Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis

Abstract

DrosophilaDBT and vertebrate CKIε/δ phosphorylate theperiodprotein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBTC/ala) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce itsin vitroactivity. Moreover, overexpression of DBTC/aladid not affect circadian behavior differently from wild-type DBT (DBTWT), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBTWTprotected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBTC/aladid not protect and was not degraded. Finally, we show that the HSP-90 cochaperonespaghettiprotein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.