Src oncogene at 64B


Posttranscriptional Modification

The kinase activities of the vertebrate src family members are repressed by phosphorylation of a tyrosine residue in the carboxy-terminal 'tail' of these molecules. To explore whether the tail of an invertebrate src homolog might also serve a regulatory function, an examination was carried out of the ability of the carboxy terminus of Src64, a Drosophila src homolog, which contains a tyrosine homologous to those in the vertebrate src family members, to regulate the following molecules in mammalian fibroblasts: (1) a chimeric protein, p60CD, containing the amino terminus and catalytic domains of chicken p60c-src joined to the C-terminus of Src64; and (2) full-length Src64 itself. By a variety of criteria p60CD appears to be a partially, rather than fully, repressed form of p60c-src. Phosphopeptide mapping indicates that partial repression correlates with partial phosphorylation of the tyrosine in the Src64 tail of the chimera. Phosphorylation of the tail may also regulate full-length Src64. Expression of Src64 in fibroblasts does not affect cell morphology or the overall abundance of tyrosine-phosphorylated proteins. The molecule is phosphorylated at its C-terminal tyrosine (Tyr-547), but not at its in vitro autophosphorylation sites, suggesting that it is catalytically repressed in fibroblasts. Expression of a truncated Src64 mutant lacking Tyr-547 is associated with a clear alteration in cellular morphology and a two- to threefold increase in cellular phosphotyrosine levels. These results suggest that phosphorylation of the C-terminal tyrosine of the tail of an invertebrate src-like kinase can repress the activity of adjacent catalytic domains (Kussick, 1992a).

The relationship between the phosphorylation state of the Drosophila Src64 gene product and its tyrosine kinase activity has been studied in Drosophila Schneider 2 cells, using wild-type and mutated Src64 constructs that were overexpressed by transient transfection. Phosphopeptide mapping shows that the putative regulatory C-terminal tyrosine (Tyr-547) of Src64 is phosphorylated in vivo. In contrast to vertebrate src family kinases overexpressed in fibroblasts, wild-type Src64 overexpressed in Schneider 2 cells is phosphorylated at additional tyrosines outside of the C-terminus. These tyrosines correspond to the major in vitro autophosphorylation sites. Overexpression of wild-type Src64 or several catalytically active Src64 mutants significantly increased the phosphorylation of numerous Schneider cell proteins on tyrosine, while expression of catalytically inactive mutants of Src64 has no such effect. Thus, in contrast to the repression of src family kinase activity in fibroblasts, Src64 is catalytically active when overexpressed in Drosophila cells, perhaps because of substoichiometric C-terminal tyrosine phosphorylation. These results raise the possibility that fly development will be sensitive to ectopic expression of Src64 (Kussick, 1992b).

Btk family kinase at 29A, a prospective Src64-interacting protein

A cDNA clone has been sequenced for the Drosophila melanogaster gene Dsrc28C (now termed Btk family kinase at 29A or Tec29), a homolog of the vertebrate gene c-src. The cDNA contains a single open reading frame encoding a protein of 66 kilodaltons that contains features highly conserved within the src family of tyrosine protein kinases. Novel structural features of the Tek29 protein include a basic pI and a polyglycine domain near the amino terminus. Cell-free translation of in vitro-transcribed RNA yields a protein of the predicted size that can be immunoprecipitated by anti-v-src antisera. RNA blot hybridization reveals that the gene is expressed predominantly during embryogenesis, in imaginal disks of third-instar larvae, and in adult females. In situ hybridization shows that expression in adult females is largely confined to nurse cells and developing oocytes (Gregory, 1987).

In situ hybridization was used to study the developmental RNA expression of dsrc29A (now termed Btk family kinase at 29A or Tec29). This gene encodes two proteins differing at their amino termini. Both gene products contain a protein-tyrosine kinase domain and resemble the protein encoded by vertebrate src. An examination was carried out of most stages of development in the Drosophila life cycle: embryos, third instar larvae, pupae and adults. Tec29 expression is specialized throughout development, being prominent at various times in neural tissue, phagocytic cells, dorsal vessel, ovaries, gut, developing salivary glands, imaginal discs and disc derivatives. These findings confirm and extend previous results for the distribution of Tec29 protein, indicating that the regulation of this gene is primarily at the level of transcription. In some tissues expression is transient, whereas in others, it is continuous, and expression occurs in proliferative, differentiating and differentiated tissue. These patterns of expression demonstrate how a single protein-tyrosine kinase might play diverse roles at different times during development. Comparison of the expression of Tec29 and other members of the protein-tyrosine kinase gene superfamily reveals that the genes are expressed in distinctive but sometimes overlapping patterns (Katzen, 1990).

Expression of the Drosophila src-related gene, Dsrc28C (now termed Btk family kinase at 29A or Tec29) has been investigated at the protein level using monoclonal antibodies. This analysis has revealed that the Tec29 gene encodes two protein forms: a 66-kD doublet predicted from the sequence of a cDNA clone and an additional 55-kD form. The 66-kD protein doublet is observed first at the cellular blastoderm stage and is not detectable in embryos after 12 hr of development. Expression of the 55-kD protein lags behind that of the 66-kD doublet and then persists throughout embryogenesis.Tec29 protein is localized to the cell periphery during cellular blastoderm and gastrulation. The cell periphery-associated staining is then resolved into ectodermal stripes along the fully extended germ band. After the stripes fade, cytoplasmic staining of the majority of cells within the central and peripheral nervous system is observed. The 66-kD protein was shown to represents the cell periphery-associated form of the protein through antibody staining of larval salivary glands expressing a heat shock promoter-driven, full-length Tec29 cDNA. The 55-kD protein is the nervous system form. Thus, the 66- and 55-kD proteins are products of the Tec29 gene, which exhibit different temporal and spatial patterns of expression in the embryo (Vincent, 1989).

The Dsrc28C gene encodes two major proteins, p66 and p55, each of which contains a tyrosine kinase domain. A detailed investigation was carried out of the spatial expression of Dsrc28C proteins during embryonic and larval development. Differentiation of a number of embryonic tissues is accompanied by the induction of Tec29 expression. With the exception of the developing salivary glands, which express high levels of p66, developing tissues express the p55 form of Tec29. Notable examples are cells of the peripheral nervous systems which express p55 from the early stages of neurogenesis through the remainder of embryogenesis and pole cells, which transiently express p55 during portions of embryonic stages 10 and 11. Nervous system expression includes the cell bodies and neuronal fibers of the central nervous system, the anterior sensory organs, and the peripheral sensory neurons. During larval development, p55 levels within the central nervous system remain high but substantial changes in the pattern of expression take place. p55 gradually disappears from the neuronal fibers of the central nervous system and from embryonic cell bodies. During the third larval instar, the birth of immature neuroblasts within the ventral and midbrain ganglia, but not within the optic ganglia, is marked by a transient high level of p55 expression. All imaginal cells that have been observed within the larva express the p66 protein. The patterns of expression that have been noted suggest that expression of the p55 form of Tec29 protein is an early event in the differentiation of neuronal cells, while expression of the p66 form is characteristic of cells committed to ectodermal cell differentiation (Wadsworth, 1990)

Tec29 encodes the only known Drosophila member of the Tec tyrosine kinases. By identifying the first mutations in Tec29 (formerly Src29A), it has been shown that Tec29 is essential for head involution during embryogenesis and for ring canal development during oogenesis. Tec29 mutant egg chambers are defective in the transfer of cytoplasm from the accessory nurse cells through the ring canals into the oocyte. Growth of the mutant ring canals is arrested, and they lack the strong phosphotyrosine localization seen in wild-type ring canals. Mutants lacking the Drosophila Src homolog Src64 show the same phenotype, and Src64 is required for the localization of Tec29 to the ring canals. This interaction is similar to that between vertebrate Src and Tec kinases and suggests that Tec29 is an effector of Src64 that modifies ring canal components required for growth (Roulier, 1998).

