Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis.
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.
University of Missouri-Kansas City, School of Biological Sciences, Kansas City, Missouri, USA.
Jin-Yuan
JY
Fan
University of Missouri-Kansas City, School of Biological Sciences, Kansas City, Missouri, USA University of Missouri-Kansas City, School of Medicine, Kansas City, Missouri, USA.
John C
JC
Means
University of Missouri-Kansas City, School of Biological Sciences, Kansas City, Missouri, USA.
Edward S
ES
Bjes
University of Missouri-Kansas City, School of Biological Sciences, Kansas City, Missouri, USA University of Missouri-Kansas City, School of Medicine, Kansas City, Missouri, USA pricejL@umkc.edu.
Jeffrey L
JL
Price
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects; D048149:Casein Kinase 1 epsilon / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D002940:Circadian Rhythm / Q000502:physiology; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004331:Drosophila melanogaster; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014466:Ultraviolet Rays
D029721:Drosophila Proteins; D018832:Molecular Chaperones; D047428:Protein Kinase Inhibitors; C000601321:SPAG protein, Drosophila; C113519:dco protein, Drosophila; D013912:Threonine; D012694:Serine; D048149:Casein Kinase 1 epsilon