoskeletal regulators results in disruption to differentiation, in that the photoreceptor cell marker, Elav, and the early cell fate gene, Dac, are not expressed, although the antennal cell fate gene, Dll, is retained in all except ab Src64B co-expressing clones. Finally, a significant correlations was found in human epithelial cancer datasets between the high expression of BTB-ZF oncogenes, Bcl6 and ZBTB7A, and low expression of Dlg2 or lgl1 cell polarity genes or high expression of ArhGef11, ArhGef12, MAP2K4, MAP2K7, MAPK8, MAPK9, MAPK10, Src or Yes1 cytoskeletal genes. This data suggests that cooperation between these genes may occur in some human epithelial cancers (Turkel, 2015).

RhoGEF2 ab or Src64B ab tumours showed overgrowth during an extended larval period resulting in giant larvae and loss of differentiation. However, unlike scribab tumours there was also non-cell autonomous proliferation and the tumours did not appear to be as invasive as scrib ab tumours, although a more detailed analysis of this is required. By contrast, co-expression of DRac1 and ab did not result in cooperative tumorigenesis, but rather non-cell autonomous proliferation. Relative to the cooperation of these cytoskeletal genes with RasV12, RhoGEF2 or Src64B cooperation with ab showed similar properties. By contrast, DRac1 RasV12 tumours showed strong cell-autonomous overgrowth and invasive properties, whereas DRac1 ab expressing cells did not overgrow relative to wild-type tissue, but instead the surrounding wild-type tissue was induced to overgrow (Turkel, 2015).

The phenomenon of non-cell autonomous overgrowth observed in DRac1 ab mosaic eye-antennal discs (and to some extent in ab RhoGEF2 and ab Src64B mosaic discs) is similar to the effect that 'undead' cells (cells where apoptosis is initiated by activation of initiator caspases, but effector caspase activation is blocked - and thus cell death - by expression of the inhibitor, p35) have upon their surrounding wild-type neighbours. This occurs by the release of Wingless (Wg) and Decapentaplegic (Dpp) and perhaps other morphogens from the undead cells, which promote compensatory proliferation in the surrounding wild-type cells. The similarity of these phenotypes suggests that DRac1 ab expressing cells might be in an 'undead' state, and release Dpp and Wg, thereby inducing proliferative overgrowth of the surrounding wild-type cells. Alternatively, these cells might be deficient in mitochondrial function, which together with expression of a cell-survival factor, such as RasV12, results in non-cell autonomous overgrowth without evidence of caspase activation. In this scenario, the mitochondrial dysfunction results in increased reactive oxygen species (ROS) that activate JNK signalling, which subsequently inactivates Hippo pathway signalling, leading to increased expression of the target genes Wingless and Unpaired (Upd) that activate Wg signalling and Jak/Stat signalling, respectively, in the neighbouring wild-type cells. However, since TUNEL-positive cells in were observed DRac1 ab, RhoGEF2 ab and Src64B ab expressing clones, it is more likely that the first of these mechanisms is responsible for the non-cell autonomous overgrowth, however this requires further investigation (Turkel, 2015).

Interestingly, in undead cells JNK activation is required for Dpp and Wg production and non-cell autonomous overgrowth. Furthermore, strong activation of JNK signalling together with RasV12results in non-cell autonomous overgrowth, although at presumably lower levels of JNK activation, cell autonomous overgrowth occurs. Therefore it is possible that the different effects on non-cell autonomous versus autonomous cell overgrowth in DRac1 ab versus RhoGEF2 abor Src64B ab-expressing cells might depend on the level of JNK activation. Nonetheless, at early stages, ab-driven RhoGEF2, Src64B or DRac1 tumours were similar in inducing non-cell autonomous effects, but at later times the RhoGEF2 ab and Src64B ab-expressing cells showed more predominant autonomous cell overgrowth, whilst the DRac1 ab expressing cells did not, suggesting that there are likely to be molecular differences between DRac1 and RhoGEF2 or Src64B in their cooperative interactions with ab that impact on cell proliferation or survival of the tumour cells (Turkel, 2015).

Profiling of Ab targets and deregulated genes revealed that dac, dan, eya and ct eye-antennal differentiation genes were repressed, along with changes in expression of cell growth/proliferation and survival genes that would be expected to promote tumorigenic growth in cooperation with scrib loss-of-function. scrib ab tumours showed downregulation of Dac, but the antennal cell fate expression domain of Dll was not affected. Similarly, ab expression with either of the cytoskeletal genes resulted in repression of Dac, however Src64B ab tumours additionally repressed Dll, in contrast to DRac1 ab, RhoGEF2 ab and scrib ab tumours where Dll was unaffected. This data suggests that Src64B expression exerts an additional effect on ab-expressing cells to inhibit Dll gene expression and differentiation. Srcupregulation activates the JNK and Stat signalling pathways, affects adherens junction function and represses Hippo signalling. Furthermore, recent studies have shown that overexpression of Src64B in the Drosophila intestinal stem cells can alter differentiation and result in amplification of progenitor cell pools. scrib mutant cells also upregulate JNK, downregulate the E-cadherin/β-catenin adhesion complex and repress Hippo signalling. Furthermore, the Jak/Stat ligand, Upd3, is also upregulated in the scrib cells, where it drives tumour overgrowth, and is also required to activate Jak/Stat signalling in the wild-type neighbouring cells in cell competition. RhoGEF2 and DRac1 also upregulate JNK signalling, and might also repress Hippo signalling to promote tissue growth, since regulators of actin cytoskeletal tension, such as activated Rok and Myosin II regulatory light chain, induce Yki target gene expression. However, in Drosophila it is unknown if RhoGEF2 or DRac1 affect Jak/Stat signalling. Since scrib loss-of-function and Src activation deregulate similar pathways, the precise mechanism by which Src64B cooperates with abto block expression of Dll in the developing eye-antennal disc remains to be determined (Turkel, 2015).

