FIG. 6.
Ser109 phosphorylation of CARMA1 with CaMKII. (A) Purified CARMA1 and CARMA3 were phosphorylated in vitro by wild-type CaMKII. (B) Purified CARMA1 and the CARMA1 S109A mutant were phosphorylated in vitro in the presence and absence of a CaMKII inhibitor as indicated. The amount of CARMA1 was determined by Coomassie staining, and then the dried gels were exposed to a phosphorimager. The remainder of each sample was analyzed by Western blotting using an antibody against CaMKII (lower panel). (C) 293T cells were transfected with Myc-CARMA1 or its S109A mutant and CaMKII1-290. The cells were lysed 18 h later. Monoclonal anti-Myc antibody was used for blotting. (D) Jurkat cells were electroporated with siRNA construct (vector or D1488) and Myc-CARMA1 construct. Three days later, the cells were incubated with anti-CD3 antibody (5 μg/ml) and anti-CD28 antibody (1 μg/ml) for 10 min and then lysed. (E) Jurkat cells were electroporated with Myc-CARMA1 or its S109A mutant. One day later, the cells were incubated with anti-CD3 antibody and anti-CD28 antibody for 10 min and then lysed. Phosphoproteins were isolated via IMAC as described in the text. Lower panels show total lysates immunoblotted with anti-Myc antibody. The experiments were performed three times, and representative results are presented. WT, wild type.