Fig. 6. SCA14 mutations in C1B reduce PKC kinase activity in living cells. (A) HeLa cells were transiently transfected with either MyrPalm-CKAR alone or cotransfected with wild-type PKC-RFP or the different SCA14 mutant PKC-RFP. Intramolecular FRET was measured by determining the ratio of the YFP/CFP fluorophore intensities of the MyrPalm-CKAR molecule. Upon phosphorylation, less FRET from CFP to YFP will be measured because conformational changes increase the distance between the N- and C-terminus. MyrPalm-CKAR shows reduced phosphorylation, as was shown by less loss of FRET in the presence of SCA14 mutant PKC (G118D, V138E and C142S) when compared with wild-type PKC upon stimulation with 500nM PMA. The data traces were corrected by the background images acquired prior to adding ligand. The corrected traces were normalized to 1 by dividing the traces by the average baseline FRET ratio. (B) Bars represent the FRET efficiencies of the MyrPalm-CKAR reporter in the absence and presence of wild-type PKC and SCA14 mutant PKC (unpaired t-test, ^***P<0.001). (C) Western blot analysis shows reduced MARCKS phosphorylation in whole cell lysates of SCA14-mutant PKC upon PMA stimulation (400 nM) compared with cells containing wild-type PKC using anti-phospho-MARCKS antibody. Top panel shows equal PKC protein levels in all cell lysates. (D) Quantification of band intensity. Bar graph represents the phosphorylation of MARCKS normalized to the amount of MARCKS and PKC, and related to the value obtained for cells expressing wild-type PKC. Data in B and D represent mean ± s.e.m.