Btk family kinase at 29A : Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Btk family kinase at 29A

Synonyms - Tec29A

Cytological map position - 29A1--3

Function - signaling

Keywords - salivary gland morphogenesis, dorsal closure, ovarian ring canal formation, oncogene

Symbol - Btk29A

FlyBase ID: FBgn0003502

Genetic map position - 2L

Classification - protein-tyrosine kinase, SH2 motif, SH3 domain, Pleckstrin homology

Cellular location - cytoplasmic



NCBI link: EntrezGene
Btk29A orthologs: Biolitmine
Recent literature
Sunouchi, K., Koganezawa, M. and Yamamoto, D. (2016). Requirement of the tec family tyrosine kinase Btk29a for courtship memory in Drosophila males. Arch Insect Biochem Physiol [Epub ahead of print]. PubMed ID: 26782301
Summary:
A male Drosophila that is not successful in courtship will reduce his courtship efforts in the next encounter with a female. This courtship suppression persists for more than 1 h in wild-type males. The Btk29AficP mutant males null for the Btk29A type 2 isoform, a fly homolog of the nonreceptor tyrosine kinase Btk, show no courtship suppression, while Btk29A hypomorphic males exhibit a rapid decline in courtship suppression, leading to its complete loss within 30 min. The males of a revertant stock or Btk29AficP males that are also mutant for parkas, a gene encoding the presumptive negative regulator of Btk29A, exhibit normal courtship suppression. Since another behavioral assay has shown that Btk29AficP mutants are sensitization-defective, it is hypothesized that the mutant flies are unable to maintain the neural excitation state acquired by experience, resulting in the rapid loss of courtship suppression.
Hamada-Kawaguchi, N. and Yamamoto, D. (2017). Ovarian polarity and cell shape determination by Btk29A in Drosophila. Genesis [Epub ahead of print]. PubMed ID: 28639397
Summary:
Drosophila Btk29A is a Tec family nonreceptor tyrosine kinase, the ortholog of which causes X-linked agammagluburinemia in humans when mutant. In Btk29AficP mutant ovaries, multiple defects are observed: extra polar cells form ectopically; osk mRNA fails to accumulate posteriorly in mature oocytes; the shape and alignment of follicle cells are grossly distorted. All these phenotypes are rescued by selectively overexpressing the type 2 isoform of wild-type Btk29A in follicle cells. Expression of certain proteins enriched in adherens junctions is markedly affected in Btk29AficP mutants; the anterior-posterior gradient normally observed in the expression of DE-Cadherin and Armadillo are lost and Canoe is sequestered from adherens junctions. Intriguingly, tyrosine phosphorylation of Canoe is reduced in Btk29AficP mutants. It is proposed that Btk29A is required for the establishment of egg chamber polarity presumably through the regulation of subcellular localization of its downstream proteins, including Cno.
Roussou, I. G., Papanikolopoulou, K., Savakis, C. and Skoulakis, E. M. C. (2019). Drosophila Bruton's Tyrosine Kinase regulates habituation latency and facilitation in distinct mushroom body neurons. J Neurosci. PubMed ID: 31530645
Summary:
Habituation is the adaptive behavioral outcome of processes engaged in timely devaluation of non-reinforced repetitive stimuli, but the neuronal circuits and molecular mechanisms that underlie them are not well-understood. To gain insights into these processes this study developed and characterized a habituation assay to repetitive footshocks in mixed sex Drosophila groups, and has demonstrated that acute neurotransmission from adult alpha/beta mushroom body (MB) neurons prevents premature stimulus devaluation. This study demonstrates that activity of the non-receptor Tyrosine kinase Btk protein is required within these neurons to prevent premature habituation. Significantly, it was also demonstrated that the complementary process of timely habituation to the repetitive stimulation is facilitated by alpha /beta MB neurons and also requires Btk activity. Hence the results provide initial insights into molecular mechanisms engaged in footshock habituation within distinct MB neurons. Importantly, Btk attenuation specifically within alpha /beta neurons leads to defective habituation, which is readily reversible by administration of the antipsychotics Clozapine and Risperidone suggesting that the loss of the kinase may dysregulate monoamine receptors within these neurons, whose activity underlies the failure to habituate.
Hamada-Kawaguchi, N., Nore, B. F., Zain, R., Engstrom, Y., Smith, C. I. E. and Yamamoto, D. (2023). Exposure to Therapeutic BTK Inhibitors Induces Phenocopying of Btk29A Mutants in the Fruit Fly Drosophila melanogaster. Front Biosci (Landmark Ed) 28(6): 124. PubMed ID: 37395037
Summary:
Bruton's tyrosine kinase (BTK) is a non-receptor type tyrosine kinase originally identified as the genetic signature responsible for X-linked agammaglobulinemia (XLA) when mutated. Its functional form is required for B lymphocyte maturation in both humans and mice, whereas loss-of-function causes a different form of developmental defect in the fruit fly, Drosophila melanogaster. Ibrutinib and other therapeutic inhibitors of BTK have been extensively used to successfully treat various leukemias and lymphomas. Btk29A type 2 is the ortholog of BTK in the fruit fly. Feeding wild-type flies an ibrutinib-containing diet induces phenocopying of Btk29A mutants, i.e., failure in the fusion of left and right halves of the dorsal cuticles, partial loss of wing tissues and dysregulation of germ cell production. It has been previously reported that Btk29A phosphorylates Drosophila Arm (β-catenin), and ibrutinib reduces phosphorylation at Tyrosine142 of endogenously expressed β-catenin in Cos7 cells transfected with Btk29A type 2 cDNA. Thus, Drosophila is suitable for screens of novel BTK inhibitor candidates and offers a unique in vivo system in which the mode of action of BTK inhibitors can be examined at the molecular, cellular, and organismal levels.
BIOLOGICAL OVERVIEW