Mutation of the Src64 gene of Drosophila results in ovarian ring canal defects and reduced female fertility. A dosage-sensitive modifier screen was used to search for downstream components of the SRC64 signaling pathway. Mutations affecting Tec29, an essential gene encoding a member of the Tec family of protein tyrosine kinases, dominantly enhance the Src64 ring canal phenotype. Loss of Tec29 function in the female germline results in a phenotype strikingly similar to that caused by the loss of Src64 function. In each case, the ring canals are reduced in size and phosphotyrosine content. Tec29 is shown to localize to the ring canal, and this subcellular localization requires Src64 function. These data suggest that Tec29 is a downstream target of SRC64, and that regulating Tec29 localization during ring canal growth may be a crucial Src64 function (Guarnieri, 1998).

The Src superfamily of non-receptor-type tyrosine kinases is composed of four families (the Src family, the Csk family, the Abl family, and the Btk family), each of which is represented by multiple family members. These kinases have been suggested to play diverse roles in cell proliferation, differentiation, survival, and death. Among these four families, the Bruton's tyrosine kinase (Btk) family is unique in that the kinases in this family have extended N-terminal sequences that are called collectively the pleckstrin homology (PH) domain. This domain is known to bind the gamma subunits of heterotrimeric G proteins as well as membrane lipids such as phosphatidylinositol-4,5-bisphosphate. Another feature unique to the Btk family resides in the restricted localization of its members in mammalian tissues. For example, Btk is present only in B cells whereas the expression of Itk, another Tek protein-tyrosine kinase family member, is confined strictly to T cells. Similarly, one of the two forms of the Tec kinase is liver specific whereas the other is hepatocyte specific. In Drosophila, Dsrc29A (Btk family kinase at 29A) is the sole kinase that represents the Btk family, and it is most similar to Btk itself in terms of overall homology. However, the reported N-terminal sequence of Dsrc29A has no similarity to the PH domain. This fact raises the possibility that Dsrc29A is not an ortholog of Btk but represents a distinct member of the Btk family. Deficits in Btk function are responsible for X chromosome-linked agammaglobulinemia in humans and X chromosome-linked immunodeficiency in mice, where B-cell maturation is blocked. The defense mechanism involving immunoglobulins secreted from B lymphocytes exists only in higher vertebrates, and the machinery for antibody production is expected to have its origin in an apparently unrelated cellular function in lower animals. Thus, functional analysis of Dsrc29A would provide insights into the evolution of Btk-like kinases (Baba, 1999 and references).

Btk family kinase at 29A/Dsrc29A is required for adult survival and male genital formation in Drosophila. fickleP (ficP), which removes the Btk homolog in Drosophila, was isolated in a screening of P-element mutants with defects in mating behavior. The mating behavior of Drosophila is made up of several discrete elementary steps. First, the male finds and tracks the female. While tracking, the male approaches the female to tap her abdomen with his forelegs. The male then performs courtship songs by using unilateral wing vibration. Provided that she is sexually receptive, the female frequently stops moving when exposed to the courtship songs, offering the male a chance to lick her genitalia and to attempt copulation. If the male is successful, he mounts on and copulates with the female for 10 to 17 min. Upon termination of copulation, the male releases his genitalia from the female's and dismounts. These behavioral acts are considered to be fixed-action patterns, i.e., instinctive behavior (Baba, 1999 and references).

There are mutations that are known to affect the unique aspects of mating behavior in D. melanogaster. One class of mutations affect copulation and postcopulatory behavior. These include stuck (sk) and coitus interruptus (coi). The sk males often fail to withdraw their genitalia after copulation, with the result that the male and female pair tug at each other, pulling in opposite directions. The coi mutation affects males, causing copulation to terminate prematurely, even before completion of the sperm transfer from the male to the female. Phenotypically, ficP seemed to belong to this class of mutations. The ficP flies exhibit an extremely variable copulatory duration ranging from 1 to 15 min, in sharp contrast to the wild-type flies. Unlike wild-type flies, ficP mutant flies tended to mate repeatedly within a short period (minutes). These behavioral phenotypes of ficP are probably a consequence of malformation of male genitalia. Another conspicuous phenotype of ficP is a reduced life span after adult emergence. The functional rescue experiments of the ficP mutant demonstrate that Drosophila Btk is required in the pupal stage for normal adult longevity and male genital formation. The longevity phenotype is believed to be linked to Btk expression in the developing brain, while the genital phenotype is associated with its expression in the developing genital disc (Baba, 1999).

Interestingly, the Btk homolog is generated by an alternative exon usage of the transcription unit for the Btk family kinase at 29A/Dsrc29A. Both the Btk-coding transcript (referred to here as type 2) and the Dsrc29A kinase transcript (type 1) are expressed in the central nervous system (CNS) and imaginal discs; the domains and/or timing of expression in these tissues are distinct from each other. Complete loss of function of the gene (i.e., loss of both types 1 and 2) causes oocyte undergrowth and embryonic death accompanied by defective head involution, while selective loss of the type 2 transcript spares life but reduces the life span in the adult and leads to malformation of the male genitalia. Thus, the single Btk/Dsrc29A kinase gene exerts pleiotropic functions in different developmental contexts in different tissues through the generation of distinct forms of protein products by means of alternative splicing (Baba, 1999, and references).

Two types of cDNA for this gene were found by library screenings. The type 1 clone is about 2.9 kb long whereas the type 2 clone is about 3.7 kb long. Although the type 1 and 2 clones had identical 3' coding sequences, they differed from each other in the 5' half. This difference appears to have resulted from alternative exon usage. Sequencing of the type 1 cDNA clone reveals a long open reading frame that encodes a protein of 603 amino acids (if the first ATG codon is chosen as the translation initiation site). A database search for similar sequences revealed that an amino acid sequence very similar to the protein has been described as Dsrc29A. An important difference between the sequence found in this study and the reported Dsrc29A sequence (Gregory, 1987) is found in their N termini; the open reading frame for the cDNA clones isolated in this study is open for an additional 45 bp upstream of the methionine start codon chosen in the previous stydy. Furthermore, there are 34 amino acid differences in the deduced protein sequences. Dsrc29A belongs to the Src superfamily, having highly conserved sequence motifs including the SH2 (Src-homology 2), the SH3 (Src-homology 3), and the catalytic (kinase) domains. Dsrc29A differs from Src in that it has a long, basic N-terminal region upstream of the SH3 domain (Baba, 1999).