The finding that there was a significant correlation between increased expression of human BTB-ZF oncogenic genes, Bcl6 or ZBTB7A, and downregulation of the cell polarity genes, Dlg2 and Llgl1, or homologs of JNKK(MAPK2K4, MAPK2K7), JNK (MAPK8, MAPK9, MAPK10), RhoGEF2 (ArhGEF11,ArhGEF12) or Src (Yes1, Src) cytoskeletal genes in various epithelial cancers, suggests that the concordant expression of these genes might be contributing to human epithelial cancer initiation and progression. Whilst this study only focused on two of the 47 BTB-ZF genes in the human genome, it raises the question of whether other BTB-ZF genes might also show correlations with the expression of cytoskeletal or cell polarity genes in human epithelial cancers. However, tissue and cancer-grade specific effects might be observed, as a recently published study revealed that ZBTB7A was commonly deleted in late stage oesophageal, bladder, colorectal, lung, ovarian and uterine cancers. Moreover, it was found that low ZBTB7A expression correlates with poor prognosis in colon cancer patients, suggesting that ZBTB7A plays a tumour suppressor function in these cancers. Interestingly, this study also found that in colon cancer xenografts, ZBTB7A represses the expression of genes in the glycolytic pathway, a metabolic pathway that is required for aggressive tumour growth, and that inhibition of this pathway reduces tumour growth. Pertinent to this finding, it was found that blocking glycolytic pathways in Drosophila polarity-impaired tumours, impedes tumour growth without substantially affecting normal tissues, suggesting that downregulation of the Scribble polarity module might upregulate glycolytic metabolic pathways and be dependent on them for tumour growth and survival. It is therefore possible that the cooperation between ab and scrib or cytoskeletal genes in Drosophila may also reflect a need for upregulation of the glycolytic pathway. In human epithelial cancers, the correlations observed between elevated ZBTB7A expression and reduced expression of the Scribble polarity module gene (or high expression of cytoskeletal genes) might also indicate a requirement for glycolytic pathway activation for tumorigenesis. Further studies are clearly required to examine the cooperative effects of Bcl6 or ZBTB7A with deregulated cytoskeletal or cell polarity genes in human epithelial cell lines and mouse models in order to discern whether the findings in Drosophila are indeed conserved in mammalian systems (Turkel, 2015).

Identifying cooperative interactions in cancer is likely to provide novel therapeutic approaches in combating the tumour. Indeed, recently a small molecule inhibitor targeting Bcl6 has been developed, and combining this with a Stat3 inhibitor resulted in enhanced cell killing in triple negative breast cancer cell lines. Since in Drosophila and human cells, Src upregulates Stat activity, tumours showing high Bcl6 and Src or Yes1 expression would be predicted to be sensitive to this combined therapeutic regime. Interestingly, a predominance of the significant correlations that were observed in the human epithelial cancer datasets with either Bcl6 or ZBTB7A involved upregulation of JNKK and JNK family genes. Since JNK signalling is central to many cooperative interactions, inhibiting the JNK pathway in addition to Bcl6 in Bcl6-driven cancers might also be a promising therapeutic approach to combat these cancers. In summary, these functional studies in Drosophila and bioinformatics analysis of human cancers has shown that cooperative tumorigenic interactions occur between BTB-ZF genes and cell polarity or cytoskeletal genes, and warrants further investigation to determine whether restoring normal expression of these genes or downstream pathways in human cancer cells can reduce tumorigenesis (Turkel, 2015).

Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling

Cancer cells demand excessive nutrients to support their proliferation but how cancer cells sense and promote growth in the nutrient favorable conditions remain incompletely understood. Epidemiological studies have indicated that obesity is a risk factor for various types of cancers. Feeding Drosophila a high dietary sugar was previously demonstrated to not only direct metabolic defects including obesity and organismal insulin resistance, but also transform Ras/Src-activated cells into aggressive tumors. This study demonstrates that Ras/Src-activated cells are sensitive to perturbations in the Hippo signaling pathway. Evidence that nutritional cues activate Salt-inducible kinase, leading to Hippo pathway downregulation in Ras/Src-activated cells. The result is Yorkie-dependent increase in Wingless signaling, a key mediator that promotes diet-enhanced Ras/Src-tumorigenesis in an otherwise insulin-resistant environment. Through this mechanism, Ras/Src-activated cells are positioned to efficiently respond to nutritional signals and ensure tumor growth upon nutrient rich condition including obesity (Hirabayashi, 2015).

The prevalence of obesity is increasing globally. Obesity impacts whole-body homeostasis and is a risk factor for severe health complications including type 2 diabetes and cardiovascular disease. Accumulating epidemiological evidence indicates that obesity also leads to elevated risk of developing several types of cancers. However, the mechanisms that link obesity and cancer remain incompletely understood. Using Drosophila, a whole-animal model system has been developed to study the link between diet-induced obesity and cancer: this model has provided a potential explanation for how obese and insulin resistant animals are at increased risk for tumor progression (Hirabayashi, 2015).

Drosophila fed a diet containing high levels of sucrose (high dietary sucrose or ‘HDS') developed sugar-dependent metabolic defects including accumulation of fat (obesity), organismal insulin resistance, hyperglycemia, hyperinsulinemia, heart defects and liver (fat body) dysfunctions. Inducing activation of oncogenic Ras and Src together in the Drosophila eye epithelia led to development of small benign tumors within the eye epithelia. Feeding animals HDS transformed Ras/Src-activated cells from benign tumor growths to aggressive tumor overgrowth with tumors spread into other regions of the body (Hirabayashi, 2013). While most tissues of animals fed HDS displayed insulin resistance, Ras/Src-activated tumors retained insulin pathway sensitivity and exhibited an increased ability to import glucose. This is reflected by increased expression of the Insulin Receptor (InR), which was activated through an increase in canonical Wingless (Wg)/dWnt signaling that resulted in evasion of diet-mediated insulin resistance in Ras/Src-activated cells. Conversely, expressing a constitutively active isoform of the Insulin Receptor in Ras/Src-activated cells (InR/Ras/Src) was sufficient to elevate Wg signaling, promoting tumor overgrowth in animals fed a control diet. These results revealed a circuit with a feed-forward mechanism that directs elevated Wg signaling and InR expression specifically in Ras/Src-activated cells. Through this circuit, mitogenic effects of insulin are not only preserved but are enhanced in Ras/Src-activated cells in the presence of organismal insulin resistance (Hirabayashi, 2015).

These studies provide an outline for a new mechanism by which tumors evade insulin resistance, but several questions remain: (1) how Ras/Src-activated cells sense the organism's increased insulin levels, (2) how nutrient availability is converted into growth signals, and (3) the trigger for increased Wg protein levels, a key mediator that promotes evasion of insulin resistance and enhanced Ras/Src-tumorigenesis consequent to HDS. This study identifies the Hippo pathway effector Yorkie (Yki) as a primary source of increased Wg expression in diet-enhanced Ras/Src-tumors. Ras/Src-activated cells are sensitized to Hippo signaling, and even a mild perturbation in upstream Hippo pathway is sufficient to dominantly promote Ras/Src-tumor growth. Functional evidence is provided that increased insulin signaling promotes Salt-inducible kinases (SIKs) activity in Ras/Src-activated cells, revealing a SIKs-Yki-Wg axis as a key mediator of diet-enhanced Ras/Src-tumorigenesis. Through this pathway, Hippo-sensitized Ras/Src-activated cells are positioned to efficiently respond to insulin signals and promote tumor overgrowth. These mechanisms act as a feed-forward cassette that promotes tumor progression in dietary rich conditions, evading an otherwise insulin resistant state (Hirabayashi, 2015).

