Differential evolutionary wiring of the tyrosine kinase Btk.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: Out of 7004-7979 transcripts expressed in the four sample groups, 5587 (70-79%) were found in all four tissues and strains. Here, we investigated the role of Btk29A type 2 on a transcriptomic level in larval CNS and adult heads. We used samples either selectively defective in Btk29A type 2 (Btk29A(ficP)) or revertant flies with restored Btk29A type 2-function (Btk29A(fic Exc1-16)). 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. CONCLUSIONS: 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.

A new versatile gene-trap vector for insect transgenics.

A new piggyBac-based gene-trap vector, pB-GT1, was constructed. pB-GT1 contains three marker genes, dsRed, Gal4, and EGFP. dsRed is under the control of the constitutive 3xP3 promoter, which induces dsRed expression wherever the vector is inserted in the host genome. The Gal4 sequence has no promoter but is preceded by the splice acceptor site so that it can be transcribed as a transcript fused with the host exon 5' to the insertion site. EGFP is driven by the constitutive ie+hr promoter but lacks a poly(A)(+) signal sequence, and thus the EGFP expression is detectable only when its transcript is fused with the host exon 3' downstream of the insertion. By the microinjection of the vector into fertilized eggs, we obtained transgenic Drosophila with a single copy of pB-GT1, which was inserted into the first intron of the ovo gene. The female flies of this transgenic line are sterile, indicating that the insertion inactivated the ovo gene, generating a new allele of this locus, ovo(pB-GT1). RT-PCR analysis demonstrated that an ovo-Gal4-fusion transcript is produced in ovo(pB-GT1) flies. The fact that UAS-EGFP reporter expression was detected in ovo(pB-GT1) germ cells in a pattern similar to that reported for wild-type ovo indicates that functional Gal4 is expressed via pB-GT1, recapitulating the endogenous expression pattern of the trapped gene. pB-GT1 is thus useful in insect genomics for the efficient assignment of functions of individual genes.

Functional replacement of Drosophila Btk29A with human Btk in male genital development and survival.

Drosophila type 2 Btk29A reveals the highest homology to Btk among mammalian Tec kinases. In Btk29A(ficP) mutant males, the apodeme holding the penis split into two pieces. Human Btk rescued this phenotype in 39% of Btk29A(ficP) males, while the Drosophila transgenes did so in 90-100% of mutants. The Btk29A(ficP) mutation reduced adult longevity to 11% that of wild-type. This effect was counteracted by Drosophila type 2, yielding 76% of the wild-type lifespan. Human Btk extended the lifespan of Btk29A(ficP) mutants only to 20% that of wild-type. Thus human Btk can partially replace Drosophila Btk29A+ in male genital development and survival.

Ras target protein canoe is a substrate for Cdc2 and Cdk5 kinases.

Mutations in the canoe locus of Drosophila lead to failure in the dorsal closure of the embryonic epidermis and pattern formation defects in imaginal eyes and wings. In the wing, the canoe mutants develop extra veins when they are heterozygous for shaggy, a mutation in the locus encoding the glycogen synthase kinase 3 beta (Gsk3 beta), which has been known to phosphorylate the Armadillo protein. Although Canoe has a putative target sequence for phosphorylation by Gsk3 beta similar to that found in Armadillo, in vitro experiments indicate that Canoe is not phosphorylated by Gsk3 beta . Instead, Canoe is demonstrated to be a good substrate of Cdc2 and Cdk5 kinases. Thus, Cdc2 and Cdk5 kinases are the potential regulators of the function of Canoe in morphogenesis. Arch.