C-terminal Src kinase: Biological Overview | References
Gene name - C-terminal Src kinase
Cytological map position - 86E13-86E13
Function - signaling
Symbol - Csk
FlyBase ID: FBgn0262081
Genetic map position - 3R: 7,459,000..7,476,418 [-]
Classification - Tyrosine kinase catalytic domain, Src homology 2 domain
Cellular location - cytoplasmic
|Recent literature||Spring, A. M., Brusich, D. J. and Frank, C. A. (2016). C-terminal Src kinase gates homeostatic synaptic plasticity and regulates Fasciclin II expression at the Drosophila neuromuscular junction. PLoS Genet 12: e1005886. PubMed ID: 26901416
Forms of homeostatic plasticity stabilize neuronal outputs and promote physiologically favorable synapse function. At the Drosophila neuromuscular junction (NMJ) impairment of postsynaptic glutamate receptor activity is offset by a compensatory increase in presynaptic neurotransmitter release. This study aimed to elucidate how this process operates on a molecular level and is preserved throughout development. A tyrosine kinase-driven signaling system was identified that sustains homeostatic control of NMJ function. C-terminal Src Kinase (Csk) was identified as a potential regulator of synaptic homeostasis through an RNAi- and electrophysiology-based genetic screen. Csk loss-of-function mutations impair the sustained expression of homeostatic plasticity at the NMJ, without drastically altering synapse growth or baseline neurotransmission. Muscle-specific overexpression of Src Family Kinase (SFK) substrates that are negatively regulated by Csk also impaired NMJ homeostasis. Surprisingly, it was found that transgenic Csk-YFP can support homeostatic plasticity at the NMJ when expressed either in the muscle or in the nerve. However, only muscle-expressed Csk-YFP was able to localize to NMJ structures. By immunostaining, it was found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII). By immunoblotting, it was found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals-but markedly increased in Csk mutant animals. Additionally, it was found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis. Based on these data, it is propose that Csk and its SFK substrates impinge upon homeostatic control of NMJ function by regulating downstream expression or localization of FasII.
The construction and maintenance of normal epithelia relies on local signals that guide cells into their proper niches and remove unwanted cells. Failure to execute this process properly may result in aberrant development or diseases, including cancer and associated metastasis. This study shows that local environment influences the behavior of dCsk-deficient cells. Broad loss of dCsk leads to enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion. Loss of dCsk in discrete patches leads to a different outcome: epithelial exclusion, invasive migration, and apoptotic death. These latter phenotypes required sharp differences in dCsk activity between neighbors; dE-cadherin, P120-catenin, Rho1, JNK, and MMP2 mediate this signal. Together, these data demonstrate how the cellular microenvironment plays a central role in determining the outcome of altered dCsk activity, and reveal a role for P120-catenin in a mechanism that protects epithelial integrity by removing abnormal cells (Vidal, 2006).
The mechanisms that regulate organ size and shape are not well understood, but recent studies have pointed to the importance of local interactions between neighboring cells. For example, in the process known as 'cell competition', cells with relatively higher proliferative rates actively eliminate their neighbors by programmed cell death. Conversely, apoptotic cells send proliferative signals to their neighbors to compensate for their loss. In this way, normal tissue size is achieved. The misregulation of such mechanisms may contribute to the development of cancer, since most solid tumors arise from intact epithelia and are resistant to size-control signals. Tumors are particularly dangerous when linked to metastasis, a process in which cells leave the primary tumor and invade distant tissues. These processes are best understood within the context of an intact epithelium, in which the full range of cell interactions is retained. Work in Drosophila has provided an important in situ view of the action of oncogenes within epithelia (Vidal, 2006).
Src family kinases (SFKs) are active in a broad range of cancer types, including tumors of the breast, colon, and hematopoietic systems. SFK activity typically increases as tumorigenesis progresses and is associated with metastatic behavior. The major inhibitor of SFK activity is C-terminal Src kinase (Csk) and its paralog Chk; these may act as tumor suppressors in, e.g., breast cancer, presumably through their ability to inhibit Src activity and perhaps other pathways. Drosophila Csk acts primarily or exclusively through Src pathway regulation (Read, 2004), and the reduction of dCsk activity by itself led to increased organ size, organismal lethality, and increased cell proliferation due to a failure to exit the cell cycle (Read, 2004; Stewart, 2003). However, neither Csk loss nor Src activation has been clearly linked to early events in tumorigenesis (Yeatman, 2004), bringing into question the role of Csk/Src in proliferation in vivo. Instead, Src is currently thought to be a major player in the metastatic events that occur later in oncogenesis. How Csk or Src promotes the metastatic behavior of cells in situ remains largely unknown (Vidal, 2006).
This study analyzed the phenotypes of dCsk in the context of developing epithelia. The outcome of a cell's loss of dCsk is linked to its cellular microenvironment. When dCsk activity is reduced broadly in the developing eye or wing, the result is overproliferation, inhibition of apoptosis, and decreased cell adhesion. Tissue integrity is retained, but dCsk cells become inappropriately mobile and fail to maintain their appropriate contacts. The outcome of these effects is an overgrown and mispatterned adult tissue. By contrast, loss of dCsk in discrete patches results in epithelial exclusion, invasive migration through the basal extracellular matrix, and eventual apoptotic death; these events occur exclusively at the boundary between dCsk and wild-type cells. Further emphasizing the unique nature of cells at this boundary, a specific requirement was found for a signal that includes Drosophila orthologs of E-cadherin, P120-catenin, RhoA, JNK, and the metalloprotease MMP2. Hence, this study explores the mechanisms by which the cellular microenvironment can direct different behaviors of cells, both in the regulation of apoptosis and epithelial integrity. It also uncovers a mechanism for the removal of abnormal cells from a normal epithelium (Vidal, 2006).