Previously work has demonstrated that Ras/Src-activated cells preserve mitogenic effects of insulin under the systemic insulin resistance induced by HDS-feeding of Drosophila (Hirabayashi, 2013). Evasion of insulin resistance in Ras/Src-activated cells is a consequence of a Wg-dependent increase in InR gene expression (Hirabayashi, 2013). This study identified the Hippo pathway effector Yki as a primary source of the Wnt ortholog Wg in diet-enhanced Ras/Src-tumors. Mechanistically, functional evidence is provided that activation of SIKs promotes Yki-dependent Wg-activation and reveal a SIK-Yki-Wg-InR axis as a key feed-forward signaling pathway that underlies evasion of insulin resistance and promotion of tumor growth in diet-enhanced Ras/Src-tumors (Hirabayashi, 2015).

In animals fed a control diet, at most a mild increase was observed in Yki reporter activity within ras1G12V;csk-/- cells. A previous report indicates that activation of oncogenic Ras (ras1G12V) led to slight activation of Yki in eye tissue. Activation of Src through over-expression of the Drosophila Src ortholog Src64B has been shown to induce autonomous and non-autonomous activation of Yki. In contrast, inducing activation of Src through loss of csk (csk-/-) failed to elevate diap1 expression. The results indicate that activation of Yki is an emergent property of activating Ras plus Src (ras1G12V;csk-/-). However, this level of Yki-activation was not sufficient to promote stable tumor growth of Ras/Src-activated cells in the context of a control diet: Ras/Src-activated cells were progressively eliminated from the eye tissue (Hirabayashi, 2013). It was, however, sufficient to sensitize Ras/Src-activated cells to upstream Hippo pathway signals: loss of a genetic copy of ex-which was not sufficient to promote growth by itself-dominantly promoted tumor growth of Ras/Src-activated cells even in animals fed a control diet. These data provide compelling evidence that Ras/Src-transformed cells are sensitive to upstream Hippo signals (Hirabayashi, 2015).

SIK was recently demonstrated to phosphorylate Sav at Serine-413, resulting in dissociation of the Hippo complex and activation of Yki (Wehr, 2013). SIKs are required for diet-enhanced Ras/Src-tumor growth in HDS. Conversely, expression of a constitutively activated isoform of SIK was sufficient to promote Ras/Src-tumor overgrowth even in a control diet. Mammalian SIKs are regulated by glucose and by insulin signaling. However, a recent report indicated that glucagon but not insulin regulates SIK2 activity in the liver. The current data demonstrate that increased insulin signaling is sufficient to promote SIK activity through Akt in Ras/Src-activated cells. It is concluded that SIKs couple nutrient (insulin) availability to Yki-mediated evasion of insulin resistance and tumor growth, ensuring Ras/Src-tumor growth under nutrient favorable conditions (Hirabayashi, 2015).

The results place SIKs as key sensors of nutrient and energy availability in Ras/Src-tumors through increased insulin signaling and, hence, increased glucose availability. SIK activity promotes Ras/Src-activated cells to efficiently respond to upstream Hippo signals, ensuring tumor overgrowth in organisms that are otherwise insulin resistant. One interesting question is whether this mechanism is relevant beyond the context of an obesity-cancer connection: both Ras and Src have pleiotropic effects on developmental processes including survival, proliferation, morphogenesis, differentiation, and invasion, and these mechanisms may facilitate these processes under nutrient favorable conditions. From a treatment perspective the current data highlight SIKs as potential therapeutic targets. Limiting SIK activity through compounds such as HG-9-91-01 may break the connection between oncogenes and diet, targeting key aspects of tumor progression that are enhanced in obese individuals (Hirabayashi, 2015).

Cortactin modulates cell migration and ring canal morphogenesis downstream of Src and PVR during Drosophila oogenesis

Cortactin is a Src substrate that interacts with F-actin and can stimulate actin polymerization by direct interaction with the Arp2/3 complex. Complete loss-of-function mutants of the single Drosophila Cortactin gene have been isolated. Mutants are viable and fertile, showing that Cortactin is not an essential gene. However, Cortactin mutants show distinct defects during oogenesis. During oogenesis, Cortactin protein is enriched at the F-actin rich ring canals in the germ line, and in migrating border cells. In Cortactin mutants, the ring canals are smaller than normal. A similar phenotype has been observed in Src64 mutants and in mutants for genes encoding Arp2/3 complex components, supporting that these protein products act together to control specific processes in vivo. Cortactin mutants also show impaired border cell migration. This invasive cell migration is guided by Drosophila EGFR and PDGF/VEGF receptor (PVR). Accumulation of Cortactin protein is positively regulated by PVR. Also, overexpression of Cortactin can by itself induce F-actin accumulation and ectopic filopodia formation in epithelial cells. Evidence is presented that Cortactin is one of the factors acting downstream of PVR and Src to stimulate F-actin accumulation. Cortactin is a minor contributor in this regulation, consistent with the Cortactin gene not being essential for development (Somogyi, 2004).

A number of specific defects were observed in cortactin mutants. One cortactin phenotype is a mild defect in 'dumping', transfer of bulk cytoplasmic material from nurse cells to the oocyte. This phenotype is similar to that observed in Src64 mutants, but is also seen in other mutants. In the case of Src64, the defect is correlated to the presence of Src64 protein at the ring canals and to reduced size of the ring canals. Cortactin is also present at ring canals. To determine whether cortactin affects ring canal morphogenesis, their size was quantified at stage 10. Ring canal size is significantly reduced in the cortactin mutant relative to wild type. Dumping defects usually result in smaller eggs. Eggs from cortactin mutant mothers and from Src64 mutant mothers are on average smaller than wild type. The hatching rate of the eggs from mutant mothers is also decreased. Thus, mutations in cortactin affect the actin rich ring canals, and processes dependent on function of ring canals, in a manner similar to mutations in Src64. Mutations in components of the Arp2/3 complex (Arpc1 or Arp3 mutants) are also known to specifically affect ring canal morphogenesis (Somogyi, 2004).

