Figure 3: ARHGAP36 inhibits PKAC.
(a) Recombinant PKAC (25 ng) was incubated with 10 μM scrambled, 36i or PKI peptide, and its activity determined by its ability to phosphorylate the substrate peptide PepTag A1. Phosphorylated and non-phosphorylated peptide were separated by agarose electrophoresis, and densitometrically analysed. Representative gel shown. Recombinant PKAC activity is shown as the ratio of phosphorylated to non-phosphorylated peptide (mean of eight repeats±s.e.m., ***P<0.001, Student’s T-test versus all three controls). (b) AKAR4-NES FRET sensor was expressed together with Cherry control, Cherry-ARHGAP36 (WT), the indicated mutants or 36i-Cherry or PKI-Cherry in HEK293T cells. Cells were serum starved for 5 h, treated with 10 μM Forskolin and 100 μM IBMX for 30 min, and subsequently imaged. Mean FRET emission ratio (acceptor intensity/donor intensity) of three independent experiments normalized to control±s.d. ***P<0.001, NS, not significant, versus control and RRV. (c) Representative images of HEK293T cells expressing AKAR4-NES FRET sensor together with Cherry control, Cherry-ARHGAP36 (WT) or Cherry-ARHGAP36-RRV. Cells were treated as in b. Shown are YFP intensity images and pseudocoloured FRET ratio images (acceptor intensity/donor intensity) reflecting the relative PKA activity levels. Scale bars, 100 μm. The histograms show the pixel distribution within the FRET emission ratio images.