Epithelial invagination is necessary for formation of many tubular organs, one of which is the Drosophila embryonic salivary gland. Actin reorganization and control of endocycle entry are crucial for normal invagination of the salivary placodes. Embryos mutant for Tec29 (Flybase designation - Btk29A), the Drosophila Tec family tyrosine kinase, show delayed invagination of the salivary placodes. This invagination delay is partly the result of an accumulation of G-actin in the salivary placodes, indicating that Tec29 is necessary for maintaining the equilibrium between monomeric actin (G-actin) and filamentous actin (F-actin) during invagination of the salivary placodes. Furthermore, normal invagination of the salivary placodes appears to require the proper timing of the endocycle in these cells; Tec29 must delay DNA endoreplication in the salivary placode cells until they have invaginated into the embryo. Taken together, these results show that Tec29 regulates both the actin cytoskeleton and the cell cycle to facilitate the morphogenesis of the embryonic salivary glands. It is suggested that apical constriction of the actin cytoskeleton may provide a temporal cue ensuring that endoreplication does not begin until the cells have finished invagination.

Tec29 has also been demonstrated to be a key downstream effector of Src64 during ring canal growth (Guarnieri, 1998; Roulier, 1998). Mutations in Tec29 enhance the ring canal size defect seen in animals with reduced Src64 expression. Src64 and Tec29 mutants also share similar phenotypes: both have small ring canals that lack phosphotyrosine content. Tec29 protein is localized to ring canals in an Src64-dependent manner, implying that Src64 activity leads to the recruitment of Tec29 to ring canals, which may then mediate the function of Src64 by phosphorylating target proteins on ring canals. Therefore, elucidating the mechanism by which Tec29 localizes to ring canals, and demonstrating how Src64 regulates this localization, have proven essential for understanding how Src64 and Tec29 coordinate to regulate actin networks in Drosophila (Lu, 2004 and references therein).

Invagination of epithelial sheets is an important process during gastrulation of many organisms and the formation of tissues such as the vertebrate lung, gut, insect trachea and the salivary glands. During invagination, the cells specified to be particular tissues break down their cell adhesion with the rest of the epithelium, reorganize their cytoskeleton and apically constrict to ingress into the embryo. The process of invagination has been extensively studied during ventral furrow formation in Drosophila embryos. During this process, cells close to the ventral midline constrict apically to produce a shallow groove, which is deepened by cell shortening along the apicobasal axis. Prior to apical constriction, nonmuscle myosin II and ßH spectrin are translocated to the apical side of the ventral furrow cells. Disruption of this accumulation prevents ventral furrow formation, suggesting that actin-myosin contractility is required for apical constriction. In addition to the cytoskeletal changes, ventral furrow formation requires coordination between the cell cycle and invagination. Normally, ventral furrow cells delay mitosis until after they are inside the embryo. As shown by tribbles mutants, premature entry into mitosis before invagination prevents ventral furrow formation. Since invagination is a fundamental process used repeatedly during organogenesis, it is of interest to understand whether the process of invagination and its genetic control are similar in other epithelial tissues (Chandrasekaran, 2005 and references therein).