This study shows that reducing dCskactivity results in a blockade of apoptosis and downregulation of cellular adhesion. The work is consistent with the view that Csk is a tumor suppressor that acts at multiple steps. Mutations in the locus encoding the Csk paralog Chk have been described in breast tumors (Biscardi, 1998; Masaki, 1999; Zrihan-Licht, 1997), and, in this study, it was observed that human Chk can functionally replace dCsk. Therefore the experimental advantages of developing Drosophila imaginal epithelia were used to explore specific aspects of dCsk function that are relevant to the behavior of tumor cells (Vidal, 2006).
Visualization studies suggest that a reduction in dCsk activity leads to a failure of cells to stably retain associations with their neighbors, resulting in prolonged cell movement as cells slide across each other in a manner not observed in wild-type tissue. This may reflect a failure to establish stable junctions, excess cell motility, or both. Recent work has demonstrated a critical and dynamic role for the cadherin-based apical junctions in patterning the Drosophila retina. Misexpressing dE-cadherin prevents patterning defects in GMR>dCsk-IR retinas, suggesting that dCsk cells have reduced dE-cadherin function. Links between Csk, Src, cadherins, and junctional integrity have been reported in mammalian cell culture, and an association has been observed between Drosophila Src42A and dE-cadherin during embryonic development (Takahashi, 2005). The data are consistent with this view: misexpression of a kinase-dead form of Src42A leads to a disruption in the localization of the dE-cadherin-associated protein Armadillo; also, reduced Armadillo levels observed in dCsk retinas is rescued by dE-cadherin misexpression. Together, these data suggest that altering dCsk/Src activity affects cell movements by decreasing dE-cadherin adhesion (Vidal, 2006).
The mechanism by which dCsk alters dE-cadherin function is not clear, but it is relevant to note that Src activation can shift cadherin-based cell adhesion from a 'strong' to a 'weak' adhesive state in mammalian cultured cells. Phosphorylation of cadherins and catenins may mediate 'inside-out' signaling that can alter the adhesive strength of the homophilic bond between cells (reviewed in Gumbiner, 2005). Evidence for such a mechanism has been provided for integrin-mediated focal adhesions (reviewed in Hynes, 2002), and Src activity can alter focal adhesions (Yeatman, 2004). However, normal basal membrane architecture was observed in dCsk cells, as assessed both by anti-integrin staining and by transmission electron microscopy, indicating that at least the gross structure is not affected (Vidal, 2006).
The ability of dCsk to influence cell proliferation, apoptosis, and cell adhesion is consistent with its ability to direct tissue overgrowth: reducing dCsk activity throughout a tissue (or the entire organism) leads to significantly enlarged tissues. This ability demonstrates that dCsk can participate in the mechanisms that set tissue size. A small number of other proteins have been implicated in this process, including Salvador, Hippo, and Lats/Warts, which show phenotypes that are strikingly similar to dCsk. Furthermore, dCsk can directly phosphorylate Lats/Warts (Stewart, 2003) in vitro (Vidal, 2006).
However, reduction of dCsk activity shows some important differences. Mutations in salvador, hippo, or lats/warts lead to an increase in Diap1 levels, which, in turn, blocks apoptotic cell death. By contrast, reductions in dCsk does not significantly alter Diap1 protein levels. Furthermore, although both Hippo and dCsk are required to exit the cell cycle, the cell cycle profile from hippo mutant cells is normal, while dCsk cells contain a significant shift toward G2/M (Read, 2004; Stewart, 2003). Perhaps the most striking difference is the effects of these factors on discrete mutant patches. While broad loss of dCsk activity leads to expanded tissues, surprisingly discrete patches of dCsk tissue are eliminated by neighboring cells. Unlike salvador, hippo, or lats/warts, clonal patches of dCsk cells fail to survive to adulthood. The effects of dCsk reduction are more similar to those reported for the tumor suppressor gene scribble. The scribble locus encodes a component of the septate junction that regulates cell polarity and proliferation; mutant cells display neoplastic overgrowth in a homotypic environment, but are removed by JNK-dependent apoptosis in discrete clonal patches abutting wild-type tissue (Vidal, 2006).
This work provides evidence that neighboring wild-type tissue provides a locally nonautonomous signal that leads to the removal of dCsk mutant cells. For example, FRT-derived clones of dCsk cells were out-competed by neighbors with normal levels of dCsk: this was most easily seen by the clonally related 'twin spot' of wild-type tissue that was consistently larger than the few surviving dCsk clones. In contrast, FRT-mediated dCsk clones that encompassed the entire eye survived and overproliferated. In the developing wing, cells at the periphery of sd>dCsk-IR or ptc>dCsk-IR expression domains were preferentially removed by apoptosis. This death is dependent not on absolute dCsk activity, but on the juxtaposition of cells that are starkly different in their levels of dCsk. Small differences, for example across the ptc>dCsk-IR or omb>dCsk-IR graded expression domains, did not trigger cell death (Vidal, 2006).