Analysis of the type 2 cDNA reveals that the protein encoded by the second form of the transcript has an amino acid sequence identical to that of the type 1 product in the C-terminal two-thirds, including the SH3, SH2, and kinase domains, but has a unique N-terminal stretch of 231 amino acids. This isoform has not been reported previously. Although the type 1 product does not contain any discernible conserved motifs in its N-terminal extension, the newly identified type 2 isoform bears typical PH and TH domains, which are regarded as hallmarks of the Btk family kinases, such as Atk, Itk, Tec, and Btk. A striking difference between the type 2 product and Btk is the presence of a polyglycine stretch insertion in the proline-rich region. Overall, the percent similarity between the Drosophila type 2 protein and mammalian Btk is 67.7%. When compared for each domain, the value is 56.9% (PH), 33.8% (TH), 78.8% (SH3), 80.4% (SH2), and 85.3% (kinase). The low value for the TH domain is ascribable to the polyglycine stretch present in the Drosophila sequence. The similarity increases to 76.9% if only the Btk motif in the TH domain is considered for comparison. Thus, the type 2 product is very likely to be the Drosophila ortholog of Btk (Baba, 1999).

Northern blot analysis using a sequence common to both type 1and 2 clones as a probe revealed that a major 3-kb transcript and a minor 4-kb transcript are expressed at constant levels throughout development. The 4-kb transcript is detectable in poly(A)+ mRNA extracted from the heads but not from the bodies of adult flies. This transcript may represent an isoform specific to neural tissue that occupies most of the head. In the wild-type Drosophila, a 4-kb transcript is detected in the adult head poly(A)+ RNA when probed with the type 2-specific sequence. In addition, the type 2-specific probe hybridized with a transcript, of about 3 kb, in both the head and body parts. On the other hand, the type 1-specific probe detects a 3-kb transcript in the head and body poly(A)+ RNA. Thus, the type 1 cDNA corresponds to a 3-kb transcript whereas the type 2 cDNA corresponds to a different 3-kb transcript and a 4-kb transcript. The 3-kb type 1 and 3-kb type 2 transcripts are expressed in both the head and body, while the 4-kb type 2 transcript is head specific (Baba, 1999).

The difference between wild-type and ficP strains was evident when a type 2-specific probe was used for Northern blot analysis; neither of the 4-kb and 3-kb transcripts are detected in ficP flies. The absence of these transcripts is associated with the fic mutation, since ficR, a phenotypic revertant obtained by excision of the inserted P element, has both the transcripts. The sole transcript detectable in ficP flies with the type 2-specific probe is distinct from any of the transcripts found in the wild type. The mutant transcript is slightly shorter than the wild-type 3-kb type 2 transcript, implying that it is a truncated version of the type 2 transcript. (Baba, 1999).

To examine whether the ficP phenotypes are causally linked to malfunction of the cloned gene, ficP mutant lines were created carrying the full-length type 1 or 2 wild-type cDNA driven by the heat shock promoter (hs-cDNA). Although the phenotypes of ficP results from the selective loss of the type 2 transcript, ubiquitous overexpression of either type 1 or 2 similarly rescues these phenotypes. This fact suggests that the specific phenotype of the ficP mutant reflects disruption of specific temporal and/or spatial expression of the type 2 transcript rather than functional specificity of the type 2 product (Baba, 1999).

Mutant flies carrying hs-cDNA, reared at 25°C, retain the ficP phenotype: the apodeme of the male genitalia remains torn. Consistent with this observation, the histogram for copulatory duration reveals a pattern similar to that for ficP flies without hs-cDNA. Subjecting the ficP;hs-cDNA males to heat shock throughout the pupal stage restores normal apodeme structure and is accompanied by a dramatic alteration in mating behavior after emergence. The pattern of distribution of copulatory duration for the ficP;hs-cDNA flies after heat shock resembles that for wild-type flies. Thus, expression of hs-cDNA in the pupal stage rescues the ficP defect in the genitalia and restores normal copulation behavior, establishing the causal relationship between the mutant phenotype and the gene. Heat shock induction of hs-cDNA on the first or second day of pupal life is sufficient for rescue of both the behavioral and genital phenotypes. Repeated remating is not observed between pairs of ficP;hs-cDNA males and wild-type females, when the males had been exposed to heat shock after the third-instar larval stage until emergence (Baba, 1999).

Apart from the defects in the male genitalia and mating behavior, it was noted that the life span after eclosion is decreased in ficP flies, when compared to wild-type flies. The longevity phenotype of ficP adult flies is also rescued by ubiquitous overexpression of either type 1 or 2 cDNA, indicating that the type 1 and 2 transcripts are functionally equivalent to each other in terms of life span regulation. This implies that a specific site at which type 2 mRNA, but not type 1 mRNA, is expressed plays a critical role for adult survival. The most effective period of hs-cDNA induction for rescue of the longevity phenotype is between the last day of the third larval instar stage and the first day of pupation. Thus, the results of the heat shock experiments suggest a requirement for Btk/Dsrc29A in the prepupal to early pupal stage for normal copulation and genital formation and in the third larval instar to early pupal stage for normal adult life span (Baba, 1999).

To study the tissue localization of the Btk/Dsrc29A transcript during the critical period, whole-mount in situ RNA hybridization was performed. Both the type 1 and 2 transcripts are detected in the male genital disc, although their expression patterns are distinctly different from each other. The expression of the type 1 transcript is confined to the middle portion of the anterior bulbus, which is known to form the internal genitalia. However, the type 2 transcript is strongly expressed along the edge of the male genital primordium facing the lumen. Fate map studies have indicated that this part of the male genital primordium contributes to the main body of the male genitalia including the penis apparatus, to which the apodeme is attached. This finding supports the notion that the specific phenotype of the ficP mutant correlates with the spatial restriction of the type 2 transcript (Baba, 1999).

