Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

Abstract

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner.

Deregulation of Translational Control of the 65-kDa Regulatory Subunit (PR65α) of Protein Phosphatase 2A Leads to Multinucleated Cells

“…We checked whether a stem-loop structure occurred in the 5Ј-UTR of the Drosophila PR65␣ mRNA (27). Residues Ϫ194 to Ϫ47 have indeed the potential to fold as a stem-loop, but this structure is less stable (⌬G ϭ -27.1 kcal/mol).…”

Protein phosphatase 2A negatively regulates aPKC signaling by modulating phosphorylation of Par-6 inDrosophilaneuroblast asymmetric divisions

“…PP2A holoenzyme functions as a heterotrimeric complex comprising a catalytic C subunit [Microtubule star (Mts) in Drosophila (Snaith et al, 1996)], a scaffolding A subunit [PP2A-29B in Drosophila (Mayer-Jaekel et al, 1992)] and a regulatory B-subunit [Twins (Tws) (Shiomi et al, 1994;Uemura et al, 1993), Widerborst (Wdb) (Hannus et al, 2002) and PP2A-BЈ in Drosophila]. The A-subunit can serve as a linker between the C-and B-subunits, and the B-subunit can influence the enzymatic activity and substrate specificity of the holoenzyme.…”

Saccharomyces cerevisiae Homologs of Mammalian B and B′ Subunits of Protein Phosphatase 2A Direct the Enzyme to Distinct Cellular Functions

“…The regulatory A subunit has been identified in cells of diverse origin. Two isoforms of the A subunits have been characterized in mammalian cells (15) and one in Drosophila (16). In S. cerevisiae, the TPD3 gene encodes a homolog of the mammalian subunit (17).…”