This translocation and death of dCsk-IR cells at the patched/wild-type boundary requires at least two steps. At boundaries with wild-type tissue, dCsk cells initially lose their apical profile, shift downward, and eventually become basally excluded from the epithelium. Such excluded cells then migrate away from the boundaries in both directions and eventually die by apoptosis. These events are strikingly reminiscent of those described for tumor cells undergoing metastasis. Altered activity of both Csk and Src has been implicated in a broad variety of tumors. Typically, however, increased Src activity is associated with later events in tumorigenesis, particularly metastasis (reviewed in Yeatman, 2004). Although the connections between high Src activity and metastases are not understood, they likely include Src's ability to break cell-cell junctions and increase cell motility. Another hallmark of metastatic behavior is the ability to degrade basal extracellular matrix: this study also demonstrate a functional requirement for MMP2 activity during the translocation of mutant cells out of the wing epithelium (Vidal, 2006).
While evidence supports the view that the activity of Csk -- and presumably Src and perhaps other effectors -- can regulate metastatic behavior, it alone is not sufficient. First, reducing dCsk activity by itself is not sufficient to allow migrating cells to survive; the data suggest that most or all eventually die. This is consistent with previous work highlighting the importance of a 'two-hit' model to allow for stable tumor overgrowth and metastasis. A second mutation that prevents apoptotic cell death would be minimally required. Second, all cells within a discrete dCsk patch are not equivalent: cells at the boundary of the clone that border cells of strongly differing dCsk levels are exclusively prone to release from the epithelium. This work predicts that cells at the borders of some human tumors are especially prone toward metastatic behavior. Metastasis is often the most serious aspect of a tumor, and approaches that address the metastatic behavior of cells may need to take into account the properties of cells at the periphery. Understanding whether and how these cells are unique may help to more effectively target therapeutic intervention (Vidal, 2006).
In addition to enabling a detailed examination of dCsk cells and their behavior within an epithelium, this model system permitted identification of signaling components that are necessary to execute the aberrant cell mobility and cell death. The results indicate important roles for dE-cadherin, dP120ctn, Rho1, dJnk, and MMP2 (Vidal, 2006).
JNK-dependent apoptosis is required for a broad palette of related mechanisms such as cell competition in developing tissues and the removal of scribble mutant cells. JNK signaling is also associated with the movement of cells within epithelia, including dorsal closure in Drosophila and in mammals. Interestingly, JNK activity is required for the synthesis of MMP2 by v-Src-transformed mammalian cells (Vidal, 2006).
JNK activity can be triggered by several upstream signaling factors, including the small GTPases of the Rho family, and genetic data provide a link between dCsk, dJnk, and Rho1. Rho family proteins are key regulators of cell shape and motility. They also promote the cytoskeletal rearrangements required for epithelial-to-mesenchymal transitions (EMTs), and it is noted that dCsk boundary cells show a number of features that are reminiscent of EMTs. In Drosophila, Rho1 was found to induce an 'invasive' phenotype in wing disc cells, but, in this study, it was demonstrated that, similar to dCsk boundary cells, ptc>Rho1 misexpressing cells also undergo apoptotic death. Most importantly, halving the genetic dose of Rho1 strongly suppresses discrete loss of dCsk, but does not appreciably affect broad loss. Thus, Rho1 activity is linked to dCsk, and activation of Rho1 is sufficient to phenocopy both the apoptotic and migratory phenotypes of dCsk cells located near wild-type tissue (Vidal, 2006).
Previous work in mammalian cell culture has provided direct links between Src and P120-catenin, between cadherins and P120-catenin, and between RhoA and P120-catenin; the latter two interactions have been reported in Drosophila tissue culture systems as well. This study further supports links between these factors in dCsk boundary cells. Interestingly, although normal levels of both dP120ctn and Rho1 were required for the efficient removal of dCsk boundary cells, they were not required for the phenotypes resulting from broad loss of dCsk. The requirement for p120ctn specifically in boundary cells may explain why, although it is the only ortholog present in Drosophila, dP120ctn (Drosophila p120-catenin) is not required for organism viability (Vidal, 2006).
Both Src and P120-catenins are known to directly interact with cadherins, and, in fact, a role was demonstrated for dE-cadherin/Shotgun in the removal of dCsk cells. A model is postulated in which the loss of dCsk results in the remodeling of the zonula adherens, presumably by the phosphorylation of catenins and dE-cadherin itself by Src. Src activation is known to switch cadherin from a strong adhesive state to a weak one, providing one potential explanation for why dCsk retinal cells displayed reduced cell adhesion in situ. One critical question regarding cadherins is whether they have signaling roles that are independent of their adhesive properties. Perhaps relevant to this point, it was surprising to find that reducing dE-cadherin function leads to a suppression of the effects of dCsk-IR at the boundary. A simple dCsk-IR-mediated reduction in dE-cadherin adhesion would be enhanced by further reducing dE-cadherin activity, suggesting that dE-cadherin may provide an active signal that promotes boundary cells' release from the epithelium. If such a signal does exist, neighboring wild-type cells must trigger it, either through their own endogenous dE-cadherin or through a separate, local signal. Why are multiple (3-4) rows affected? The results are consistent with the creation of a successive new boundary as the previous row of cells descends, although other longer-range signals cannot be ruled out (Vidal, 2006).
It is noted that reducing dCsk activity by itself is not sufficient to direct stable tumor overgrowth, supporting the importance of a 'two-hit' model in Drosophila. Loss of the junction protein Scribble showed similar phenotypes to dCsk, including apoptosis, but was found to confer survival and metastatic-like behavior to cells in the presence of an activated Ras isoform. Interestingly, coexpression of dE-cadherin prevents this metastatic behavior (Vidal, 2006).