The other tissue that is rich in Btk/Dsrc29A mRNA is the CNS: particularly strong expression is detected in the cells of the inner optic anlage. Several hours after puparium formation, strong Btk/Dsrc29A mRNA expression is noted in the cells above the mushroom bodies. Hybridization with type-specific probes demonstrate that the transcript expressed in the mushroom body region consists exclusively of the type 2 transcript. In other areas of the brain, type 1 transcript predominates. These cells are thought to contribute to the development of mushroom bodies. The expression is detected in many mushroom body ganglion cells but not in neuroblasts. Soon thereafter, weak expression of type 1 is noted in many scattered cells in the central brain and ventral ganglion while expression in the optic lobe ceases. In the ventral ganglia, weak Btk/Dsrc29A expression is detected in many scattered cells in a manner similar to that in the central brain. Strong expression is observed in the cells on the midline of the ventral ganglion. Since these are irregularly shaped cells that enfold the anterior and posterior comissures in each segment, they are likely to be midline glia. The expression in the ventral ganglia consisted exclusively of the type 1 transcript. No expression of the type 2 transcript is detected in this region. At around 72 h after puparium formation, nearly half of the cells in the brain express type 1 mRNA. At this stage, expression of the type 2 mRNA is observed only in a cluster of cells just above each antennal lobe and cells near the mushroom body. The level of type 2 mRNA expression in these cells is remarkably high. Most of the cells expressing this mRNA in the antennal lobe region appear to be neurons because the cell bodies are round. The antennal lobe is known to consist of approximately 1,200 cells. Of these, only a very small portion exhibit strong Btk/Dsrc29A expression. Interestingly, there are other clusters of cells above the antennal lobes that do not express Btk/Dsrc29A. Four clusters of cells expressing the mRNA per hemisphere are observed in the mushroom body region. Each cluster apparently corresponds to the progeny of one of four mushroom body neuroblasts. Thus, it is concluded that the type 2 transcript is selectively expressed in the cells of the mushroom body and the antennal lobe whereas the type 1 transcript is expressed in dispersed neurons and the midline glial cells in the CNS during the larval and pupal stages. The localizations of types 1 and 2 mRNA appear to be mutually exclusive in both the CNS and the genital disc. It is emphasized that the temporal correlation between the genital and behavioral phenotypes revealed by the rescue experiments with time-restricted Btk/Dsrc29A expression does not exclude the possibility of a neural origin of the behavioral phenotype (Baba, 1999).

The Btk/Dsrc29A gene is distinctly different from other members of the Btk family genes in two respects: (1) it is alternatively spliced so as to give rise to two different proteins, one of which has, at its N terminus, a unique domain without sequence homology to any other known protein, in place of the PH domain in the sibling protein. (2) The N-terminal segments in both isomers are separated from the main body of the protein by an intervening polyglycine stretch. This structural complexity may be necessary for the support of the pleiotropic functions of the Btk/Dsrc29A kinase in Drosophila, in which no other members of the Btk kinase family exist. It is of interest that the PH domain is dispensable for normal copulation and survival. It should be pointed out, however, that the adult life span of the rescued mutant flies is not identical to that of wild-type flies. Such partial rescue may indicate that the function of the type 1 product only partially overlaps that of the type 2 product (Baba, 1999).

Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation

The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. Kelch functions to cross-link actin fibers. Biochemical studies using purified, recombinant Kelch protein show that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Kelch is tyrosine phosphorylated in a Src64-dependent pathway at tyrosine residue 627. A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescues the actin disorganization phenotype of kelch mutant ring canals, but fails to produce wild-type ring canals. Phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the nonphosphorylatable KelY627A protein phenocopies Src64 ring canals. KelY627A protein in ring canals also dramatically reduces the rate of actin monomer exchange. The phenotypes caused by Src64 mutants and KelY627A expression suggest that a major function of Src64 signaling in the ring canal is the negative regulation of Kelch-dependent actin cross-linking (Kelso, 2002).

In Drosophila, 15 syncytial nurse cells and 1 oocyte are enveloped by a monolayer of somatic follicle cells and constitutes an egg chamber, the structural and functional unit of the Drosophila ovary. A ring canal is a gateway through which mRNAs, proteins, and nutrients flow from nurse cells into the oocyte during the entire course of oogenesis. Ring canals are derived from arrested mitotic cleavage furrows that are modified by the addition of several proteins. These include abundant F-actin, at least one protein that is recognized by antiphosphotyrosine antibodies (PY protein), a mucin-like glycoprotein, the Hts ring canal protein (HtsRC), ABP280/filamin, Tec29 and Src64 tyrosine kinases, and Kelch (Kelso, 2002 and references therein).

As nurse cell cytoplasm transport proceeds, the diameter of ring canals grows from <1 µm to 10-12 µm. This represents the addition of over one inch of filamentous actin during a period in which the filament density remains constant. Near the end of oogenesis, the ring canal actin transforms from a single continuous bundle into several interwoven actin cables. Ring canal expansion probably involves the nucleation of new actin filaments and an increase in actin filament length, coupled with filament reorganization that requires the establishment of reversible actin cross-links (Kelso, 2002).

A signaling cascade that leads to malformed ring canals involves the Src family kinases (SFKs) Src64 and Tec29 (Btk family kinase at 29A). SFKs are associated with the phosphorylation of several important proteins involved in regulating F-actin-rich structures, including cell-substrate adhesions, cell-cell adhesions, and actin regulatory proteins such as p190 RhoGAP, cortactin, and ABP280/filamin. Src mutations in mice result in osteoclasts deficient in the formation of ruffled borders and defective in forming the peripheral actin ring. Mutations in Drosophila Src64 or tec29 lead to small ring canals that lack most phosphotyrosine staining, and egg chambers that have incomplete nurse cell cytoplasm transport (Kelso, 2002 and references therein).

Using a series of two-dimensional (2D) gel electrophoresis experiments, it has been determined that Kelch is phosphorylated in an SFK-dependent manner. Site-directed mutagenesis has been used to map the phosphorylated tyrosine residue. Thin section electron microscopy has revealed striking differences in actin organization and filament number in lines expressing wild-type Kelch when compared with src64Delta17 and the nonphosphorylatable form of Kelch. This shows that phosphorylation of Kelch is necessary for normal filament organization. Binding studies show that the phosphorylated form of Kelch does not interact with actin. Therefore, Src64-mediated phosphorylation probably dissociates Kelch cross-links in ring canals. The nonphosphorylatable mutant also causes a reduction in actin monomer turnover kinetics. This suggests that reversible cross-links are required to allow dynamic actin monomer turnover and maintain overall ring canal morphology. These observations suggest that a major cytoskeletal target of Src64 signaling at the ring canal is the actin-cross-linking protein Kelch (Kelso, 2002).

The regulation of Kelch-actin cross-links could be accomplished by Src64 directly phosphorylating Kelch. Alternatively, Src64 may activate another protein tyrosine kinase, such as Tec29, which in turn phosphorylates Kelch. However, the shared phenotype seen by electron microscopy of the src64 and P[kelY627A] ring canals is strongly suggestive of Kelch being the major downstream component of a Src64 cascade. Analysis of Kelch phosphorylation in tec29 mutants is difficult because available tec29 alleles are lethal. SFKs have been shown to signal rearrangements in the actin cytoskeleton in other contexts. In Drosophila, embryos mutant for src64 or tec29 fail to complete epidermal closure at the end of gastrulation. This is, in part, because the leading edge cells contain reduced quantities of F-actin, and the cells only partially elongate and fail to migrate completely. SFKs are also known to interact directly with cytoskeletal proteins, as in the case of c-Src and cortactin. Phosphorylation of cortactin by c-Src tyrosine kinase decreases its ability to cross-link F-actin in vitro. These examples suggest that there could be a critical role played by tyrosine phosphatases to ensure that F-actin does not become disorganized due to excessive phosphorylation of cross-linking proteins. There are several candidate phosphatases in Drosophila; however, their roles in ring canal development have not been studied (Kelso, 2002 and references therein).

