Btk family kinase at 29A


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 partner (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).

Differential evolutionary wiring of the tyrosine kinase Btk

A central question within biology is how intracellular signaling pathways are maintained throughout evolution. Btk29A is considered to be the fly-homolog of the mammalian Bruton's tyrosine kinase (Btk), which is a non-receptor tyrosine-kinase of the Tec-family. In mammalian cells, there is a single transcript splice-form and the corresponding Btk-protein plays an important role for B-lymphocyte development with alterations within the human BTK gene causing the immunodeficiency disease X-linked agammaglobulinemia in man and a related disorder in mice. In contrast, the Drosophila Btk29A locus encodes two splice-variants, where the type 2-form is the more related to the mammalian Btk gene product displaying more than 80% homology. In Drosophila, Btk29A displays a dynamic pattern of expression through the embryonic to adult stages. Complete loss-of-function of both splice-forms is lethal, whereas selective absence of the type 2-form reduces the adult lifespan of the fly and causes developmental abnormalities in male genitalia. Out of 7004-7979 transcripts expressed in the four sample groups, 5587 (70-79%) were found in all four tissues and strains. This study investigated the role of Btk29A type 2 on a transcriptomic level in larval CNS and adult heads. Samples either selectively defective in Btk29A type 2 (Btk29AficPa) or revertant flies with restored Btk29A type 2-function (Btk29A(ic Exc1-16) were used. The whole transcriptomic profile for the different sample groups revealed Gene Ontology patterns reflecting lifespan abnormalities in adult head neuronal tissue, but not in larvae. It is concluded that in the Btk29A type 2-deficient strains there was no significant overlap between transcriptomic alterations in adult heads and larvae neuronal tissue, respectively. Moreover, there was no significant overlap of the transcriptomic changes between flies and mammals, suggesting that the evolutionary conservation is confined to components of the proximal signaling, whereas the corresponding, downstream transcriptional regulation has been differentially wired (Nawaz, 2012).

Btk29A promotes Wnt4 signaling in the niche to terminate germ cell proliferation in Drosophila

Btk29A is the Drosophila ortholog of the mammalian Bruton's tyrosine kinase (Btk), mutations of which in humans cause a heritable immunodeficiency disease. Btk29A mutations stabilized the proliferating cystoblast fate, leading to an ovarian tumor. This phenotype was rescued by overexpression of wild-type Btk29A and phenocopied by the interference of Wnt4-β-catenin signaling or its putative downstream nuclear protein Piwi in somatic escort cells. Btk29A and mammalian Btk directly phosphorylate tyrosine residues of β-catenin, leading to the up-regulation of its transcriptional activity. Thus, this study identified a transcriptional switch involving the kinase Btk29A/Btk and its phosphorylation target, β-catenin, which functions downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through up-regulation of piwi expression. This signaling mechanism likely represents a versatile developmental switch (Hamada-Kawaguchi, 2014).

Stem cell maintenance and differentiation are not entirely autonomic, but instead are under strict control by supporting cells that form the 'niche'. Recent studies in Drosophila have shown that the dynamics of Piwi and its associated piRNAs, a protein-RNA complex for gene silencing, are required in not only germ cells but also distinct niche-forming somatic cells (escort cells for germ cell development); however, their regulatory mechanisms remain largely unknown. This study identified a transcriptional switch involving the factor Bruton's tyrosine kinase (Btk) and its phosphorylation target, β-catenin, operating downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through modulation of piwi expression (Hamada-Kawaguchi, 2014).

Drosophila Btk29A type 2 is the ortholog of human BTK. The type 1 isoform is present and the type 2 is absent in Btk29AficP mutants. Germ stem cells (GSCs) and transit amplifying cystoblasts (CBs) are localized in the germarium situated at the anterior tip of an ovariole, posteriorly flanked by region 2, in which each CB divides twice and differentiates into cystocytes. The 16 cystocytes originating from a single CB remain interconnected by the fibrous structure fusome, a derivative of the spectrosome. GSCs and CBs both carry the spectrosome, a round, tubulin-enriched structure. The Btk29A mutant germarium contains significantly more germ cells than does the wild-type germanium. Although supernumerary cells were observed with spectrosomes in the Btk29AficP germarium, many of the excess cells appear to be cystocytes, as they were accompanied by a branched fusome structure. A large excess of cystocytes in grossly deformed ovarioles has been observed in female Drosophila that are mutant for mei-P26, a gene encoding a TRIM-NHL (tripartite motif and Ncl-1, HT2A, and Lin-41 domain) protein that binds to the argonaute protein Ago-1 for microRNA regulation. In mei-P26 mutants, an ovarian tumor 'cystocytoma' is formed because cystocytes regain the ability to self-renew after they enter the differentiation path. This suggests that mei-P26 normally terminates CB proliferation. Intriguingly, the following phenotypes of mei-P26 were recapitulated in Btk29AficP. First, phospho-histone H3-positive mitotic germline cells, which were restricted to the anterior tip of the wild-type germarium, were detected throughout the ovarioles. Second, the expression of Bam, a protein that induces differentiation of GSCs into CBs in the wild type, was markedly increased in CB-like GSC daughters. Third, oo18 RNA-binding protein (Orb) remained expressed in multiple cells in a cyst, contrasting to a wild-type cyst, where Orb expression becomes restricted to an oocyte (Hamada-Kawaguchi, 2014).

