Increased levels of the cell cycle inhibitor protein, dacapo, accompany 20-hydroxyecdysone-induced G1 arrest in a mosquito cell line

When treated with the steroid hormone 20-hydroxyecdysone (20E), C7-10 cells from the mosquito, Aedes albopictus, arrest in the G1 phase of the cell cycle. To explore whether 20E-mediated cell cycle arrest proceeds through increased levels of cell cycle inhibitor (CKI) proteins, we cloned the Ae. albopictus homolog of dacapo, the single member of the Cip/Kip family of CKI proteins known from Drosophila melanogaster. The Ae. albopictus dacapo cDNA encoded a 261-amino acid homolog of the Aedes aegypti protein XP-001651102.1, which is encoded by an ∼23kb gene containing three exons. Like dacapo from D. melanogaster, the ∼27kDa protein from Aedes and Culex mosquitoes contained several S/TXXE/D motifs corresponding to potential protein kinase CK2 phosphorylation sites, and a binding site for proliferating cell nuclear antigen (PCNA). When extracts from cells treated with 20E were analyzed by western blotting, using a primary antibody to synthetic peptides from the mosquito dacapo protein, up-regulation of an ∼27kDa protein was observed within 24h, and the abundance of the protein further increased by 48h after hormone treatment. This is the first investigation of a cell cycle inhibitory protein in mosquitoes. The results reinforce growing evidence that 20E affects expression of proteins that regulate cell cycle progression.