Drosophila Src-family kinases function with Csk to regulate cell proliferation and apoptosis

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).

Localization of Tec29 to ring canals is mediated by Src64 and PtdIns(3,4,5)P3-dependent mechanisms.

Two tyrosine kinases, Src64 and Tec29, regulate the growth of actin rich-ring canals in the Drosophila ovary. Src64 directs the localization of Tec29 to ring canals, but the mechanism underlying this process has remained unknown. This study shows that Tec29 localizes to ring canals via its Src homology 3 (SH3) and Src homology 2 (SH2) domains. Tec29 activity is required for its own ring canal localization, suggesting that a phosphotyrosine ligand for the SH2 domain is generated by Tec29 itself. Src64 regulates this process by phosphorylating Y677 within the kinase domain of Tec29, an event required for Tec29 activation. The pleckstrin homology (PH) domain of Tec29 has dual functions in mediating Src64 regulation. In the absence of Src64, the PH domain prevents Tec29 ring canal localization. In the presence of Src64, it enhances membrane targeting of Tec29 by a PI(3,4,5)P3-mediated mechanism. In the absence of its PH domain, Tec29 constitutively localizes to ring canals, but still requires Src64 for full activation (Lu, 2005).

Thus SH3 and SH2 protein-protein interaction domains of Tec29 are necessary and sufficient for ring canal localization. Localization of a truncated protein that contains these two domains (DeltaKinase), however, is dependent on endogenous Tec29 activity. A likely reason for this result is that endogenous Tec29 activity can generate ring canal binding sites for the SH2 domain of DeltaKinase. The fact that Tec29 activity is directly correlated with phosphotyrosine contents on ring canals is consistent with this model. Alternatively, endogenous Tec29 may phosphorylate DeltaKinase, thus allowing it to bind to an SH2-domain-containing protein on the ring canal. A tyrosine residue within the SH3 domain of Btk has been shown to be a major autophosphorylation site (Rawlings, 1996). When the corresponding tyrosine is mutated in Tec29, however, the mutant protein localizes to ring canals and fully rescues all defects associated with Tec29 mutants, indicating that this residue is not important for the function of Tec29 on ring canals. In addition, no phosphotyrosine content was detected within the DeltaKinase protein by immunoblotting with an antiphosphotyrosine antibody. Therefore, the most likely scenario is that Tec29 phosphorylates a ring canal protein, thus generating binding sites for its own SH2 domain (Lu, 2005).

Since Src64 protein is localized to nurse cell cortical membrane, as well as ring canals, it is interesting to consider why localization of Tec29, a process regulated by Src64, is directed to ring canals. One possible explanation is that Src64 activates Tec29 everywhere on the membrane, but the substrate (and therefore binding partner) of Tec29 is only present on ring canals. Alternatively, Tec29's substrate may be present on membranes and ring canals, but Src64 may be activated only at ring canals to ensure local activation of Tec29. A third possibility is that activation of Src64 and Tec29's substrates are both restricted to ring canals. The data that overexpressed type 1 Tec29 localizes to ring canals in the absence of Src64 are consistent with Tec29 substrates being on ring canals only. Therefore a model is proposed for how type 2 Tec29 may localize to ring canals. Interactions between the PH domain and PIP3 can target type 2 Tec29 to the cortical membrane, thus allowing it to be phosphorylated and activated by Src64. Once activated, the Tec29 protein that is localized to the membrane region adjacent to ring canals might access and phosphorylate substrate proteins, and bind to them via its SH3 and SH2 domains. In addition, ring-canal-localized Tec29 can phosphorylate additional substrate proteins, thus generating ring canal binding sites and directly recruit undocked Tec29 protein from the cytoplasm through a positive feedback mechanism (Lu, 2005).

The results suggest that Tec29's binding partner on the ring canal is one of its substrates. This substrate may be a scaffolding protein, on which a signaling complex can assemble, or it may be an effector that is directly involved in the formation and rearrangement of actin networks on ring canals. Several proteins that interact with either the SH3 (hnRNP-K, Vav, WASp, etc.) or the SH2 domain (BLNK, BRDG-1, SLP-76) of other TFKs have been identified. Although none of these proteins has been shown to be a direct substrate of TFKs, their Drosophila homologs remain attractive candidates for downstream effectors of Tec29. For example, the SCAR protein, which has functions similar to WASp in promoting actin polymerization, has been shown to localize to actin-rich structures during Drosophila development. SCAR mutant ring canals have abnormal morphology and exhibit growth defects. Analysis of the phosphorylation of these and other candidate proteins, including Kelch, in response to Tec29, their mutant phenotypes in the ovary and the functional consequences of their potential association with Tec29, may lead to the identification of Tec29's ring canal binding partnerĀ (Lu, 2005).

TFKs are the only group of nonreceptor protein tyrosine kinases that contain PH domains. The existence of Tec29 splicing variants that differ in this domain provide an opportunity to analyze the in vivo significance of its function and how it affects Tec29 localization. The results show that the PH domain can accentuate Src64 regulation of Tec29 by inhibiting Src64-independent ring canal localization of Tec29. Interestingly, a recent study has shown that truncation of the PH domain increases the basal activity of Btk in vitro, but eliminates PIP3-dependent regulation (Saito, 2001). This suggests that an inhibitory function by the PH domain may be more universal among other TFKs, perhaps to confer a more stringent regulatory mechanism by activators such as SFKs. However, these results also raised questions as to the biological significance of type 1 Tec29. The fact that type 1 Tec29 lacks a PH domain dictates that it can be regulated by Src64 but not PIP3. This may be important in other tissues and developmental processes, where temporal and spatial coordination of multiple signaling pathways may be critical. Interestingly, Tec29 has been shown to be involved in dorsal closure and male genital formation (Baba, 1999a; Tateno, 2000). Type 1 or type 2 Tec29-specific expression in the CNS has also been described (Baba, 1999a). Further experiments will be necessary to address the physiological importance of type 1 and type 2 Tec29 proteins in these tissues (Lu, 2005).

Raf activation is regulated by tyrosine 510 phosphorylation in Drosophila

The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, this study has demonstrated that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf), which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. It was further shown that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, it was shown that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved (Xia, 2008; full text of article).

Drosophila Abelson kinase, acting downstream of the Src kinases, mediates cell invasion and proliferation through two distinct MAPK pathways

The Abelson (Abl) family of non-receptor tyrosine kinases has an important role in cell morphogenesis, motility, and proliferation. Although the function of Abl has been extensively studied in leukemia, its role in epithelial cell invasion remains obscure. Using the Drosophila wing epithelium as an in vivo model system, this study shows that overexpression (activation) of Drosophila Abl (dAbl) causes loss of epithelial apical/basal cell polarity and secretion of matrix metalloproteinases, resulting in a cellular invasion and apoptosis. The in vivo data indicate that dAbl acts downstream of the Src kinases, which are known regulators of cell adhesion and invasion. Downstream of dAbl, Rac GTPases activate two distinct MAPK pathways: c-Jun N-terminal kinase signaling (required for cell invasion and apoptosis) and ERK signaling (inducing cell proliferation). Activated Abl also increases the activity of Src members through a positive feedback loop leading to signal amplification. Thus, targeting Src-Abl, using available dual inhibitors, could be of therapeutic importance in tumor cell metastasis (Singh, 2010).

