Inhibitor-1 variant (G147D) exhibits reduced binding and inhibition of PP1 upon PKA phosphorylation.

<p>(A) Coomassie blue stained gel of purified GST-Inhibitor-1 (I-1) recombinant proteins. (B) Blot overlay assay with MBP-PP1 protein and immunodetection with the MBP antibody determined similar levels of PP1 binding to both WT and G147D inhibitor-1 proteins under basal conditions. PKA phosphorylation resulted in significant enhancement of PP1's interaction with WT but no such effect was observed for G147D. (C) Quantification of PP1 binding to WT and G147D inhibitor-1 under basal and PKA conditions. n = 4; <i>t</i>-test, two-tailed, *<i>P</i><0.05 vs WT (–PKA); #<i>P</i><0.05 vs WT (+PKA). (D) Phosphorylation of G147D at Thr35 is reduced compared to WT. (E) PP1 activity measurements indicate impaired regulation of PP1 by G147D variant after PKA-phosphorylation. n = 4; <i>t</i>-test, two-tailed, *<i>P</i><0.05 vs WT (–PKA); #<i>P</i><0.05 vs WT (+PKA). WB: Western blot