Figure 7: Ser177 phosphorylation of Pdlim5 suppressed Rac1 activity.
From: Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5
(a–c) Activities of Rac1 (a), RhoA (b) and Cdc42 (c) in KDR cells were quantitated using G-LISA specific for Rac1, RhoA and Cdc42, respectively. Numbers in the bars indicate n. Data are representative of means±s.e.m. from three independent experiments. Significance of differences between series of results was assessed using one-way analysis of variance, followed by a post-hoc comparison with Dunnett’s method for multiple comparisons. *P<0.01 compared with KDR/EGFP-WT-Pdlim5. (d) The KDR system was established in vSMCs stably expressing FRET probes specific for Rac1 (Raichu-Rac1/vSMCs). Raichu-Rac1/vSMCs were transfected with either siCTL or siPdlim5-2. siPdlim5-2-resistant Pdlim5-T2A-mCherry (WT, S177A and S177D) was introduced via adenoviral-mediated gene delivery (Raichu-Rac1/KDR-Pdlim5-T2A-mCherry cells). TCLs were subjected to immunoblotting. (e) Imaging of Rac1 activity. Each type of Raichu-Rac1/KDR-Pdlim5-T2A-mCherry cell (WT, S177A and S177D) was imaged for YFP and CFP. FRET efficiencies are shown as YFP/CFP ratio images. Scale bars, 20 μm.