This is the first study to provide in vivo evidence for the role of Abl in cell invasion. Cells expressing dAbl (in the dpp-domain) become invasive and migrate into the area of the posterior compartment, where they are located basally to the basement membrane. Although during this process many cells die, those that 'resist' cell death can be visualized by the presence of GFP at the base of the epithelium in either compartment. Furthermore, mechanistic evidence is provided for an Src-Abl signaling cascade and an Abl/Src signal amplification loop in epithelial cell invasion. Targeting both kinase types using dual Abl/Src inhibitors in cancer patients could thus be of clinical significance. It was also shown that increased cell proliferation associated with Abl can be separated from its cell invasion function by distinct downstream effectors. Different MAPKs are activated downstream of dAbl and Rac, and mediate the cell proliferation and cell invasion phenotypes, respectively (Singh, 2010).

Loss of cell polarity has been linked to tumor growth and cell invasion. The mechanism(s) by which dAbl downregulates cell adhesion/polarity genes like DE-Cadherin, β-Catenin/Armadillo and Dlg are not known. This could be a direct effect of dAbl on junctional complexes or a consequence of the cell invasive behavior. Downregulation of E-cadherin has been linked to several types of tumors. Furthermore, Src family members have been shown to increase the turnover of AJs, which in turn would cause an increase in cell mobility, a possible mechanism by which Abl can mediate loss of cell polarity. This hypothesis is further supported by the observation that overexpression of DE-cadherin suppresses the effects induced by Src upregulation (through Csk reduction, using UAS-dCsk-RNAi) in the retina. Consistent with this notion, overexpression of DE-Cadherin rescues the dAbl-induced cell invasion phenotype. Moreover, removing a genomic copy of mmp1 and mmp2 results in suppression of the dAbl cell invasion phenotype. On the basis of these data it is concluded that loss of cell polarity and MMP secretion are the key factors in contributing to cell invasive behavior of dAbl-expressing cells. However, the possibility of minor contributions of unknown factors in this process cannot be completely ruled out (Singh, 2010).

A complicated question is how dAbl causes cell proliferation in epithelial cells (dAbl lacks nuclear localization signals). The effect of dAbl expression results in cell-autonomous and non-autonomous cell proliferation. In Drosophila, cells destined to undergo apoptosis express specific growth factors (Wingless and Dpp; their upregulation is mediated by JNK activation), inducing non-autonomous compensatory proliferation in neighboring cells. This compensatory proliferation is important for maintaining proper tissue homeostasis and may also be relevant for the induction of tumor cell proliferation. As dAbl activation results in cell death in migrating cells, one argument could be that cell proliferation associated with dAbl activation is a consequence of compensatory proliferation. Interestingly, dAbl expression results in an increase in Wg expression, suggesting that compensatory proliferation takes place in response to dAbl. Taken together, these data suggest that at least some aspects of dAbl-mediated cell proliferation (mediated by activation of ERK) are cell-autonomous independent of such compensatory proliferation, as Bsk-DN co-expression in a dAbl overexpression background (which blocks JNK signaling and thus induction of Wg and Dpp expression), does not block excessive proliferation within the dAbl expression domain (Singh, 2010).

The cell invasion phenotype of dAbl overexpression is similar to Csk reduction (dCsk-RNAi) and the data indicate that dAbl acts downstream of dCsk. As Csk negatively regulates Abl/Src family kinases (SFKs), this suggested that Src mediates the effect of dCsk on dAbl. Previous studies have shown that Abl can act as a substrate of SFKs, though other studies have shown that the opposite can also be true. The data indicate that Src acts upstream of Abl and that Abl can feed back and amplify the signal through its positive effect on Src. How is the dAbl-Src feedback loop working mechanistically? From the in vivo experiments it is not possible to conclude whether dAbl acts directly on Src, dCsk, or unknown upstream components. dAbl does not co-immunoprecipitate either dCsk or the Src kinases in Drosophila S2 cells. Since binding between kinases can be of very transient nature, it is possible that even if dAbl would bind Src or dCsk in vivo, it may not be possible to detect it. However, in vivo data suggest that dCsk does not mediate the dAbl effect: if dAbl would act through dCsk (by inhibiting it), phospho-Src (pSrc) levels should be similar with dCsk-IR or dAbl expression, which is not the case. dAbl expression results in a much more robust activation Src with pSrc detected in all dAbl/GFP-positive cells, whereas dCsk-IR does not result in such strong activation. This observation suggests that dAbl does not act through dCsk in this process. Although the possibility cannot be excluded that dAbl could modulate an unknown component upstream of dCsk, the fact that co-expression of dCsk-IR and dAbl (dCsk-IR; UAS-Abl at 18°C) shows a synergistic effect (even at 18°C, where neither individual transgene has a phenotype on its own) suggests that dAbl and Csk act in parallel on Src. As Abl can phosphorylate Src kinases, a direct effect of dAbl on the Src kinases is favored (Singh, 2010).

JNK signaling is activated in response to environmental stress and by several classes of cell surface receptors, including cytokine receptors and receptor tyrosine kinases. In mammalian cells, JNK has been implicated in oncogenic transformation in fibroblasts and hematopoietic cells, and in cell invasion. In oncogenic transformation, JNK signaling can promote tumor growth, while it can also act as a tumor suppressor. It also functions in basement membrane remodeling during imaginal disc eversion and tumor invasion. This study provides evidence for a link between Src and JNK during cell invasion, mediated through dAbl. The cell invasion and apoptosis phenotypes induced by dAbl require JNK activity, whereas the cell proliferation function of dAbl appears to be mediated by ERK signaling. dAbl does not affect expression levels of JNK but instead causes an increase in active JNK (phospho-JNK). It is worth noting that removing a genomic copy of each of the Drosophila Rac genes suppresses all phenotypes associated with dAbl overexpression (cell invasion, death, and proliferation). These data are consistent with the study of BCR-Abl-mediated cell growth, which requires Rac function, suggesting a general relevance of Rac GTPases as Abl effectors (Singh, 2010).

It is not established how Abl mediates Rac activation. A possible link can be Crk, which primarily consists of SH2 and SH3 domains, serving as an adaptor. Crk-I can associate with and be phosphorylated by c-Abl. Furthermore, ectopic expression of Crk can result in JNK activation. As overexpression/activation of dAbl results in JNK activation, Crk may provide a missing link between dAbl and Rac for JNK activation. Another candidate to mediate an interaction between dAbl and Rac GTPases can be Trio, a guanine exchange factor. Trio has two putative Rac and Rho-binding domains. In Drosophila, Trio function has been studied extensively in the context of axon guidance where it has been shown to interact with dAbl. Interestingly, a recent report has identified Trio as one of the guanine exchange factors responsible for invasive behavior of glioblastoma. Thus, a potential role of Trio in the context of Abl-mediated cell invasion warrants further investigation (Singh, 2010).

