Src oncogene at 64B
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).
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).
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).
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).
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 partnerç (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). Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
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