Genetic investigation of cAMP-dependent protein kinase function in Drosophila development.

Abstract

The cAMP-dependent protein kinase (PKA) has been shown to mediate the vast majority of cellular responses to the intracellular second messenger, cAMP, in eukaryotes. To study the role of cAMP signal transduction in Drosophila development, we have isolated and molecularly characterized mutations of varying severity in the Drosophila PKA gene, DC0. Biochemical measurements indicate that DC0 is either the sole or the major PKA catalytic subunit gene in Drosophila. Adult females heterozygous for a strong and a weak DC0 allele fail to lay eggs and show a striking and novel defect in oogenesis that includes the formation of egg chambers containing multinucleate nurse cells. Females heterozygous for two weak DC0 alleles are fertile but produce offspring showing a variety of defects in embryogenesis, including preblastoderm arrest and alterations in cuticular patterning. Animals zygotically null for DC0 die as morphologically normal first-instar larvae, implying that maternally encoded protein, which perdures for at least 12 hr, suffices for embryogenesis. Animals hemizygous for weak DC0 alleles survive for several days as larvae but grow slowly. Mitotic recombination experiments in the adult eye indicate that the DC0 gene is not required autonomously either for cell viability or normal growth rates. These results argue that cAMP-mediated signal transduction is essential at a variety of stages during the development of a metazoan.

Protein kinase A inhibits a consolidated form of memory in Drosophila

“…All DCO alleles assayed in this article, DC0 B3 , DC0 H2 , DC0 H3 , DCO B10 , and DC0 B12 , are homozygous-lethal, and PKA assays of head extracts of heterozygotes of all 5 alleles show a reduction of PKA activity to Ϸ50% of wild-type activity as assayed biochemically from crude extracts (20,24). However, previous studies have revealed notable differences between these different alleles.…”

“…In a previous study, we determined that heterozygotes of lethal point mutations in DC0, the gene encoding the major catalytic subunit of cAMP-dependent protein kinase (PKAc), dramatically delay AMI onset (20). These heterozygotes all reduce PKA activity to Ϸ50% of wild type and delay AMI onset Ͼ2-fold (20,24). While characterizing these mutants further, we made the startling observation that some of them also improve memory retention in young flies.…”

“…However, previous studies have revealed notable differences between these different alleles. DC0 B3 and DC0 H2 have been characterized as null and strong alleles, respectively, whereas DC0 H3 , DC0 B10 , and DC0 B12 have been classified as weaker alleles (24). The DC0 B3 allele contains a nonsense mutation that truncates the last 54 aa of PKAc, whereas the DC0 H2 allele contains a missense mutation that changes a nonpolar amino acid to a charged residue (Gly 203 3 Asp).…”

“…DC0 H3 , DC0 B10 , and DC0 B12 (data not shown) are classified as medium and weak alleles based on complementation studies and the lethal phase of hemizygotes. Severity of alleles is based on classification by Lane and Kalderon (24). Note that the null allele improves memory to a greater extent than the strong allele, whereas the medium and weak alleles do not improve memory at all.…”

Development of apical membrane organization and V-ATPase regulation in blowfly salivary glands

“…Antisera against the following proteins were used: bovine VATPase subunit A and subunit E (Roussa et al, 1998), Culex quinquefasciatus V-ATPase subunit B (Filippova et al, 1998), Manduca sexta V-ATPase subunits d, C and D (Merzendorfer et al, 2000;Tiburcy et al, 2013) and Drosophila melanogaster PKA catalytic subunit and regulatory subunit II (Lane and Kalderon, 1993;Skoulakis et al, 1993). Cross-reactivity of these antibodies with the homologous proteins in C. vicina has been demonstrated previously (Zimmermann et al, 2003;Dames et al, 2006;Voss et al, 2007;Voss et al, 2009;Baumann and Walz, 2012).…”