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

• Published in: Molecular and cellular biology 2015-07, Vol.35 (14), p.2414-2424
• Main Authors: Fan, Jin-Yuan, Means, John C., Bjes, Edward S., Price, Jeffrey L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Apoptosis - physiology; Apoptosis - radiation effects; Casein Kinase Iepsilon - antagonists & inhibitors; Casein Kinase Iepsilon - genetics; Casein Kinase Iepsilon - metabolism; Casein Kinase Iepsilon - physiology; Cell Line; Circadian Rhythm - physiology; Drosophila; Drosophila melanogaster; Drosophila Proteins - antagonists & inhibitors; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Drosophila Proteins - physiology; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Molecular Sequence Data; Mutation; Phosphorylation; Protein Kinase Inhibitors - metabolism; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Serine - metabolism; Threonine - metabolism; Ultraviolet Rays
• ISSN: 1098-5549; 0270-7306; 1098-5549
• DOI: 10.1128/MCB.00390-15

Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (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 (DBT C/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 its in vitro activity. Moreover, overexpression of DBT C/ala did not affect circadian behavior differently from wild-type DBT (DBT WT ), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT WT protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT C/ala did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (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.