The embryonic salivary glands of Drosophila provide a good system to study epithelial invagination. The salivary glands are derived from a disc of columnar, postmitotic epithelial cells in parasegment 2 of the Drosophila embryo known as the salivary placodes. The salivary placodes are specified at stage 10 by three positive regulators – the homeotic gene Sex combs reduced, extradenticle and homothorax (reviewed by Abrams, 2003). The dorsal and ventral boundaries of the salivary placodes are determined by the decapentaplegic and Egfr signaling pathways, respectively. Following their specification, the salivary placodes invaginate into the embryo to form the tubular salivary glands. The process of invagination begins with a wave of apical constriction and basal movement of nuclei that progresses from the dorsal posterior cells to the dorsal anterior cells and finally to the ventral cells of the salivary placodes. The order of invagination follows the apical constriction wave, beginning with the dorsal posterior cells, followed by the dorsal anterior cells and then the ventral cells. This sequential internalization of the cells results in a narrow tubular gland where the first invaginated cells form the distal tip of the gland and the last cells to ingress into the embryo form the proximal part of the salivary gland (Chandrasekaran, 2005 and references therein).

The genetic control of salivary invagination is beginning to be understood. Interestingly, at least one of the signaling pathways used during ventral furrow formation is also involved in salivary invagination. Signaling by folded gastrulation (fog) activates RhoGEF2, a RhoGTPase exchange factor, in the ventral furrow and the salivary placodes. Both fog and RhoGEF2 are necessary for the invagination of the ventral furrow and the salivary glands (Nikolaidou, 2004). In the ventral furrow, RhoGEF2 causes the apical myosin II localization that is necessary for invagination of the ventral furrow (Nikolaidou, 2004). It is therefore possible that RhoGEF2 has a similar function in the salivary glands, thereby facilitating apical constriction of the placode cells. In addition to the fog-RhoGEF2 signaling pathway, the apical constriction of the salivary placodes cells requires fork head, a winged helix transcription factor; in its absence, the salivary primordium fails to invaginate (Chandrasekaran, 2005).

Once the salivary placode cells invaginate into the embryo, they enter a modified cell cycle known as the endoreplication cycle or endocycle, in which the cells alternate between G and S phase without cell division, leading to an increase in ploidy. The salivary gland cells are the first cells to enter the endocycle in the embryo, and endoreplication in the gland reliably progresses from the distal tip of the gland to the proximal part during stages 12-14. Thus, the wave of endoreplication in the invaginated glands follows the same order as the preceding wave of apical constriction and invagination. This endoreplication is the first of many in the salivary cells, leading to the giant polytene chromosomes present in mature larvae (Chandrasekaran, 2005).

The early events in salivary morphogenesis, including the localized initiation of invagination, the orderly progression of invagination to other placode cells and the beginning of endoreplication, appear to be carefully coordinated. Thus far, however, the mechanisms coordinating these processes have remained elusive (Chandrasekaran, 2005).

This study finds that the Tec29 tyrosine kinase is necessary to coordinate two essential processes: actin cytoskeletal organization and regulation of the cell cycle. Tec29 is a member of the Tec family of non receptor tyrosine kinases, which includes BTK, TEC, ITK, ETK and TXK (reviewed by Mano, 1999). Mutations in BTK are known to cause X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. The human disorder results from the absence of mature B lymphocytes, whereas mice with the immunodeficiency have abnormal B cells (Maas, 2001; Satterthwaite, 2000). Other Tec kinases regulate many processes during development of lymphocytes, including cell cycle, cell death, cell adhesion and migration (Mano, 1999; Takesono, 2002). By contrast, the Drosophila Tec kinase Tec29 has only been linked to the actin cytoskeleton during Drosophila embryogenesis and oogenesis (Guarnieri, 1998; Roulier, 1998; Tateno, 2000; Thomas, 2004). Tec29 is needed for actin filament reorganization and bundling of actin during early cellularization and dorsal closure in embryos, as well as in the ring canals of the ovary. The current study shows that lack of Tec29 causes a delay in invagination of the salivary glands because of a shift in the equilibrium between F-actin and G-actin, and because of premature endoreplication in the salivary placode cells. Thus, like ventral furrow formation, invagination of the salivary placodes requires both the reorganization of the actinmyosin cytoskeleton and a cell cycle delay (Chandrasekaran, 2005).

