Fig. 6: 14-3-3 influences cellular localization and scaffolding range of PEAK3.
From: Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3
a Immunofluorescence-based imaging of FLAG-tagged PEAK3 transiently expressed in COS-7 cells. Representative confocal microscopy images show cells transfected with an empty vector (EV) control or PEAK3 variants: WT, S69A, R147A, S225A, K293A, W298A, or K293A/W298A. PEAK3 was detected with an anti-FLAG antibody (green), and cells were further stained with DAPI (blue, nucleus) and iFluor-647 conjugated phalloidin (red, actin). Scale bars: 10 µm. b Quantification of relative nuclear enrichment of PEAK3 under conditions of impaired 14-3-3 binding. The ratio of fluorescence intensity in the green channel after background subtraction measured in the nucleus to the fluorescence intensity of the non-nuclear portion of the cell is plotted for each PEAK3 variant; see Methods for details. Data are plotted as the mean with standard deviation, combining all cells from at least 3 independent experiments (n = 57, 65, 60, 69, 71, 53, and 48 total cells for WT, S69A, R147A, S225A, K293A, W298A, and K293A/W298A, respectively). Statistical significance was determined using One-way ANOVA Dunnett’s multiple comparisons test, **p < 0.01, ***p < 0.001, ****p < 0.0001. c Spectrum of protein-protein interactions engaged by the WT or S69A PEAK3 analyzed by immunoprecipitation/mass spectrometry approach. Edges represent significant specific interactions of PEAK3 WT or S69A as determined by SAINTexpress (BFDR < 0.05) compared to negative control (non-transfected cells). Node colors represent the log2 fold change enrichment determined by MSstats between WT and S69A PEAK3 (bold node border represents significant adjusted p value < 0.05). CORUM complexes are indicated by yellow circles. Source data are provided as a Source Data file.