The reduction in mei-P26 transcription in Btk29AficP places mei-P26 downstream of Btk29A. Notably, mei-P26 functions cell-autonomously in germ cells. However, the almost complete rescue of germ cell defects in Btk29AficP was attained by overexpression of Btk29A+ type 2 via bab1-Gal4, which showed high levels of expression in terminal filament cells and cap cells (TF and CPC, respectively) and lower levels of expression in escort cells (EC). bab1-Gal4 was effective in inducing germ cell overproduction when used to knockdown Btk29A. hh-Gal4 with expression in the terminal filament cells and cap cells and c587-Gal4 with expression in escort cells were also used to target UAS-Btk29ARNAi expression; c587-Gal4, but not hh-Gal4, led to the overproduction of spectrosome-bearing cells, and therefore, the escort cells were considered as likely sites of Btk29A action. These observations imply that Btk29A is required in the escort cells for soma-to-germ signaling to control the switch from proliferation to differentiation in germ cells, where mei-P26 functions as a core component of the switch (Hamada-Kawaguchi, 2014).

Bone morphogenetic protein (BMP) signaling and piwi-dependent signaling compose two different pathways in the niche to control proliferation and differentiation of GSCs and their daughters. BMPs are secreted morphogens, and Piwi is an argonaute protein regulating gene expression. The Btk29AficP mutation abrogated piwi expression with little effect on decapentaplegic (dpp) or glass bottom boat (gbb) expression, two BMPs operating in the germarium, and the BMP downstream component Mothers against Dpp (Mad) was normally phosphorylated in Btk29AficP GSCs. Furthermore, somatic piwi knockdown mimicked the Btk29AficP ovarian phenotypes (Hamada-Kawaguchi, 2014).

Immunohistochemistry revealed that the Btk29AficP mutation or somatic Btk29A knockdown abrogated Piwi expression in the niche, but not in germ cells. This reduction in Piwi expression was reversed by the somatic Btk29A+ overexpression. Furthermore, the loss-of-function piwi allele dominantly enhanced the Btk29A mutant phenotype. Moreover, somatic overexpression of piwi+ in Btk29AficP alleviated the germ cell hypertrophy and reduced Bam expression to the normal level. It is therefore considered that Btk29A regulates the Piwi-dependent pathway in the niche to control germ cell proliferation (Hamada-Kawaguchi, 2014).

Piwi and piRNAs constitute a major transposon-silencing pathway. Somatic knockdown of Btk29A resulted in an increase in the expression of gypsy-lacZ that monitored the activity of the gypsy transposon. Also, transcript levels of the ZAM, DM412, and mdg1 transposons were significantly increased in Btk29AficP. It is therefore concluded that the Piwi deficiency due to the impairment of Btk29A results in derepression of transposon activities (Hamada-Kawaguchi, 2014).

Genome instability associated with transposon mobilization may lead to the activation of a DNA double-strand break (DSB) checkpoint. A mutation in DSB signaling, mnk, did not ameliorate the germ cell phenotype induced by somatic Btk29A knockdown, indicating that the germ cell hypertrophy by the Btk29A deficiency is not a consequence of the DSB checkpoint activation (Hamada-Kawaguchi, 2014).

Next, potential substrates of Btk29A in the niche were sought. Btk29A type 2 was enriched in the interface between cells where Drosophila melanogaster epithelial (DE)-cadherin and associated Arm, the β-catenin ortholog, are the major structural components. No sign were found of tyrosine phosphorylation of DE-cadherin, whereas Arm contained a high level of phosphotyrosine, which was almost entirely absent from Btk29AficP ovaries. However, Arm immunoprecipitated from Btk29AficP was strongly phosphorylated in vitro by the exposure of Arm to active Btk29A protein that had been immunoprecipitated from wild-type ovaries. These results demonstrate that Btk29A mediates the tyrosine phosphorylation of Arm in vivo (Hamada-Kawaguchi, 2014).