There were several reasons to suspect that the effect of Cortactin on the actin cytoskeleton could be related to effects of RTKs. Border cell migration is guided by two RTKs, namely PDGF/VEGF receptor (PVR) and EGFR. Also, an activated form of PVR induces robust formation of actin-rich extensions in follicle cells in a Rac dependent manner. Finally, mammalian Cortactin has been suggested to act as a link between RTKs such as PDGF receptor and the actin cytoskeleton. Therefore the effect of PVR signaling on Cortactin protein was examined in the follicular epithelium. Overexpression of wild type PVR is sufficient to increase signaling in follicle cells slightly, resulting in a small increase in F-actin accumulation in the cell. This is most visible at the basal F-actin network. PVR overexpression also results in clear recruitment and/or stabilization of Cortactin at the cell cortex. Cortactin protein is not simply recruited by the increased amount of F-actin; the subcellular localization of Cortactin is distinct from that of F-actin. In addition, the level and the localization of other actin-associated proteins such as moesin and alpha-spectrin are not visibly affected by PVR overexpression. Expression of a constitutive active form of PVR (lambda-PVR) results in more robust F-actin accumulation but also disruption of the normal cell shape. The activated receptor is not restricted to the cell cortex but is present in vesicles throughout the cell. The constitutive active PVR also induces accumulation of Cortactin throughout the cell. Thus, PVR activation in follicle cells affects the accumulation and subcellular localization of Cortactin protein, primarily resulting in more Cortactin at the cell cortex of normal epithelial cells (Somogyi, 2004).

The ability of Cortactin to induce F-actin accumulation and filopodia formation in conjunction with the effect of PVR on Cortactin protein suggested that Cortactin might act downstream of PVR with respect to control of the actin cytoskeleton. To determine if this might be the case, an epistasis experiment was performred. The effect of activated PVR (lambda-PVR) on F-actin accumulation and cell shape in follicle cells was scored using three categories of severity. Quantification was done by blindly scoring severity of the phenotype in many egg chambers. In each experiment follicle cells that were mutant for cortactin were compared to a control, wild type background. A small, but statistically significant decrease was seen in the severity of the lambda-PVR induced phenotypes in the cortactin mutant background. This result is consistent with Cortactin acting downstream of PVR, but also shows that the effect of PVR on the actin cytoskeleton does not strictly require Cortactin. Another factor that appears to act downstream of PVR is the Rac activator Mbc (related to DOCK180 and Ced-5). In the same assay, removal of mbc has a much more pronounced effect. Activation of Rac can cause translocation of Cortactin to the cell periphery in mammalian cells. Thus, cortical Cortactin accumulation could be one of the downstream effects of Rac activation by PVR. In conclusion, Cortactin appears to contribute to the effects of PVR on actin, but is largely redundant with other factors. A contributing, but not essential, function is also consistent with cortactin not being an essential gene (Somogyi, 2004).

Expression of an activated form of Src (Src42CA) in border cells and other follicle cells shows a phenotype similar to that of activated PVR. It completely blocks border cell migration and disrupts cell shape and the actin cytoskeleton of follicle cells. Src64 has a specific function in the female germ line, which coincides with a function of Cortactin as discussed above. Src42 appears to function more generally in somatic cells and is required for viability. Unfortunately, it is technically not feasible to make Src42 mutant clones to determine whether Src42 function is required in border cells. However, whether Cortactin might act downstream of activated Src42 could be examined by performing an epistasis experiment equivalent to that with activated PVR. Removal of cortactin results in a small, but significant, decrease in the severity of the activated Src-induced phenotype. This is consistent with a role of Cortactin downstream of Src. It also shows that Src can affect the cytoskeleton independently of Cortactin (Somogyi, 2004).

It is perhaps surprising that Cortactin in not an essential gene. Many modulators of essential processes in the cell such as dynamics of the cytoskeleton, cell adhesion or cell signaling are very well conserved in higher eukaryotes. They may add to the robustness and fidelity of the regulation, but they may only be essential to the organism if their absence completely changes the behavior or fate of specific, important cells. In mammals, there are often multiple closely related genes and simple redundancy between these gene products may explain an absence of phenotypes in knockout mice. In Drosophila, this type of simple redundancy is less frequent. For example, there is no evidence for another Cortactin gene in the sequenced Drosophila genome. However, more distantly related genes may have overlapping functions. Subtle phenotypes may also reflect that one process can be regulated in multiple ways. Combining multiple mutations can then be used to genetically help define which genes and pathways overlap in function (Somogyi, 2004).

src64 and tec29 are required for microfilament contraction during Drosophila cellularization

Formation of the Drosophila cellular blastoderm involves both membrane invagination and cytoskeletal regulation. Mutations in src64 and tec29 (Btk29A - FlyBase) reveal a novel role for these genes in controlling contraction of the actin-myosin microfilament ring during this process. Although membrane invagination still proceeds in mutant embryos, its depth is not uniform, and basal closure of the cells does not occur during late cellularization. Double-mutant analysis between scraps (a mutation in anillin that eliminates microfilament rings) and bottleneck suggests that microfilaments can still contract even though they are not organized into rings. However, the failure of rings to contract in the src64 bottleneck double mutant suggests that src64 is required for microfilament ring contraction even in the absence of Bottleneck protein. These results suggest that src64-dependent microfilament ring contraction is resisted by Bottleneck to create tension and coordinate membrane invagination during early cellularization. The absence of Bottleneck during late cellularization allows src64-dependent microfilament ring constriction to drive basal closure (Thomas, 2004).

tec29, a non-receptor tyrosine kinase, is, like src64, required for the morphogenesis of ovarian ring canals and interacts with Src64 protein to control ovarian ring canal growth. Cellularizing embryos derived from tec29k00206 germline clones have large and non-rounded microfilament rings like those of src64 embryos. tec29 microfilament rings have a circularity index of 0.82, similar to that of src64 but very different from that of wild-type microfilament rings. Tec29 protein is expressed at the cellularization front and more weakly along the lateral cellular membrane in both wild-type embryos and src64Delta17 embryos, suggesting that src64 does not act to localize Tec29 protein during cellularization. This is in contrast to the situation in the ovary where Src64 protein acts to localize Tec29 protein to the ring canal. Phosphotyrosine staining is observed at the cellularization front in both wild-type and src64 mutant embryos. This suggests that Src64 is not the major source of phosphotyrosine during cellularization as it is in the egg chamber. Thus both src64 and tec29 are required for microfilament ring contraction during cellularization, but tec29 is localized to the cellularization front independent of src64 activity (Thomas, 2004).

