Figure 10.
14-3-3 ζ preferentially binds to phosphorylated K18 and is required to KIF network remodeling. (A) Cell lysates from static control (CT) and shear stress (30 dyn/cm2; 60 min, SS) cells were incubated with GST-14-3-3ζ (lanes 1 and 2). Alternatively, recombinant K18 (rK18) was phosphorylated in vitro by recombinant PKC ζ (rPKC ζ) and incubated with GST-14-3-3 ζ (lanes 3 and 4). Proteins bound to GST-14-3-3ζ were recovered using GSH-Sepharose, separated on 10% SDS-PAGE, transferred and immunoblotted with anti-K8/K18 and anti-GST antibodies. Lane 5 is negative control without GST-14-3-3 ζ, whereas lane 6 is GST control without 14-3-3 ζ. (B and C) Confocal micrographs showing immunofluorescence staining of A549 cells pretreated with R18 peptide under static control (B) and shear stress (30 dyn/cm2; 60 min) conditions (C), processed with anti-K18 antibody. Arrow in B indicates direction of flow. (D) Recovery of fluorescence in the FRAP zones of GFP-K18 stably transfected A549 cells pretreated with R18 peptide for 60 min before shear stress (SS) or time matched controls (CT). Cells were subject to shear stress (15 dyn/cm2; 120 min) before photobleaching; shear stress was continued throughout fluorescence recovery. Each data point is mean ± SEM of at least four different cells. White scale bar, 10 μm.