A novel conserved phosphotyrosine motif in the Drosophila fibroblast growth factor signaling adaptor Dof with a redundant role in signal transmission

The fibroblast growth factor receptor (FGFR) signals through adaptors constitutively associated with the receptor. In Drosophila melanogaster, the FGFR-specific adaptor protein Downstream-of-FGFR (Dof) becomes phosphorylated upon receptor activation at several tyrosine residues, one of which recruits Corkscrew (Csw), the Drosophila homolog of SHP2, which provides a molecular link to mitogen-activated protein kinase (MAPK) activation. However, the Csw pathway is not the only link from Dof to MAPK. This study identified a novel phosphotyrosine motif present in four copies in Dof and also found in other insect and vertebrate signaling molecules. These motifs are phosphorylated and contribute to FGF signal transduction. They constitute one of three sets of phosphotyrosines that act redundantly in signal transmission: (1) a Csw binding site, (2) four consensus Grb2 recognition sites, and (3) four novel tyrosine motifs. Src64B binds to Dof and Src kinases contribute to FGFR-dependent MAPK activation. Phosphorylation of the novel tyrosine motifs is required for the interaction of Dof with Src64B. Thus, Src64B recruitment to Dof through the novel phosphosites can provide a new link to MAPK activation and other cellular responses. This may give a molecular explanation for the involvement of Src kinases in FGF-dependent developmental events (Csiszar, 2010).

Mutational analysis of Dof, which was used as an indirect approach to map tyrosines that are phosphorylated in the presence of an activated FGFR, showed that consensus tyrosine motifs for PI3K and Csw binding at amino acid positions 486 and 515 were substrates of phosphorylation. In addition, three tyrosine residues at positions 592, 613, and 629 were identified as phosphorylation targets, but these do not conform to known conserved tyrosine motifs. Finally, the last 200 amino acids of Dof also contain several phosphorylation target sites. Mutational analyses of this type do not prove that the same residues are phosphorylated in the wild-type situation, but the tyrosine at position 515 is required for the binding of Csw upon FGFR activation, and this study has shown that mutation of most of the identified sites resulted in impaired activity of the molecule in vivo, supporting the notion that these tyrosines are physiologically relevant phosphorylation targets in Dof (Csiszar, 2010).

The three tyrosine-containing motifs at positions 592, 613, and 629 do not resemble known conserved tyrosine motifs, but their positions and their sequences are conserved in Anopheles Dof. In Drosophila the sites are very close together, so that they could act as tandem interaction surfaces, but the fact that they are separated by longer insertions in Anopheles makes this unlikely. The motifs at 613 and 629 resemble each other, and the same sequence motif is present two more times in the C termini of both Drosophila and Anopheles Dof. Protein database searches with the consensus sequence motif Y-X3-P-X3-P, generated from these eight related sites, showed that this motif is present in several signaling molecules, in many cases as known phosphorylation target sites (e.g., in Shc and the mammalian FGFR-1). Mammalian Shc contains two consensus Grb2 binding sites. One, conserved in vertebrate Shc proteins, has been shown to bind Grb2 and activate the Ras-MAPK pathway. The other, part of a double-phosphorylation site of two adjacent tyrosines and conserved in all members of the Shc family from insects to vertebrates, does not interact with Grb2, and its function in mitogenic activation and apoptosis protection does not depend on Ras. This site is part of the novel tyrosine motif. In vitro kinase assays showed that this pair of adjacent tyrosines can be phosphorylation targets for EGFR and Src as well, but no proteins have been identified as binding partners. The highly conserved sequence patch around these two adjacent phosphorylated tyrosines in Shc goes beyond the Grb2 consensus site and outlines exactly the novel Y-X3-P-X3-P motif, suggesting that the whole motif is important for an evolutionarily conserved biological function (Csiszar, 2010).

In the mammalian FGFR-1, the novel tyrosine motif surrounds the one conserved autophosphorylation site at position Y583 and thus is located in the loop separating the small and large lobes of the kinase domain (28). This is the most variable intracellular sequence part within the FGFR family, and no other mammalian FGFRs share this motif. In spite of the fact that most autophosphorylation sites in the FGFR are conserved, many of these tyrosines are dispensable for signal propagation and only a few proteins that associate with these sites have been identified to date. Phosphorylated Y583 has no known binding partners, and no signaling function has been linked to it so far (Csiszar, 2010).

These findings lead to the speculation that the mammalian FGF-R1 has the ability to recruit a molecule directly that in the case of the Drosophila FGFR is recruited indirectly via Dof and, in the other mammalian FGF receptors, perhaps via other interactors, such as Shc (Csiszar, 2010).

The rescue experiments used to assay the functional relevance of the tyrosine mutations that influenced the phosphorylation levels of Dof in vitro yielded two important findings: first, three independent functional units in Dof were identified that contribute to signal propagation, and second, these units act redundantly, in that any one of them is sufficient to provide significant biological activity. However, it cannot be ruled out that the overexpression system used might have masked potential minor qualitative differences and therefore exaggerated the redundancy. Similarly, two of the phosphorylated tyrosines showed no functional relevance in this or previous studies. One is the tyrosine of a PI3K consensus site at position 486 for which there was no requirement in any of the assays, employing either full-length Dof or mutant forms retaining only the first 600 amino acids. The other identified phosphotyrosine site without an identified function is located at position 592. Though it is conserved in Anopheles Dof, including several surrounding amino acids, the mutation of this tyrosine alone or in combination with other tyrosines did not affect the biological function of Dof. Again, perhaps subtle effects of the loss of these sites might have been missed. Nevertheless, the presence of several copies of docking sites for downstream signaling molecules and the availability of alternative routes to activate the same signaling cascade may provide Dof with options for fine-tuning of signaling strength and duration (Csiszar, 2010).

Csw has previously been shown to interact with Dof. The phosphotyrosine of the Csw consensus site was required for efficient interaction and for MAPK activation in the context of a Dof construct that lacked any of the other phosphorylation sites. This study shows that the Csw site is indeed important only if other parts of Dof with MAPK activating capacity are deleted or mutated. The same is true for the phosphorylation sites in the C-terminal domain, which this study shows to be the consensus Grb2 binding sites (Csiszar, 2010).

The four novel phosphotyrosine motifs contributed to the activation of the MAPK cascade, although they were essential only if other parts of the molecule with MAPK activation capability were deleted or mutated. Phosphorylation of these tyrosines was essential for the efficient interaction of Dof with the protein kinase Src64B, and Src activity was in turn required for Dof-dependent MAPK activation in S2 cells. Src kinases can activate mitogenic signaling in many different ways. Recently, Drosophila Src64B has been shown to be essential in the regulation of Raf activity by phosphorylating a regulatory tyrosine residue in Raf, which is also conserved in mammalian B-Raf. Thus, it is reasonable to postulate that upon FGFR-dependent phosphorylation the novel tyrosine motif in Dof is utilized to recruit Src64B, which can then contribute to MAPK activation via Raf activation (Csiszar, 2010).