To determine whether other actin-binding proteins affect microfilament ring contraction in a manner similar to src64, the actin-binding protein anillin, which is expressed at the cellularization front in a domain similar that of Src64, was examined. During later development the anillin protein also localizes to contractile rings during cytokinesis. Anillin is encoded by the gene scraps, which is defined by a maternal effect lethal mutation. The phenotype of embryos from mothers trans-heterozygous for two strong reduction-of-function mutations of scraps was examined. The most informative phenotype of scraps mutants is the absence of microfilament rings. This can be readily seen by observing the density and continuity of myosin staining around the basal openings in the cellularization front. The furrow canals are collapsed and lack the early bulb-like or the late flask-like morphology of the wild type. Only some furrow canals show strong myosin staining. Myosin is seen in dense rod-like clumps lying between some of the basal cellular openings. This myosin distribution is similar to that of F-actin in the septin mutant peanut during cellularization, suggesting that both anillin and certain septins play a role in the assembly or maintenance of the microfilament rings (Thomas, 2004).

During early cellularization in scraps mutant embryos, the basal cytoplasmic openings are angular and resemble polygons with relatively straight sides. Because the sides of these polygons are somewhat uniform in length, they approximate circles and have a circularity index of 0.89, differing only slightly from wild-type microfilament rings. Unlike in src64 mutants, they are not convoluted or wavy, and indentations are not observed. During late cellularization, the scraps phenotype becomes more severe, significantly differing from wild type. This decrease in circularity reflects an increase in the length of some of the sides of each polygon and a decrease in the length of others so that the basal openings more closely resemble polygons with fewer sides. The sides remain straight and show no waviness that would indicate a lack of tension. Instead, the gradual distortion of the polygons in scraps mutants is consistent with stretching due to microfilament contraction in the absence of an organizing structure. The scraps phenotype is therefore distinct from that of src64 mutants, which appear to lack microfilament tension and maintain similar, but deviant, circularities throughout cellularization. This interpretation implies that microfilament rings are not required for microfilament contraction. The low circularity index of later basal openings in scraps mutants suggests that microfilament ring structure provides a stabilizing framework for microfilament contraction during the late phase of cellularization (Thomas, 2004).

Mutations in bottleneck (bnk) have been used to define more clearly the roles that src64 and scraps play in cellularization. Bnk is a small, highly basic protein that regulates the dynamic restructuring of the actin cytoskeleton so as to control the timing of microfilament ring contraction during late cellularization. It is expressed during early cellularization and its level drops precipitously during the transition to the late phase (Schejter, 1993). During early cellularization, Bnk co-localizes with myosin, but extends further apically in the furrow canal (Thomas, 2004).

The bnk phenotype is distinct to that of src64 or scraps in that embryos homozygous for a bnk deficiency have a hypercontractile phenotype. The microfilament rings are prematurely constricted during early cellularization (Schejter, 1993). The rings squeeze the nuclei into dumbbell shapes during early cellularization, trapping and dragging some of them along with the advancing cellularization front during late cellularization. The microfilament rings of early cellularization and late cellularization bnk embryos have circularity indices of 0.92 and 0.91, respectively, values that do not differ from those of similarly staged wild-type embryos, even though initially they enclose a much smaller area of open cytoplasm (Thomas, 2004).

scraps; bnk double-mutant embryos show a mixture of the phenotypes of both scraps and bnk embryos. Like scraps embryos, scraps; bnk embryos fail to form actinmyosin rings, and instead show dense rod-like aggregates of myosin II lying between some of the non-rounded basal cellular openings. In spite of the absence of contractile rings, scraps; bnk embryos still display the premature hypercontraction phenotype characteristic of bnk embryos. The cytoskeleton surrounding cells in scraps; bnk embryos is more contracted, in terms of both area and circularity, than the cytoskeleton surrounding cells in scraps embryos. Microfilaments are constricted around dumbbell-shaped nuclei that are trapped and dragged out of the periphery of the embryo by the cellularization front in scraps; bnk double-mutant embryos, as is characteristic of bnk embryos. The area enclosed by the microfilaments of scraps; bnk embryos is significantly less than that of scraps embryos, but is still larger than that of bnk embryos. During late cellularization, the circularity index of the basal openings of scraps; bnk embryos is 0.90, similar to that of bnk embryos, but significantly different from the 0.73 value of scraps embryos. This difference suggests that the actin-myosin network can still contract in the absence of microfilament rings, but without the efficiency that is conferred by the organization of the cytoskeleton into rings (Thomas, 2004).

The hypercontraction caused by the absence of Bnk protein, coupled with the loss of structural integrity of the cellularization network caused by the absence of anillin and microfilament rings, leads to the apparent tearing of parts of the cellularization network. Several regions of the cytoskeleton are either stretched thin or broken, leaving large gaps in the cellularization. This suggests that the loss of anillin and microfilament rings results in a fragile cytoskeletal structure that unravels in the absence of Bnk. These double-mutant results suggest that Bnk and anillin both play structural roles in the cellularization front, but that neither are necessary for microfilament contraction itself (Thomas, 2004).

In restructuring the cytoskeleton during cellularization, Bnk controls the timing of microfilament ring contraction so that basal closure does not occur until after the cellularization front has passed the bases of the nuclei. bnk mutants have a prematurely hyperconstricted ring phenotype opposite that of the non-constricted ring phenotype of src64 mutants. src64 bnk double-mutant embryos look like src64 mutant embryos. The src64 bnk embryos have the large, non-constricted microfilament rings that appear to be under no tension. They have a circularity index of 0.85, similar to that of src64 embryos but different from that of bnk embryos. A few double-mutant embryos showed some degree of microfilament ring contraction during late cellularization; it is likely that these embryos are the result of some residual activity of the reduction-of-function src64Delta17 allele. The analysis of src64 bnk double-mutant embryos demonstrates that the premature hypercontraction of bnk requires src64 activity. The interaction of bnk mutation with src64 and scraps reveals the difference between the two genes: src64 is required for microfilament contraction and scraps (anillin) is not. This suggests that bnk regulates cytoskeletal contractility during cellularization by counteracting the src64-mediated contraction of the microfilament rings (Thomas, 2004).

These analyses suggest that src64 and tec29 are required for tension in the cellularization front during early cellularization, and for the constriction of the basal microfilament rings during late cellularization. Src64 and Tec29, which are present at higher levels in the microfilament rings, might activate actin-myosin contraction or be essential for the ability of the actin-myosin network to contract. Despite a general similarity of form, the cellularization microfilament ring and the oocyte-nurse cell complex ring canal differ substantially in structure and dynamics. In the ovary, src64, and presumably tec29, control ring canal expansion by regulating actin polymerization and cross-linking. It is unlikely that myosin can play a role in this process since myosin-driven sliding of actin filaments would lead to contraction rather than expansion. Although myosin is localized to the ring canal, and null mutations in regulatory myosin light chain cause defects in the ring canals, these defects are not severe and do not prevent ring canal assembly or expansion. Thus, despite a similar involvement of src64 and tec29, it is unlikely that microfilament ring constriction and ring canal expansion are mechanistically similar (Thomas, 2004).

