Autophosphorylation of DBT Occurs in its C-terminal Domain and is required for its Antiapoptotic Function. John C Means, Jin-Yuan Fan, Ed Bjes, Jeffrey Price. University of Missouri-Kansas City, Kansas City, MO.

   DOUBLETIME (DBT), the key circadian protein kinase responsible for PERIOD (PER) protein phosphorylation, undergoes autophosphorylation in Drosophila S2 cells. Several of the autophosphorylation sites were mapped to the C-terminal domain of DBT by mass spectrometry and analysis of DBT mutant proteins. In particular, a mutant form of DBT (DBT-Cala), in which 6 serines and threonines in a part of the C-terminal domain evolutionarily conserved in the Drosophilids are mutated to alanine, exhibits reduced autophosphorylation and enhanced stability in S2 cells treated with the general phosphatase inhibitor okadaic acid. Vertebrate orthologs of DBT (casein kinase I /) also autophosphorylate their C-terminal domains, leading to reduced kinase activity in vitro. However, autophosphorylated DBT does not exhibit reduced kinase activity in vitro. Studies addressing a role for DBT autophosphorylation in the circadian clock are thus far not conclusive. Analysis of DBT electrophoretic mobility in circadian mutants demonstrates an accumulation of slow-mobility forms of DBT, suggesting that alterations in circadian proteins with which DBT is known to interact can lead to autophosphorylation of DBT. Since autophosphorylation of DBT in S2 cells leads to reduced DBT levels, it is possible that it may trigger degradation of DBT in some circadian cells when its circadian partner protein (PER) is eliminated. The phosphatase(s) which normally antagonize DBT autophosphorylation are potentially important circadian clock components. In addition, DBTWT prevented UV induced apoptosis in S2 cells by direct binding and phosphorylation of caspases, with degradation of DBT also produced in response to UV. The catalytic activity of DBT is required to inhibit apoptosis, because expression of a catalytically inactive DBT dominant negative induced caspase activation. This antiapoptotic function was dependent on phosphorylation of the C-terminal domain of the protein, since the DBT-Cala was unable to protect S2 cells from apoptosis and was not degraded in response to UV treatment.