Salivary placode cells must constrict apically and move their nuclei basally in order to invaginate into the embryo (Myat, 2000). Therefore, the process of salivary gland invagination is expected to require actin-myosin contractility. Tec29 is responsible for regulating the actin reorganization in the salivary placodes prior to invagination. Lack of Tec29 in the salivary placodes results in a shift of actin in the salivary placodes from F-actin to more G-actin, leading to incomplete invagination of the salivary placodes cells. This change in the balance between F-actin and G-actin is observed only on the apical surfaces of these cells, suggesting that the delayed invagination is due to aberrant apical constriction of the salivary placodes cells in Tec29 embryos. Increasing actin depolymerization further in Tec29 mutants by a mutation in chic increases the salivary invagination delay, whereas promoting actin polymerization in Tec29 mutants by mutating twinstar partially rescues the invagination delay. Thus, these results provide the first evidence that actin reorganization is necessary for salivary gland invagination (Chandrasekaran, 2005).

The shift in the equilibrium towards more G-actin in Tec29 mutants might be due either to decreased polymerization of actin or decreased stability of F-actin. Tec kinases affect both actin polymerization and actin crosslinking in vertebrates and in Drosophila (Finkelstein, 2004). ITK-deficient mice show impaired actin polymerization in response to T-cell activation (Labno, 2003). In addition, both BTK and ITK interact with WASP, a protein that can activate the Arp2/3 complex and promote new actin filament formation (Baba, 1999b; Bunnell, 1996; Guinamarda, 1998). During early Drosophila oogenesis, accumulation of G-actin has been observed in the cortex of Tec29 mutants, leading to defective fusome formation (Djagaeva, 2005). In addition, Tec29 has been suggested to affect actin crosslinking by phosphorylating KELCH, an actin-bundling protein in the ring canals of the ovary. The phosphorylation of KELCH is necessary to decrease actin crosslinking, thereby allowing expansion of the ring canals (Kelso, 2002). It is therefore possible that in the salivary placodes, Tec29 might be needed for new F-actin formation or bundling of the actin filaments to promote invagination (Chandrasekaran, 2005).

Besides salivary gland invagination, Tec29 affects actin organization during two other processes in Drosophila embryogenesis: dorsal closure and cellularization. Tec29 in conjunction with Src42A is necessary for dorsal closure and lack of both these kinases results in a dorsal open phenotype. So, during dorsal closure as with salivary invagination, there is a positive interaction between Tec29 and Src42A. However, Src42 is the main player during dorsal closure, whereas Tec29 is more important for salivary invagination. Interestingly, there is a reduction of F-actin observed at the leading edge of the dorsal epidermal cells in these double mutants, causing delayed dorsal closure (Tateno, 2000). Thus, the lack of these tyrosine kinases causes changes in actin dynamics that result in delayed migration of both the salivary placodes and during dorsal closure. Similarly, the process of basal closure during cellularization resembles apical constriction prior to invagination and requires an actin-myosin based contraction at the base of the cellularization front. In embryos arising from Tec29 germline clones, the actin microfilament ring does not contract, resulting in membrane invagination of varying depths and impairment of basal closure during late cellularization (Thomas, 2004). It is possible that, similar to the salivary invagination and dorsal closure, the absence of the contractile ring during cellularization is due to decreased F-actin and/or increased G-actin. In general, Tec29 appears to be needed for regulation of actin during periods of extensive and rapid reorganization of the actin cytoskeleton as observed during migration and contraction of cells (Chandrasekaran, 2005).