Anillin, which localizes to the cellularization front and shows higher concentration in the contractile microfilament rings, is required for proper cellularization. Anillin bundles actin filaments and may stabilize these filaments during actin-myosin contraction. On the basis of these observations, it is concluded that in the absence of anillin, stable contractile microfilament rings do not form; instead the contractile protein myosin is irregularly distributed in aggregates throughout the cellularization front. Strikingly, loss of anillin in bnk embryos does not suppress the severe early contraction defect seen in bnk embryos. In the absence of the structure provided by these rings, the contraction of the microfilaments is uneven, leading to increasing defects in the shape of the basal openings as cellularization progresses. This suggests that anillin is not required for the ability of the microfilaments of the cellularization network to contract, only for their organization into stable rings (Thomas, 2004).

The phenotypes presented in this study support a model in which src64 and bnk oppose each other to control contraction of the early cellularization network. Double-mutant analysis reveals that src64 is epistatic to bnk. Bnk acts only to restrain and partially redirect Src64-mediated ring constriction. The fact that cellularization proceeds in src64 and tec29 mutants suggests that a force other than microfilament ring contraction is sufficient to drive cellularization front invagination. This force may be a result of the insertion of membrane, or may be due to the action of plus-end directed microtubular motors, or some combination of both (Thomas, 2004).

Most models for cellularization invoke a role for myosin contraction during the ring constriction and basal closure that occurs during late stages of the process; a role during early stages is more controversial. The early phenotype of src64 mutants, if the above interpretation is correct, suggests a role for microfilament ring contraction in the early stages as well, acting both to coordinate the invagination of the furrow canals by maintaining tension along the cellularization front and to direct their invagination inward. This force is a product of the interaction of src64-dependent, myosin-mediated contraction of the microfilament rings and resistance to this contraction exerted by Bnk protein, which acts as a linker between the rings. These forces oppose each other at all points along the contractile microfilament ring network, generating a dynamic tension over the entire network, keeping it taut and driving the minimization of its surface area. The addition of these force vectors acting on a cross-section of one ring on a curved surface produces a resultant vector directed both toward the interior of the embryo (the center of the circular cross-section) and toward the center of the microfilament ring. The first component of the resultant force vector is the src64-mediated force that provides direction to the invagination that follows the increase in surface area produced by membrane insertion during early cellularization. The other component of the resultant force vector is in the plane of the microfilament ring, coordinating constriction about the entire circumference of the embryo and driving a small degree of constriction consistent with the decrease in cellularization front surface area during invagination (Thomas, 2004).

As the cellularization front passes the bases of the nuclei and cellularization shifts into its late phase of rapid progression, Bnk expression is shut off and the protein is rapidly degraded and removed from the cellularization network. In the absence of Bnk protein, there is no force resisting microfilament ring contraction, so it no longer contributes to driving cellularization front invagination. The src64-mediated force is now directed along the radii of the rings, leading to their constriction. This constriction pulls the membrane toward the center of the base of the cell, expanding the furrow canals and leading to basal closure. The src64-independent force (membrane addition or microtubular motors) may be the only force now driving the inward invagination of the cellularization front (Thomas, 2004).

In conclusion, these data define the differing roles that src64, tec29 and anillin play in the cytoskeletal dynamics of Drosophila cellularization, and reveal more precisely the role that the cytoskeleton plays in the formation of the cellular blastoderm. These data establish that microfilament ring organization and contraction are crucial to basal closure of the blastoderm cells during cellularization. However, these data also suggest that membrane invagination can proceed, though abnormally and less efficiently, in the absence of microfilament organization or contraction. It will be interesting to determine what the comparative roles and contributions of membrane insertion and microtubular motors are to the progression of the cellularization front (Thomas, 2004).

Src64 and Tec29 are involved in fusome development and karyosome formation during Drosophila oogenesis

Src family tyrosine kinases respond to a variety of signals by regulating the organization of the actin cytoskeleton. During early oogenesis Src64 mutations lead to uneven accumulation of cortical actin, defects in fusome formation, mislocalization of septins, defective transport of Orb protein into the oocyte, and possible defects in cell division. Similar mutant phenotypes suggest that Src64, the Tec29 tyrosine kinase, and the actin crosslinking protein Kelch act together to regulate actin crosslinking, much as they do later during ring canal growth. Condensation of the oocyte chromatin into a compact karyosome is also defective in Src64, Tec29, and kelch mutants and in mutants for spire and chickadee (profilin), genes that regulate actin polymerization. These data, along with changes in G-actin accumulation in the oocyte nucleus, suggest that Src64 is involved in a nuclear actin function during karyosome condensation. These results indicate that Src64 regulates actin dynamics at multiple stages of oogenesis (Djagaeva, 2005).

Drosophila C-terminal Src kinase negatively regulates organ growth and cell proliferation through inhibition of the Src, Jun N-terminal kinase, and STAT pathways

Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. The Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk–/– overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. The data provide in vivo evidence that Src activity requires JNK and STAT function (Read, 2004).

Partial reduction of Src64B, Src42A, or Btk29A activity suppresses the dCsk–/– phenotype, providing functional data to support the view that the imaginal disc overgrowth, defective larval and pupal development, and lethality of dCsk–/– mutants results from inappropriate activation of the Src-Btk signal transduction pathways. Mutations in Btk29A more strongly suppress dCsk phenotypes than either Src42A or Src64B mutations, perhaps reflecting that (1) Src paralogs act redundantly to each other in Drosophila as in mammals and (2) Btk29A has been shown to act downstream of Src family kinases (SFK) in flies and in mammals. In vivo evidence is provided that loss of Csk function hyperactivates Btk to drive cell cycle entry in development, demonstrating that Tec-Btk family kinases are critical to SFK-mediated proliferation. The data raise the possibility that partial reduction of Tec-Btk kinase activity could reduce proliferation in other cellular contexts in which overgrowth is driven by hyperactivated SFKs, such as in colon tumors (Read, 2004).

