Mutation of Lys216 results in inhibition of full length RSK2 activity ex vivo.

Wildtype RSK2 induced NFAT3 activity in a dose-dependent manner. K216A mutant showed reduced activity. The 3×NFAT-luciferase reporter plasmid was co-transfected with pcDNA3-Flag-NFAT3, pcDNA4-RSK2, or pcDNA4-RSK2-K216A plasmids into HEK 293 cells. The firefly luciferase activity was analyzed after 36 h and normalized against Renilla luciferase activity. Data are presented as the mean±S.D. of values from triplicate experiments. The differences were evaluated using the Student's t-test (*, p<0.05). The equivalent level of each expressed protein was confirmed by Western blot and shown in Figure S3.