cDNA cloning of calcineurin heterosubunits from the pheromone gland of the silkmoth, Bombyx mori

Abstract

Pheromone biosynthesis activating neuropeptide (PBAN) stimulates the step of fatty acyl reduction in the pheromone biosynthetic pathway of the silkmoth, Bombyx mori. It has been suggested that the intracellular signal transduction of PBAN in B. mori involves Ca(2+), calmodulin, and calcineurin (also known as protein phosphatase 2B). We have cloned two cDNAs encoding calcineurin heterosubunits from a pheromone gland cDNA library of B. mori. The 2,996-bp clone predicts a 495-amino acid protein homologous to the catalytic subunit calcineurin A (CnA) with a molecular mass of 55,968. The deduced amino acid sequence well conserves the calcineurin B (CnB)-binding domain and two subdomains, a calmodulin-binding and an autoinhibitory domain, showing 77-85% and 82% identities to the isoforms of Drosophila melanogaster CnA and human CnA, respectively. On the other hand, the 820-bp clone predicts a 170-amino acid protein homologous to the regulatory subunit CnB with a molecular mass of 19,357. The deduced amino acid sequence well conserves four EF-hand type calcium-binding structures, showing 95% and about 85% identities to D. melanogaster CnB and mammalian CnBs, respectively. A yeast two-hybrid system has demonstrated the molecular interaction between B. mori CnA and CnB. Northern blot analyses revealed that both CnA and CnB genes were expressed in various larval and adult tissues of B. mori. Both transcripts detected in the pheromone gland three days before adult eclosion increased by the day before eclosion and the mRNA levels were found to be high even two days after adult eclosion. Immunohistochemical analysis has revealed that B. mori calcineurin is localized in the cytoplasm of the pheromone-producing cells.