Finally, how can dP120ctn and Rho1 promote release of dCsk near wild-type boundaries but not act similarly with other dCsk cells? One source of information is the cadherins themselves: the boundary creates an interface of cadherins that have been exposed to different levels of Csk and, presumably, Src activity. This unusual interface may generate the needed dE-cadherin signal. Importantly, recent work has noted a change in the subcellular localization of P120-catenin and E-cadherin specifically at the border of human tumor tissues. At the time that ptc>dCsk-IR boundary cells lose their apical profiles, this study found that dP120ctn is relocalized to the cytoplasm. These results again emphasize the possibility that cells at tumor boundaries pose a special risk of undergoing epithelial-to-mesenchymal-like transitions and metastatic behavior. Metastasis is often the most serious complication of progressing tumors. Targeting therapies to this aspect of cancer may benefit from considering boundary cells and their potentially distinctive properties (Vidal, 2006).
Src-family kinases (SFKs) control a variety of biological processes, from cell proliferation and differentiation to cytoskeletal rearrangements. Abnormal activation of SFKs has been implicated in a wide variety of cancers and is associated with metastatic behavior (Yeatman, 2004). SFKs are maintained in an inactive state by inhibitory phosphorylation of their C-terminal region by C-terminal Src kinase (Csk). Drosophila Ankyrin-repeat, SH3-domain, and Proline-rich-region containing Protein (dASPP) has been identified as a regulator of Drosophila Csk (dCsk) activity. dASPP is the homolog of the mammalian ASPP proteins, which are known to bind to and stimulate the proapoptotic function of p53. dASPP is shown to be a positive regulator of dCsk. First, dASPP loss-of-function strongly enhances the specific phenotypes of dCsk mutants in wing epithelial cells. Second, dASPP interacts physically with dCsk to potentiate the inhibitory phosphorylation of Drosophila Src (dSrc). These results suggest a role for dASPP in maintaining epithelial integrity through dCsk regulation (Langton, 2007).
The Src protein tyrosine kinase was first identified as the viral oncogene of the Rous-Sarcoma virus, v-src. Src-family kinases (SFKs), which include c-Src, Fyn, and Lck, are implicated in different cellular processes, and abnormal activation of SFKs has been associated with tumor development and with metastatic behavior (Yeatman, 2004). C-terminal Src kinase (Csk) maintains SFKs in an inactive state by an inhibitory phosphorylation (Tyr527 in avian c-Src) (Cole, 2003). This phosphorylation event triggers autoinhibition of the Src kinase domain through binding of Src's SH2 domain to the phospho-Tyrosine. Tyr527 is deleted in the v-src oncogene, underlining the biological significance of Src regulation by Csk. The csk mutant mouse phenotype is partially suppressed by loss of src, suggesting that these proteins do indeed act in concert in vivo (Thomas, 1995). Importantly, the regulation of Csk is not well understood at present (Langton, 2007).
Drosophila has emerged as a useful genetic model system in which many aspects of tumor formation can be studied, including excess proliferation, evasion of apoptosis, and tumor cell invasion and metastasis. Drosophila Csk (dCsk) has been reported to function as a tumor-suppressor gene. dCsk mutants display excess cell proliferation and overgrowth defects primarily due to activation of targets of Drosophila Src kinases (Src64B and Src42A), including c-Jun N-terminal kinase (JNK), Stat, and Btk29A (Bruton's Tyrosine Kinase29A/Tec29A) (Pedraza, 2004, Read, 2004). More recently, it has been shown that local inactivation of dCsk in small patches surrounded by normal cells surprisingly does not cause overgrowth (Vidal, 2006). Instead, these cells move to a basal position in the epithelium and spread among the wild-type cells while simultaneously undergoing apoptosis, which may reflect the function of dSrc in promoting motility and invasion (Langton, 2007).
dASPP as a regulator of dCsk activity. dASPP is the homolog of mammalian ASPP1 and -2 (Ankyrin-repeat, SH3-domain and Proline-rich-region containing Protein). ASPP1 and -2 have been reported to bind to p53 via their ankyrin and SH3 motifs (Iwabuchi, 1994, Samuels-Lev, 2001). Binding of ASPP1 or -2 to p53 is thought to specifically potentiate its transcriptional activity on proapoptotic targets such as the Bcl-family gene Bax, but not on cell cycle targets like p21 (Samuels-Lev, 2001). Both the postnatal lethality of ASPP2-/- mice and the tumor-prone phenotype of ASPP2 heterozygotes are enhanced by loss of p53, suggesting that the p53-ASPP interaction is biologically significant (Vives, 2006). Deregulation of ASPP gene expression has been reported in several different cancers (Trigiante, 2006), underlining the importance of these as tumor suppressor loci (Langton, 2007).
This study generated and characterized dASPP mutants, which are homozygous viable and exhibit an overgrowth phenotype due to an increased number of cells of normal size. Several lines of evidence are presented suggesting that dASPP positively regulates dCsk activity. First, dCsk mutant phenotypes are strongly enhanced by dASPP loss of function. Second, dASPP physically interacts with dCsk. Third, dCsk kinase activity on dSrc is potentiated in the presence of dASPP (Langton, 2007).
