Broad-complex transcription factor mediates opposing hormonal regulation of two phylogenetically distant arginine kinase genes in Tribolium castaneum.

The phosphoarginine-arginine kinase shuttle system plays a critical role in maintaining insect cellular energy homeostasis. Insect molting and metamorphosis are coordinated by fluctuations of the ecdysteroid and juvenile hormone. However, the hormonal regulation of insect arginine kinases remain largely elusive. In this report, we comparatively characterized two arginine kinase genes, TcAK1 and TcAK2, in Tribolium castaneum. Functional analysis using RNAi showed that TcAK1 and TcAK2 play similar roles in adult fertility and stress response. TcAK1 was detected in cytoplasm including mitochondria, whereas TcAK2 was detected in cytoplasm excluding mitochondria. Interestingly, TcAK1 expression was negatively regulated by 20-hydroxyecdysone and positively by juvenile hormone, whereas TcAK2 was regulated by the opposite pattern. RNAi, dual-luciferase reporter assays and electrophoretic mobility shift assay further revealed that the opposite hormonal regulation of TcAK1 and TcAK2 was mediated by transcription factor Broad-Complex. Finally, relatively stable AK activities were observed during larval-pupal metamorphosis, which was generally consistent with the constant ATP levels. These results provide new insights into the mechanisms underlying the ATP homeostasis in insects by revealing opposite hormonal regulation of two phylogenetically distant arginine kinase genes.