Although Tec kinases are known to alter the actin cytoskeleton in many systems, this study is the first to shown a relationship between Tec29 and the endocycle. The data support a previous observation that the salivary placodes in wild-type embryos enter endoreplication only after they invaginate into the embryo. In Tec29 mutants, however, the wave of endoreplication is disrupted, such that the ventral cells in the placodes initiate endoreplication prior to invagination. As a result, these cells fail to invaginate on schedule, resulting in the long salivary glands. Therefore, delaying endoreplication appears to be necessary to allow invagination, and coordinating the two events is crucial for normal development of the salivary glands. Similar coordination between the cell cycle and morphogenesis is observed during ventral furrow formation in Drosophila embryos and in the paraxial mesoderm in Xenopus embryos. In the ventral furrow and the paraxial mesoderm, the cell cycle delay is established by maintaining the Cdks in their phosphorylated form. In the ventral furrow, this is accomplished by tribbles inhibiting the Drosophila Cdc25 homolog string, a protein that dephosphorylates and activates Cdks. In the paraxial mesoderm, the localized phosphorylation of Cdks by Wee2 is sufficient to prevent the cells from entering mitosis prior to invagination. In both these cases, as in the salivary gland, the coordination between cell cycle and invagination is crucial for normal morphogenesis of the embryo. An important difference is that unlike the salivary glands, it is the mitotic cycle that is regulated with invagination of the ventral furrow and the paraxial mesoderm. This study provides the first indication of a link between the endocycle and morphogenesis, and suggests that coordination of endoreplication with invagination is crucial for normal development (Chandrasekaran, 2005 and references therein).

As shown in the ventral furrow and the paraxial mesoderm, mitosis and invagination use the same cytoskeletal components and require opposing levels of cell adhesion, making these two processes incompatible with each other. However, a normal endocycle in flies does not involve nuclear membrane breakdown or other processes that might require the same cytoskeletal components as invagination. Thus, a normal endocycle might not interfere with invagination. But this may not be a normal endocycle. Unlike the other endocycling cells in the embryo, which enter the endocycle from G1, it has been suggested that the salivary placodes may enter the endocycle from G2. It is therefore possible that the endocycle in the salivary placodes retains some aspects of mitosis that would interfere with invagination. If so, this endocycle would be similar to those in some endocycling mammalian cells that enter the endoreplication cycle after G2 during early M phase. Perhaps because this first salivary gland endocycle is unusual, it must be delayed until after the salivary cells are safely inside the embryo (Chandrasekaran, 2005).

The data indicate that Tec29 is necessary for actin remodeling during apical constriction in the salivary placodes and for endocycle progression as the glands invaginate. In addition, manipulating the actin cytoskeleton in Tec29 mutants can have effects on endoreplication in these cells. When the actin defects are enhanced by eliminating chic in Tec29 embryos, there is also an enhancement of the endoreplication defects of Tec29, suggesting that the actin cytoskeleton and endoreplication cycle are linked. The following model is proposed to explain how these two events might be coordinated in the salivary placodes. The actin remodeling during apical constriction and endoreplication follow each other such that the first cells to apically constrict are the first cells to invaginate and endoreplicate, and the remaining cells follow in sequence. These processes might be causally linked; the apical constriction wave might trigger the wave of endoreplication. This coupling and a lag between the two events would ensure that endoreplication does not occur prematurely, while the cells are still on the surface of the embryo. With this model, Tec29 would then regulate the endocycle indirectly, rather than independently regulating both actin and endoreplication. Disruption of the apical constriction wave in Tec29 mutants would lead to subsequent disruption of the endoreplication wave. An invagination delay due to inadequate actin polymerization would be compounded by placode cells endoreplicating prior to invagination. This model also explains the ability of cyclin E to partially rescue the Tec29 phenotype. Cyclin E overexpression would delay endoreplication and relieve some of the effects of inadequate actin polymerization. Studies in progress to identify TEC29 targets are likely to provide further insight into its effects on endoreplication and invagination, and should enable better understanding of the link between these processes (Chandrasekaran, 2005).


GENE STRUCTURE

cDNA clone length - 3618 (type 2, which includes the PH domain)

Bases in 5' UTR - 234

Exons - 12

Bases in 3' UTR - 1023

PROTEIN STRUCTURE

Amino Acids - 785

Structural Domains

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 (Gregory, 1987).

Most Tec family kinases have an N-terminal pleckstrin homology (PH) domain, followed by Tec homology (TH), SH2, SH3 and kinase domains. In Drosophila, there are two isoforms of Tec29 RNA. The longer form (type 2) is similar to the vertebrate Tec kinases and includes the PH domain, whereas the shorter type 1 isoform lacks the PH and part of the TH domain at its N terminus (Baba, 1999a)


Btk family kinase at 29A : Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 November 2023

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