Fig. 4.

Phosphorylation in vitro of different SMRT subdomains by purified CK2. (A) ³²P-incorporation. GST fusion proteins representing different portions of SMRT, as indicated below the Fig., were synthesized in E. coli, were immobilized on glutathione-agarose, and were incubated with [γ-³²P] ATP in the presence (right panel) or absence (left panel) of a purified protein kinase CK2 preparation obtained from recombinant E. coli. The proteins were resolved by SDS-PAGE and the ³²P-radiolabel was visualized by PhosphorImager analysis. The positions of molecular weight markers are indicated on the left. The position of the most heavily phosphorylated SMRT protein is indicated by an arrow on the right. (B) Coommassie blue staining. The same SDS-PAGE employed for panel A was stained with Coommassie blue to visualize the locations and amounts of the different GST-SMRT polypeptides. The GST fusion polypeptides synthesized by each construct are indicated by arrows.