In Drosophila dASPP funcions as an important regulator of dSrc by binding to and potentiating the kinase activity of dCsk. dASPP and dCsk mutants show similar phenotypes, namely excess proliferation, increased mass, developmental delay, and an alteration in cell-cell adhesion properties that results in mispatterning of the retina. dCsk has a stronger phenotype than dASPP. For example, dCsk mutants die as enlarged pupae, whereas dASPP mutant flies are viable with a more modest increase in size. This suggests that dASPP is not absolutely required for dCsk function but is necessary for maximal signaling (Langton, 2007).
How does dASPP promote dCsk activity? Mammalian Csk's ability to phosphorylate Src is believed to be primarily determined by its translocation to lipid rafts, where Src is tethered by virtue of its myristylated N terminus (Cole, 2003). The transmembrane protein Cbp (Csk-binding protein or PAG) has been reported to recruit Csk to lipid rafts (Kawabuchi, 2000). It will be interesting to determine whether dASPP can regulate dCsk localization. Alternatively, dASPP could control dCsk activity through a conformational change or by recruiting other proteins to dCsk (Langton, 2007).
Human ASPP proteins have been shown to directly bind to and regulate the apoptotic function of p53 (Samuels-Lev, 2001). Therefore whether dASPP is capable of binding to Drosophila p53 (Dmp53) was examined. No interaction was shown between dASPP and Dmp53 in coimmunoprecipitation experiments. Additionally, it was found that dASPP is not required for radiation-induced cell death, which is mediated via Dmp53 activation. These results suggest that the human p53-activating function of ASPP is not conserved in Drosophila and may have evolved later. Indeed, neither the four human p53 residues shown in crystallography studies to contact ASPP2 (His178, Arg181, Met243 and Asn247) (Gorina, 1996) nor the proline-rich region shown to be a second site for binding between the two proteins (Bergamaschi, 2003) are conserved in Dmp53 (Langton, 2007).
Vidal (2006) has shown that dCsk mutant cells are susceptible to apoptosis only when in contact with wild-type tissue. Perturbation of dASPP/dCsk signaling in discrete areas of the wing disc induces apoptosis of mutant cells. However, the data shows that broad loss of dCsk in wing discs also results in considerable apoptosis. This suggests that epithelial extrusion and cell death may not only occur at mutant/wild-type clone boundaries but is more a general phenotype of dCsk mutant cells. Accordingly, it was found that dCsk1jd8 mutant eyes are often overgrown but occasionally present a small eye phenotype, presumably as a result of massive apoptosis. Since this phenotype is sensitive to the dosage of btk and levels of JNK signaling, it is likely to be a consequence of ectopic activation of dSrc in dCsk mutant discs. Indeed, dSrc overexpression leads to JNK activation and apoptosis (Langton, 2007).
JNK activation in response to loss of apico-basal polarity has been reported to promote invasion and growth in cells expressing oncogenic Ras (Igaki, 2006). Protection of dCsk mutant cells from apoptosis (for example, by oncogenic Ras) might promote their metastatic potential. Such collaboration between multiple oncogenes/tumor suppressors is a necessary step in tumor progression in humans (Langton, 2007).
dCsk likely functions to maintain epithelial polarity by preventing dSrc from dissolving adherens junctions and inducing JNK activation, as suggested by mammalian and Drosophila studies (Vidal, 2006, Yeatman, 2004). Src activation has been reported to promote epithelial-mesenchymal transition, a process whereby epithelial cells lose polarity and become invasive (Avizienyte, 2005). This process is similar to the loss of polarity and cell spreading observed in Drosophila epithelial cells where dASPP/dCsk signaling is disrupted (Vidal, 2006). Interestingly, ASPP2 has been reported to be downregulated in invasive and metastatic breast carcinoma cells (Sgroi, 1999). The current results provide a potential mechanism for ASSP's role in tumor cell invasion (Langton, 2007).
In summary, this study has shown that dASPP and dCsk interact physically and genetically and that this interaction is important for maintenance of cells within the developing wing epithelium. These results provide a link between two previously unrelated tumor suppressors (Langton, 2007).
Adherens junctions (AJs) provide structure to epithelial tissues by connecting adjacent cells through homophilic E-cadherin interactions and are linked to the actin cytoskeleton via the intermediate binding proteins beta-catenin and alpha-catenin. Rather than being static structures, AJs are extensively remodeled during development, allowing the cell rearrangements required for morphogenesis. Several 'noncore' AJ components have been identified that modulate AJs to promote this plasticity but are not absolutely required for cell-cell adhesion. dASPP has been identified as a positive regulator of dCsk (Drosophila C-terminal Src kinase) (Langton, 2007). This study shows that dRASSF8, the Drosophila RASSF8 homolog, binds to dASPP and that this interaction is required for normal dASPP levels. genetic and biochemical data suggest that dRASSF8 acts in concert with dASPP to promote dCsk activity. Both proteins specifically localize to AJs and are mutually required for each other's localization. Furthermore, abnormal E-cadherin localization is observed in mutant pupal retinas, correlating with aberrant cellular arrangements. Loss of dCsk or overexpression of Src elicited similar AJ defects. Because Src is known to regulate AJs in both Drosophila and mammals, it is proposed that dASPP and dRASSF8 fine tune cell-cell adhesion during development by directing dCsk and Src activity. The dASPP-dRASSF8 interaction is conserved in humans, suggesting that mammalian ASPP1/2 and RASSF8, which are candidate tumor-suppressor genes, restrict the activity of the Src proto-oncogene (Langton, 2009).