In addition to Src64B, a tyrosine phosphorylated protein of 29 kDa (p29) was found that coprecipitated with Dof802 and required the phosphorylated tyrosines of the novel motif for this interaction. This raises the possibility that other proteins might use these sites as docking surfaces as well, although no evidence was found that this protein directly binds these phosphosites (Csiszar, 2010).

Since the residues surrounding the tyrosines in the novel motifs are conserved, it was expected that they would be found to be important for function. However, while mutating the tyrosines had measurable effects on the function of Dof in vivo, replacing the prolines had only moderate or no effects in the same assays. Similarly, Src64B binding to Dof802 in S2 cells was strongly reduced when the tyrosines of the novel motifs were mutated (in the background of mutated Csw and PI3K sites) but not when the prolines were mutated (Csiszar, 2010).

The finding that the impact of tyrosine mutations in the novel motifs on Dof function was greater than that of the proline mutations agrees with the known general characteristics of the interaction of phosphotyrosine motifs with phosphotyrosine binding domains. The driving force of these interactions is the phosphorylated tyrosine itself, with additional lower-affinity interactions of surrounding residues contributing to specificity for the different phosphotyrosine binding domains, as has also been found for dissociation constants when measuring interactions of SH2 domains in phosphopeptide library interaction screens. Indeed, it has been proposed that the modest selectivity of SH2 domains to phosphotyrosine containing linear peptides (5- to 20-fold) is not sufficient to explain selectivity of signaling pathways in living cells. Recent work has identified additional components of these type of interactions and shows that the selectivity of phospholipase Cγ binding and signaling via activated FGFR-1 are determined by interactions between a secondary binding site on an SH2 domain and a region in the FGFR-1 kinase domain in a phosphorylation-independent manner. These data suggest that the mutation of two amino acids in a tyrosine motif might have only mild consequences compared to the loss of the phosphotyrosine site in the context of whole protein-protein interactions, based on the complexity of different binding interfaces and their different affinities of this interaction (Csiszar, 2010).

Since SH2 domains preferentially interact with residues C-terminal to the tyrosine, and these are the conserved residues in the novel motif, the motif is expected to interact with SH2-type domains. Why has the motif described in this study not been found in the extensive searches for SH2 target motifs? The answer may lie in the fact that no motifs with important conserved amino acids at positions +4 and +8 are known at all, and this may be primarily because of the way they have been screened for. The degenerate phosphotyrosine peptide libraries that have been used to determine SH2 domain specificities screened only positions +1 to +3, and the furthest that other studies have gone was up to position +5 (Csiszar, 2010).

It is not known if the SH2 domain of Src64B is involved in the interaction with the novel phosphomotif of Dof, but if so, it is not clear whether this motif could be accommodated by the same interaction surface as the one that binds to the consensus Src SH2 domain recognition sequence pYEEI, which has been defined by phosphopeptide library screening and structural studies on peptide-bound Src SH2 domains (Csiszar, 2010).

Finally, little is known about the interaction of Src family kinases with other vertebrate signaling molecules bearing the novel tyrosine motif. For example, the motif in Shc is a phosphorylation target of Src, but no interaction studies have been performed, and there are conflicting reports about direct interaction between FGFR-1 and Src. The results of this study suggest that Src might be a good candidate for interacting with mammalian FGFR-1 and other vertebrate signaling molecules via the novel motif. It should be worth probing the general validity of Src binding to this novel phosphotyrosine motif in the future (Csiszar, 2010).

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

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

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

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

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

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

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

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

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

Src64B phosphorylates Dumbfounded and regulates slit diaphragm dynamics: Drosophila as a model to study nephropathies

Drosophila nephrocytes are functionally homologous to vertebrate kidney podocytes. Both share the presence of slit diaphragms that function as molecular filters during the process of blood and haemolymph ultrafiltration. The protein components of the slit diaphragm are likewise conserved between flies and humans, but the mechanisms that regulate slit diaphragm dynamics in response to injury or nutritional changes are still poorly characterised. This study shows that Dumbfounded/Neph1, a key diaphragm constituent, is a target of the Src kinase Src64B. Loss of Src64B activity leads to a reduction in the number of diaphragms, and this effect is in part mediated by loss of Dumbfounded/Neph1 tyrosine phosphorylation. The phosphorylation of Duf by Src64B, in turn, regulates Duf association with the actin regulator Dock. Diaphragm damage induced by administration of the drug puromycin aminonucleoside (PAN model) directly associates with Src64B hyperactivation, suggesting that diaphragm stability is controlled by Src-dependent phosphorylation of diaphragm components. These findings indicate that the balance between diaphragm damage and repair is controlled by Src-dependent phosphorylation of diaphragm components, and point to Src family kinases as novel targets for the development of pharmacological therapies for the treatment of kidney diseases that affect the function of the glomerular filtration barrier (Tutor, 2013).

Many of the vertebrate slit diaphragm proteins are tyrosine phosphorylated in normal glomeruli. Furthermore, tyrosine phosphorylation of nephrin and Neph1 by the SFK Fyn is crucial for the stability of this protein complex and therefore for glomerular filtration function. Thus, upon phosphorylation, the cytoplasmic regions of nephrin and Neph1 recruit the intracellular adaptors Nck and Grb2, among others, that in turn regulate actin cytoskeleton reorganisation. This study found that the post-translational regulation by phosphorylation of Duf, the orthologue of Neph1, is conserved and that Src64B, a member of the non-receptor Src family tyrosine kinases, is responsible for Duf tyrosine phosphorylation in nephrocytes. Furthermore, Src64B function is necessary for the structural integrity of the filtration diaphragm and for normal nephrocyte morphology. In Src64B loss-of-function or knockdown conditions, there is a reduction in the density of filtration diaphragms at the nephrocyte cell membrane. Presumably, this is due in part to their internalisation, as suggested by the accumulation of both Duf and Pyd at protrusions extending inwards from the membrane and the presence in these locations of structures dense to electrons reminiscent of filtration diaphragms. In addition, delocalisation was observed of Duf/Pyd complexes to cell contact membranes that, at the ultrastructural level, are rich in adherens junctions. The presence of adherens junctions in apposed membranes is never found in wild-type mature nephrocytes but is characteristic of embryonic nephrocytes prior to the formation of filtration diaphragms. All these alterations are concomitant with changes in nephrocyte architecture, revealed by their smoother surface and their agglutination. As the loss of filtration diaphragms result in regression of labyrinthine channels, and this is always associated with nephrocyte agglutination, these morphological changes are interpreted as being due to reallocation of Duf/Pyd complexes from SD to adherens junctions. It is suggested that these morphological alterations are the nephrocyte equivalent of podocyte foot process effacement, a feature common to all proteinuric diseases and believed to be initiated by changes in the actin cytoskeleton (Tutor, 2013).

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

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