Tissue culture models show that constitutively activated SFK signal transduction modulates the function of numerous downstream effector molecules and pathways. Using a loss-of-function approach to identify effectors that mediate the dCsk overgrowth phenotypes, some of these pathways were not implicated in dCsk function. For example, SFKs up-regulate the SOS-Ras-ERK pathway in multiple tissue culture studies and Drosophila overexpression models. However, although dRas1 signaling is active throughout retinal development, reduced dEGFR, Sos, and Jra (c-jun) gene dosage failed to affect the dCsk phenotype. dCsk mutations also failed to modify a hypermorphic allele of dEGFR. Levels of doubly phosphorylated and activated ERK appeared unaltered in dCsk–/– tissue. Moreover, the dCsk phenotype failed to phenocopy defects caused by Ras pathway hyperactivation. For example, constitutively active dRas1 causes increased cell size and patterning defects in the developing imaginal discs, defects that were not observed in dCsk mutant eye tissues. These data argue that not every signal transduction pathway implicated in SFK tissue culture models necessarily functions as predicted within a developing epithelial tissue (Read, 2004).

These studies emphasize the importance of two signaling pathways in dCsk and SFK function. Since certain defects in dCsk–/– animals, such as a split notum, resembled those of hep (JNKK) mutants, it is suspected that JNK pathway activity is involved in dCsk function. Phenotypic and FACS analysis established that reduced JNK (bsk) function suppresses the phenotypes and cell cycle defects caused by loss of dCsk. These results confirm studies indicating that JNK functions downstream of the Src-Btk pathway in Drosophila and mammalian tissue culture cells. Components of the JNK pathway are required for Src-dependent cellular transformation, but the exact role of JNK in these cells is unknown. Importantly, the data show that the JNK pathway mediates proliferative responses to Src signaling in vivo. Further work will be needed to precisely understand its role in proliferation (Read, 2004).

Genetic studies also highlight the importance of the Jak/Stat signal transduction pathway. dCsk proves a negative regulator of Jak/Stat signaling; for example, dCsk mutant tissues show up-regulation of Stat92E protein, a hallmark of Jak/Stat activation in Drosophila. Stat92E, the sole Drosophila STAT ortholog, is most similar to mammalian STAT3. In mammalian cells, Src directly phosphorylates and activates STAT3 and STAT3 function and activation are required for Src transforming activity. Conversely, overexpression of Csk blocks STAT3 activation in v-Src transformed fibroblasts. Activating mutations in STAT3 can also promote oncogenesis in mice. However, the physiological significance of these interactions within developing epithelia remains unclear (Read, 2004).

dCsk; Stat92E double mutant clones reveal that blockade of STAT function in dCsk mutants severely reduces Src-dependent overgrowth and promoted apoptosis of mutant tissue. dCsk–/–; Stat92E–/– EGUF adult eyes (the EGUF method produces genetically mosaic flies in which only the eye is exclusively composed of cells homozygous for the mutation) are nearly identical to phenotypes caused by overexpression of Dacapo, the fly ortholog of the cdk inhibitor p21, and PTEN, a negative regulator of cell proliferation and growth. Importantly, removing Stat92E function in dCsk mutant tissue led to a synthetic small eye phenotype and did not simply rescue the dCsk–/– proliferative phenotype. This outcome distinguishes Stat92E from mutations in Src64B, Btk29A, or bsk, which rescue dCsk-mediated defects toward a normal phenotype. The loss of tissue in dCsk–/–; Stat92E–/– clones indicates that Src-Btk signaling provokes apoptosis in the absence of Stat92E function. Consistent with this interpretation, reduced Btk29A function rescued the dCsk–/–; Stat92E–/– EGUF phenotype to a more normal phenotype, demonstrating that the reduced growth and increased apoptosis observed in the dCsk–/–; Stat92E–/– tissues is indeed Src-Btk pathway dependent (Read, 2004).

The data suggest the existence of a Src-dependent proapoptotic pathway that is normally suppressed by STAT. One possible component of this pathway is JNK, given that JNK signaling is an important activator of apoptosis in both flies and mammals. Perhaps Src-dependent hyperactivation of Bsk (JNK) in dCsk–/–; Stat92E–/– tissue contributes to cell death in the absence of proliferative and/or survival signals provided by Stat92E. However, a number of other candidate pathways may also mediate this response. The further characterization and identification of these pathways may have important implications for interceding in Src-mediated oncogenesis (Read, 2004).

Together, these observations indicate that, in tissue that contains hyperactive Src or reduced Csk, blocking STAT function is sufficient to trigger apoptosis and decrease proliferation in the absence of any further mutations or interventions. Reduced STAT3 function can promote apoptosis within breast and prostate cancer cells that show elevated SFK activity, but the molecular pathways driving apoptosis in these cells are unknown. These cells may require survival signals provided by STAT3 to counteract apoptosis due to chromosomal abnormalities or other defects. Alternatively, these cells may die because of proapoptotic signals provided by hyperactive SFKs in the absence of STAT3 function. The data argue that the latter may be true, which suggests the intriguing possibility that therapeutic blockade of STAT function in tumors with activated Src may actively provoke Src-dependent apoptosis and growth arrest in tumor tissues (Read, 2004).

Csk differentially regulates Src64 during distinct morphological events in Drosophila germ cells

Many studies have focused on roles for Src family kinases (SFKs) in regulation of proliferation, differentiation and dynamic changes in cellular morphology. In this report, Src64 is shown to be dispensable for proliferation and differentiation of both germ cells and follicle cells in the Drosophila ovary. Instead, Src64 is required for morphological changes at the ring canal and contributes to the packaging of germline cysts by follicle cells during egg chamber formation. The results demonstrate that Csk regulates Src64 function during packaging, but is dispensable during ring canal growth control. Thus, regulation of Src64 activity levels during these two morphological events is distinct (O'Reilly, 2006).

Actin polymerization is a crucial component of ring canal growth regulation, and mutation of genes that control actin dynamics causes dramatic ring canal defects. Src64Delta17 ring canals are smaller than wild type and exhibit diminished actin polymerization. Recent work has shown that Src64-mediated phosphorylation of the actin-bundling protein Kelch is crucial for regulating actin polymerization during ring canal growth. Whereas the Src64Delta17 ring canal defects are strikingly similar to those observed in germ cells expressing only [Kelch YA], which cannot be tyrosyl phosphorylated by Src64, it was found that Src64KO ring canal growth defects are more severe than those in Src64Delta17 or, by inference, [KelchYA] mutants. This result suggests that Src64 may control additional signals during this process. Cortactin or members of the WASP/SCAR protein family promote actin polymerization through Arp2/3 complex activation and are required for ring canal growth regulation. Both types of protein are known vertebrate SFK substrates, suggesting the possibility that several Src64-dependent routes may drive the actin polymerization required for ring canal growth (O'Reilly, 2006).