Cell-cell contacts are essential for development and adult life of multicellular organisms. The best-characterized form of cell-cell contact is the adherens junction (AJ), which links neighboring cells via homotypic E-cadherin (E-Cad) interactions. The highly conserved intracellular domain of E-Cad binds to β-catenin, which itself binds to α-catenin. Transient interactions between α-catenin and actin filaments link AJs to the cytoskeleton, though the exact nature of this connection remains controversial. AJs are particularly important for the integrity of epithelial tissues. In addition to establishing and maintaining cell-cell adhesion, AJs regulate several aspects of cellular behavior, including cytoskeletal rearrangement and transcription. Inappropriate disruption of cell-cell contacts can lead to excess proliferation and is a hallmark of the metastatic process (Langton, 2009).
Dynamic remodeling of AJs occurs during all major morphogenetic events involving movement and rearrangement of epithelial cells, including convergent extension and gastrulation. AJ remodeling is necessary for the generation of epithelial structures with extremely precise patterns, such as the hexagonal array of ommatidia in the Drosophila compound eye (Langton, 2009).
SRC signaling is a major cellular pathway known to promote AJ remodeling in development and metastasis. Cellular SRC (c-SRC) is a member of the SRC family kinases (SFKs), which include c-SRC, FYN, and YES. Activated c-SRC is known to regulate AJs by several mechanisms. For example, c-SRC can induce the ubiquitylation of E-Cad by an E3 ubiquitin ligase called Hakai, promoting E-Cad internalization or degradation. In Drosophila, Src42A (one of two c-Src homologs) genetically interacts with E-Cad (encoded by shotgun [shg] in Drosophila), localizes to AJs, and forms a ternary complex with E-Cad and Armadillo (Drosophila β-catenin). Furthermore, Src42A activation leads to decreased E-Cad protein levels and concurrent stimulation of E-Cad transcription by Armadillo and TCF, which is thought to be important for AJ turnover during morphogenesis (Langton, 2009).
The C-terminal region of c-SRC and other SFKs is targeted by C-terminal SRC kinase (CSK), which negatively regulates c-SRC by phosphorylating a conserved tyrosine residue (Tyr527 in avian c-SRC). Drosophila CSK (dCsk) appears to function analogously to mammalian CSK as a negative regulator of SFKs. dCsk is a negative regulator of tissue growth; mutants die as giant pupae and imaginal discs are enlarged as a result of increased proliferation. These observations are seemingly at odds with studies showing that Src activation in Drosophila tissues stimulates proliferation but also leads to considerable apoptosis. A recent report attempted to reconcile this discrepancy, suggesting that lower levels of Src activation induce proliferation and protection from apoptosis, whereas high levels lead to apoptosis and invasive migration (Langton, 2009 and references therein).
It has been shown that dCsk activity is modulated by dASPP, the Drosophila homolog of mammalian ASPP1 and ASPP2, which physically interacts with dCsk and enhances its capacity to phosphorylate Src42A (Langton, 2007). Accordingly, dASPP phenotypes are enhanced by reducing dCsk gene dosage and are rescued by complete removal of Src64B, which functions redundantly with Src42A. This study identifies dRASSF8 as a new dASPP regulator. dRASSF8 is the homolog of mammalian RASSF7/8 (Ras association domain family 7/8). Ras association (RA) domain-containing proteins are putative Ras effectors; they specifically bind the activated (GTP-bound) form of Ras family GTPases, which function in numerous signal transduction pathways regulating proliferation, apoptosis, and differentiation. Mammalian RASSF family members 1–6 are characterized by their domain structure, with a C-terminal RA domain, a C1-like zinc finger, and a SARAH (Salvador-RASSF-Hippo) domain. Mammalian RASSF7–10 are atypical RASSF proteins because they contain an N-terminal RA domain and lack a C1-like or SARAH domain. Recently, Xenopus RASSF7 was shown to be required for completing mitosis. Human RASSF8 is a putative tumor-suppressor gene; when expressed in lung cancer cells, RASSF8 inhibits anchorage-independent growth. Importantly, the molecular function of RASSF8 has not been elucidated (Langton, 2009).
Two RASSF family proteins are encoded by the Drosophila genome. dRASSF is similar to human RASSF1–6 and has been linked to the Hippo pathway. dRASSF8 is similar to human RASSF7 and RASSF8, having an N-terminal RA domain. Published genome-wide yeast two-hybrid data suggested that dRASSF8 interacts with dASPP, prompting an investigation of the relationship between these proteins. Based on genetic and biochemical data, it is suggested that the dASPP-dRASSF8 complex regulates AJs by directing the activity of dCsk and Src (Langton, 2009).
dRASSF8 is the sole Drosophila homolog of mammalian RASSF7 and RASSF8, which are so-called N-terminal RASSF proteins and the least-studied members of the RASSF family. This study demonstrates that dRASSF8 binds to dASPP in Drosophila cells and that RASSF8 binds to ASPP1 and ASPP2 in human cells, indicating that an evolutionarily conserved relationship between these proteins has been uncovered. The function of RASSF8 is currently unknown, and this study thus provides new insights into the function of N-terminal RASSF proteins (Langton, 2009).