The anti-Arm labeling intensity of cell adhesion sites was stronger in Btk29AficP than in the wild type. Immunoprecipitation assays revealed that the relative amount of Arm associated with DE-cadherin was greater in Btk29AficP than in the wild type , suggesting that the tyrosine phosphorylation of Arm facilitates its release from the membrane to the cytoplasm, as in mammalian cells (Hamada-Kawaguchi, 2014).

Mammalian β-catenin is tyrosine-phosphorylated at residues Y86, Y142, and Y654. When transfected into mammalian Cos7 cells, Drosophila Btk29A type 2 phosphorylated all these tyrosine residues of β-catenin. Moreover, the antibodies against phosphorylated Y142 (anti-pY142) and anti-pY654 recognized Arm phosphorylated at the conserved site Y150 and Y667, respectively, in the immunoprecipitates from ovarian lysates (Hamada-Kawaguchi, 2014).

Expression of unphosphorylatable Arm-Y150F in the escort cells via c587-GAL4 or bab1-GAL4, but not hh-Gal4, induced germ cell hypertrophy, whereas another unphosphorylatable mutant, Y667F, or wild-type Arm exerted little effect. In addition, somatic arm knockdown resulted in an increase in spectrosome-containing cells, reduced piwi expression in escort cells, and increased Bam expression in germ cells. Considering these results together, it is proposed that Btk29A acts on Arm, which in turn regulates piwi in the niche (Hamada-Kawaguchi, 2014).

Arm functions in the canonical Wnt pathway. Therefore the ovaries of wg, Wnt2, Wnt4, and Wnt5 mutants were examined; the germ cell overproduction was detected only in Wnt4. Somatic knockdown of Wnt4 aided by bab1-GAL4 resulted in a reduction in the expression of Piwi, accompanied by an accumulation of germ cells carrying spectrosomes with an increase in germline Bam expression. These findings support the hypothesis that Arm in the escort cells regulates germ cell proliferation under the control of Wnt4, which was likely derived from somatic cells other than cap cells and terminal filament cells, as hh-GAL4 selective for these cells was least effective to induce germ cell overproduction when used to drive Wnt4RNAi expression (Hamada-Kawaguchi, 2014).

To evaluate the ability of Arm to activate transcription, T cell factor (TCF) reporter assays were used with Cos7 cells transiently transfected with human Btk (hBtk). The wild-type hBtk alone was sufficient to induce phosphorylation at Y142 and Y654 of β-catenin, whereas the kinase-dead hBtk (Btk-K430E) was not. Tyrosine phosphorylation of β-catenin was completely blocked by two antagonists of hBtk. Similarly, Btk29A type 2 phosphorylated Y142 and Y654 of mammalian β-catenin. Notably, the TCF reporter activity was six times as high when hBtk was transfected into Cos7 cells compared with the mock-transfected control, indicating that hBtk modulates the TCF-dependent transcriptional activation mechanism, in which Arm-β-catenin is involved as a coactivator (Hamada-Kawaguchi, 2014).

The expression of an arm-dependent Ubx-lacZ reporter was examined in the embryonic midgut. Btk29A knockdown abrogated the expression of this reporter, demonstrating that Btk29A supports Arm-dependent transcription in vivo (Hamada-Kawaguchi, 2014).

Btk29A was shown to phosphorylate Arm-β-catenin on conserved tyrosine residues, one of which (Arm-Y150) is pivotal for the niche function to prevent GSC daughters from overproliferating. Notably, most GSCs in Btk29A mutants do not express Bam (fig. S1R). This suggests that the presumptive Btk29A-Arm-Piwi pathway selectively regulates the proliferation of differentiating GSC daughters without interfering with GSC maintenance. Without Btk29A type 2, cystoblasts fail to exit the cell cycle, leading to the overproduction of germ cells, many of which are unable to complete differentiation and contribute to the genesis of an ovarian tumor (Hamada-Kawaguchi, 2014).

β-Catenin exerts multiple functions through its promiscuous binding abilities in cell-to-cell interactions and transcription. This protein plays critical roles in stem cell biology, and β-catenin malfunction results in a variety of cancers. These findings add a new dimension to the study of β-catenin by highlighting the pivotal role of the tyrosine phosphorylation of β-catenin in the control of transcription in the nucleus, in addition to the regulated control of the stability and motility of cell adhesion (Hamada-Kawaguchi, 2014).

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

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