FIG. 9.
Expression of DBT in S2 cells produces a CKI activity which is reduced by the dbtS and dbtL mutations. MYC-tagged DBT was transiently coexpressed in a Drosophila S2 cell line from a metallothionein promoter, which was induced with 0.5 mM CuSO4 for 40 h, with HA-tagged-PER expressed from the actin promoter. The expressed DBT protein was immunoprecipitated with an anti-MYC antibody. (A) Recovery of the tagged DBT was assessed by immunoblot analysis of the reactions analyzed in panel B. The rat anti-DBTCim antibody was employed for detection, and the anti-rat IgG secondary antibody employed for chemiluminescent detection weakly cross-reacted with the mouse anti-MYC antibody (IgG) in the immunoprecipitate. Coexpression of PER and DBT in Drosophila S2 cells produced higher levels of mutant DBT (S, DBTS; L, DBTL) than wild-type DBT (W) in these immunoprecipitates. The signal was specific for tagged DBT produced by the transgene, because expression of untagged DBT from the same metallothionein promoter (“no tag”) did not produce a DBT antigen in the immunoprecipiate. (B) Phosphorylation of casein by the immunoprecipitates assayed in panel A was assessed in the same order as panel A, as described for Fig. 7. The dbtS and dbtL mutations both reduced the activity of the immunoprecipitated enzyme, while no activity was detected in the immunoprecipitates of extracts containing untagged DBT. Note that the lower activity for DBTS and the equivalent activity for DBTL relative to DBT+ were both produced by recovered enzyme levels that were higher than the wild-type level used in this experiment, as shown in panel A. (C) Wild-type DBT-MYC expressed alone (i.e., without PER) was immunoprecipitated. It phosphorylated casein and was sensitive to CKI-7 (DBT+DMSO+CKI-7) in comparison with the vehicle alone (DBT+DMSO), demonstrating that it was bonafide CKI activity.