Future experiments will determine whether RASSF7 also binds ASPP family proteins or whether this function is specific to RASSF8. RASSF7 has been studied in Xenopus and was found to associate with centrosomes and to be required for completing mitosis. In contrast, the current data suggest that dRASSF8 is not required for cell-cycle progression because null mutants for dRASSF8 are viable. These findings are suggestive of divergent functions for RASSF7 and RASSF8 in vertebrates, with dRASSF8 being functionally analogous to RASSF8 rather than RASSF7. Indeed, GFP-tagged RASSF7 localizes to the nucleus and centrosomes in Xenopus embryos, whereas this study never observed nuclear localization of dRASSF8. Further studies of N-terminal RASSF proteins in vertebrates should clarify whether RASSF7 and RASSF8 have overlapping or independent functions (Langton, 2009).
In vivo data point at a close relationship between dRASSF8 and dASPP, which colocalize and are required for each other's presence at AJs in epithelial cells. dRASSF8 posttranscriptionally regulates the levels of dASPP protein in epithelia. Thus, it seems likely that binding to dRASSF8 stabilizes dASPP and prevents its degradation, which can be observed for many protein complexes. Overall, these data provide compelling evidence for a functional link between dRASSF8 and dASPP, which is likely to be conserved through to their closest mammalian counterparts, RASSF8 and ASPP1/2 (Langton, 2009).
The data suggest that dRASSF8 has some dASPP-independent roles. For example, dRASSF8 mutant wings are large and broadened, whereas dASPP mutant wings are large but of normal shape. In addition, the dRASSF8 adult eye phenotype is more marked than that of dASPP mutants. Accordingly, it was found that dRASSF8, but not dASPP, is required for apoptosis of excess IOCs in the developing pupal retina. It therefore appears that the dRASSF8 eye phenotype results from both reduced apoptosis of IOCs and cell-cell adhesion defects. The subtle differences between the dASPP and dRASSF8 phenotypes indicate unknown functions for dRASSF8, which are not due to its effects on dASPP. Future efforts will be aimed at elucidating these functions (Langton, 2009).
These data are consistent with a model in which dRASSF8 binds to and positively regulates dASPP and, in this way, promotes dCsk activity indirectly. Coimmunoprecipitation experiments support this idea, showing that dRASSF8 and dASPP associate and that dASPP and dCsk associate. However, no detect interaction was detected between dRASSF8 and dCsk, indicating that dRASSF8 does not directly associate with dCsk. The proposed model is also supported by genetic data; the dRASSF8-dCsk genetic interaction is weaker than the dASPP-dCsk interaction, suggesting that dASPP is the primary regulator of dCsk. The weaker genetic relationship between dRASSF8 and dCsk can be explained by the observation that some dASPP protein persists in dRASSF8 mutant tissue. These observations suggest that dRASSF8 regulates dCsk via dASPP (Langton, 2009).
Retinal morphogenesis involves dynamic changes in cell-cell contacts to create the final ordered array of photoreceptors and accessory cells. dASPP and dRASSF8 are required for normal E-Cad localization in 26-27 hr APF retinas, providing an explanation for the patterning defects in mutant eyes. It is proposed that the abnormal E-Cad localization in dASPP mutant eyes results from increased Src activity based on several lines of evidence. dASPP binds to and positively regulates dCsk, leading to Src inhibition; therefore, loss of dASPP increases Src activity, which is known to reduce cell-cell adhesion by promoting the internalization and degradation of E-Cad. In agreement with this, it was shown that loss of dCsk or overexpression of either Drosophila Src leads to loss of AJ material in 26-27 hr APF retinas. This claim is further supported by the fact that the dASPP eye phenotype is suppressed by loss of Src64B. Thus, the presence of the dASPP-dRASSF8 complex at AJs may be required to locally prevent inappropriate Src activation and dissolution of AJs (Langton, 2009).
The fact that dASPP and dRASSF8 mutants are homozygous viable implies that these genes are dispensable for the majority of morphogenetic processes occurring during development. Therefore, the regulation of AJs by dASPP and dRASSF8 may be restricted to the eye. However, as they are expressed in other epithelial tissues, a closer examination of dASPP and dRASSF8 mutants may reveal subtle defects in other morphogenetic processes (Langton, 2009).
It is suggested that dASPP and dRASSF8 are new noncore AJ components and part of the machinery that ensures the fine regulation of AJs by Src during development. This regulation is crucial to provide precisely the right amount of junctional plasticity to allow cell-cell rearrangements and patterning to take place while limiting this plasticity to maintain epithelial coherence and prevent cell delamination. Because the interaction between these proteins is conserved in mammals, this finding is likely to be relevant to mammalian development and to the metastatic process, which is associated with downregulation of E-Cad and loss of cell-cell adhesions. Indeed, ASPP1 knockout mice present defects in the assembly of lymphatic vessels consistent with a potential adhesion defect. This suggests that regulation of cell-cell adhesion may underlie the function of ASPP1/2 and RASSF8 as mammalian tumor suppressors (Langton, 2009).