Src64 is active on ring canals throughout oogenesis, consistent with known requirements for Src64 kinase activity during ring canal growth. The ring canal-specific pattern of activated Src64 staining contrasts with the localization of Src64 protein to all germ cell membranes, suggesting that Src64 activators are present specifically at ring canals. SFKs can be activated either through SH3-SH2 domain binding to ligand or PTP-mediated dephosphorylation of the C-terminal regulatory tyrosine. Csk opposes PTP action by phosphorylating the SFK C-terminal tyrosine, thus promoting the inactive state. If the primary mechanism that determines Src64 activation at the ring canal is PTP-mediated dephosphorylation, it would be expected that loss of Csk should have dramatic effects on ring canal growth. However, no significant effects were found on ring canal growth in germ cells lacking Csk or that express a version of Src64 that cannot be regulated by Csk (Src64Y547F). The results suggest that a minimum threshold of Src64 activity is required for regulation of ring canal growth and, once this threshold is reached, the Src64-mediated response is saturated. Consistent with this idea, reduction of Csk function can suppress Src64 mutant defects and partially restore Src64 activation under limiting Src64 conditions. Taken together, these results suggest that Src64 is predominantly regulated by SH3-SH2 domain engagement at the ring canal and that Csk plays a minor role in this process (O'Reilly, 2006).

In addition to Src64 ring canal defects, deviation from wild-type Src64 activity levels leads to the formation of egg chambers containing aberrant germ cell numbers surrounded by a normal follicular epithelium. Egg chambers containing incorrect germ cell numbers can arise due to germ cell or follicle cell proliferation defects, failure to properly differentiate the stalk cells that separate adjacent egg chambers, or as a result of defective packaging of germline cysts by follicle cells within the germarium. This work shows that both Src64LOF and Src64GOF mutants exhibit normal proliferation patterns in both follicle cells and germ cells, and that follicle cell polarity and differentiation are unaffected by Src64 mutation. Instead, defects in the initial separation of germline cysts by invading follicle cells are responsible for Src64 mutant packaging defects (O'Reilly, 2006).

Two previously identified genes, egghead (egh) and brainiac (brn) are required in the germline to regulate the migration of follicle cell precursors during packaging. When germ cells lack egh or brn, follicle cell precursors frequently fail to extend projections, leading to the packaging of multiple germline cysts into one compound egg chamber. Mutations in egh or brn also affect follicle cell polarity and later migration events. Similarly, genes such as Delta, toucan or BicD are involved in germline-derived signals that affect follicle cell differentiation or morphogenesis. These results suggest that instructive cues generated by the germ cells direct follicle cell morphogenesis during packaging (O'Reilly, 2006).

Although Src64 is required in the germ cells, Src64 mutant phenotypes are inconsistent with a similar role for Src64 in regulating follicle cell morphogenesis. No defects in follicle cell proliferation, process extension, migration, differentiation or polarity are observed in Src64 mutants. Importantly, Src64 is activated at contact points between germ cells and follicle cells while packaging occurs. This finding implies that contact between follicle cells and germ cells leads to changes in the germ cell surface over which follicle cells migrate, indicating that germ cells actively respond to follicle cell-derived signals. Roles for SFKs in dynamic regulation of endothelial cell surfaces that act as substrata for attachment and migration of leukocytes or metastatic tumor cells have been previously proposed. In endothelial cells lacking SFK activity, leukocyte attachment and migration is defective, and metastatic colon cancer cells fail to penetrate the endothelial barrier. These results demonstrate crucial roles for SFKs in establishing an appropriate substratum for cell migration (O'Reilly, 2006).

It is proposed that Src64 functions in an analogous manner during packaging. In this model, Src64 is activated by contact between follicle cell projections and germ cells. The precise Src64 activity levels are determined by the balance between contact-dependent activators and Csk. Src64-dependent activation of downstream pathways may then establish the germ cell surface as an appropriate substratum for follicle cell attachment and migration. Defects in adhesion or the underlying cytoskeleton resulting from inappropriate Src64 activation levels would lead to defective adhesion by invading follicle cells, resulting in packaging defects (O'Reilly, 2006).

E-cadherin and Arm/ß-catenin are important regulators of adhesion between germ cells within an individual cyst as well as adhesion between germ cell and follicle cell surfaces. Germline mutation of arm or shotgun (shg), which encodes E-cadherin, leads to ring canal attachment defects, failure of germline cysts to flatten across the germarium, packaging defects and oocyte mislocalization. These phenotypes overlap with Src64 mutant defects, suggesting that Src64 might function within germ cells to regulate E-cadherin complexes. Vertebrate SFKs can dynamically alter the adhesive strength of E-cadherin-mediated complexes through catenin phosphorylation, supporting the idea that Src64 may function similarly during oogenesis. Although direct regulation of E-cadherin-mediated adhesion by Src64 is an attractive model, no changes were observed in the levels of E-cadherin or Arm at germ cell or follicle cell membranes in Src64 mutants, shg is dispensable for Src64 activation, and the most prominent phenotype observed in shg or arm mutants is oocyte mislocalization, a phenotype that occurs in less than 1% of Src64 mutant egg chambers. It is possible that Src64 selectively regulates E-cadherin complexes that mediate ring canal attachment and the germ cell-follicle cell interactions that occur during packaging without affecting oocyte localization. Alternatively, Src64 may target a different adhesion complex, the disruption of which indirectly affects E-cadherin-dependent events. Further analysis of the relationships between Src64 and E-cadherin complex members is required to distinguish between these possibilities (O'Reilly, 2006).

The incomplete penetrance of packaging defects in Src64 mutants suggests that follicle cells can package germline cysts properly even when an ideal substratum is lacking, that Src64 plays a modifying role in this process, or that additional unidentified mechanisms function redundantly with Src64-controlled events. Future identification of upstream activators and downstream consequences of Src64 activation will contribute significantly to the understanding of its role in regulating the germ cell surface during packaging (O'Reilly, 2006).

Mutations in the catalytic loop HRD motif alter the activity and function of Drosophila Src64

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. Mutations were generated in a Drosophila src gene, src64, that alter the three HRD amino acids. The mutants were analyzed for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. The phenotypic effects are described of other mutations in the SH2 and tyrosine kinase domains of src64, and these were compared to the phenotypic effects of the src64 null allele (Strong, 2011; full text of article).

Doherty, J., Sheehan, A. E., Bradshaw, R., Fox, A. N., Lu, T. Y. and Freeman, M. R. (2014). PI3K signaling and Stat92E converge to modulate glial responsiveness to axonal injury. PLoS Biol 12: e1001985. PubMed ID: 25369313

PI3K signaling and Stat92E converge to modulate glial responsiveness to axonal injury

Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. This study shows glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. Evidence is provided for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. It is proposed that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury (Doherty, 2014: PubMed).


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Src oncogene at 64B: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 5 October 2020

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