The Hippo signaling pathway is involved in regulating tissue size by inhibiting cell proliferation and promoting apoptosis. Aberrant Hippo pathway function is often detected in human cancers and correlates with poor prognosis. The Drosophila C-terminal Src kinase (d-Csk) is a genetic modifier of warts (wts)
Disrupting mechanisms that control cell proliferation, cell size and apoptosis can cause changes in animal and tissue size and contribute to diseases such as cancer. The LATS family of serine/threonine kinases control tissue size by regulating cell proliferation and function as tumor suppressor genes in both Drosophila and mammals. In order to understand the role of lats in size regulation, a genetic modifier screen was performed in Drosophila to identify components of the lats signaling pathway. Mutations in the Drosophila homolog of C-terminal Src kinase (dcsk) were identified as dominant modifiers of both lats gain-of-function and loss-of-function phenotypes. Homozygous dcsk mutants have enlarged tissue phenotypes similar to lats and FACS. An immunohistochemistry analysis of these tissues revealed that dcsk also regulates cell proliferation during development. Animals having mutations in both dcsk and lats display cell overproliferation phenotypes more severe than either mutant alone, demonstrating these genes function together in vivo to regulate cell numbers. Furthermore, homozygous dcsk phenotypes can be partially suppressed by overexpression of lats, indicating that lats is a downstream mediator of dcsk function in vivo. It was shown that dCSK phosphorylates LATS in vitro at a conserved C-terminal tyrosine residue, which is critical for normal LATS function in vivo. Taken together, these results demonstrate a role for dCSK in regulating cell numbers during development by inhibiting cell proliferation and suggest that lats is one of the mediators of the dcsk phenotype (Stewart, 2003).
Elevated Src protein levels and activity are associated with the development and progression of a variety of cancers. The consequences of deregulated Src activity have been studied extensively in cell culture; however, the effects of this deregulation in vivo, as well as the mechanisms of Src-induced tumorigenesis, remain poorly understood. In this study, the effect of expressing wild-type and constitutively active Drosophila Src-family kinases (SFKs) in the developing eye was examined. Overexpression of either wild-type Drosophila SFK (Src64 and Src42) is sufficient to induce ectopic proliferation in G1/G0-arrested, uncommitted cells in eye imaginal discs. In addition, both kinases trigger apoptosis in vivo, in a dosage-dependent manner. Constitutively active mutants are hypermorphic; they trigger proliferation and death more potently than their wild-type counterparts. Moreover, SFK-induced proliferation and apoptosis are largely independent events, since blocking ectopic proliferation does not block cell death. Further, Csk (the Drosophila C-terminal Src kinase) phosphorylates and interacts genetically with the wild-type SFKs, but not with the constitutively active mutants in which a conserved C-terminal tyrosine was mutated to phenylalanine, providing the first in vivo evidence that Csk regulates SFKs during development through phosphorylation of their C-terminal tyrosine (Pedraza, 2004).
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).
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).
Search PubMed for articles about Drosophila Csk
Bergamaschi, D., Samuels, Y., O'Neil, N.J., Trigiante, G., Crook, T., Hsieh, J.-K., O'Connor, D.J., Zhong, S., Campargue, I. and Tomlinson, M.L. (2003). iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human. Nat. Genet. 33: 162-167. PubMed ID: 12524540
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Kwon, H. J., Waghmare, I., Verghese, S., Singh, A., Singh, A. and Kango-Singh, M. (2014). Drosophila C-terminal Src kinase regulates growth via the Hippo signaling pathway. Dev Biol 397(1): 67-76. PubMed ID: 25446534
Langton, P. F., Colombani, J., Aerne, B. L. and Tapon, N. (2007). Drosophila ASPP regulates C-terminal Src kinase activity. Dev. Cell 13: 773-782. PubMed ID: 18061561
Langton, P. F., et al. (2009). The dASPP-dRASSF8 complex regulates cell-cell adhesion during Drosophila retinal morphogenesis. Curr. Biol. 19(23): 1969-78. PubMed ID: 19931458
Masaki, T., et al. (1999). Reduced C-terminal Src kinase (Csk) activities in hepatocellular carcinoma, Hepatology 29: 379-384. PubMed ID: 9918913
O'Reilly, A. M., et al. (2006). Csk differentially regulates Src64 during distinct morphological events in Drosophila germ cells. Development 133(14): 2627-38. 16775001
Pedraza, L. G., Stewart, R. A., Li, D. M. and Xu, T. (2004). Drosophila Src-family kinases function with Csk to regulate cell proliferation and apoptosis. Oncogene 23(27): 4754-62. 15107833
Read, R. D., Bach, E. A. and Cagan, R. L. (2004). Drosophila C-terminal Src kinase negatively regulates organ growth and cell proliferation through inhibition of the Src, Jun N-terminal kinase, and STAT pathways. Mol. Cell. Biol. 24: 6676-6689. 15254235
Samuels-Lev, Y., O'Connor, D. J., Bergamaschi, D., Trigiante, G., Hsieh, J.-K., Zhong, S., Campargue, I., Naumovski, L., Crook, T. and Lu, X. (2001). ASPP proteins specifically stimulate the apoptotic function of p53. Mol. Cell 8: 781-794. PubMed ID: 11684014
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Takahashi, M., et al. (2005). Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila. Development 132: 2547-2559. PubMed ID: 15857910
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Trigiante, G. and Lu, X. (2006). ASPPs and cancer. Nat. Rev. Cancer 6: 217-226. PubMed ID: 16498444
Vidal, M., Larson, D. E. and Cagan, R. L. (2006). Csk-deficient boundary cells are eliminated from normal Drosophila epithelia by exclusion, migration, and apoptosis. Dev. Cell 10: 33-44. PubMed ID: 16399076
Vives, V., Su, J., Zhong, S., Ratnayaka, I., Slee, E., Goldin, R. and Lu, X. (2006). ASPP2 is a haploinsufficient tumor suppressor that cooperates with p53 to suppress tumor growth. Genes Dev. 20: 1262-1267. PubMed ID: 16702401
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date revised: 15 April 2011
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