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. 2013 Sep 25;110(41):E3937–E3944. doi: 10.1073/pnas.1315022110

Fig. 5.

Fig. 5.

Cytoskeletal turnover is regulated by PKCζ- and PP2A-dependent phosphorylation and dephosphorylation, respectively, with 14-3-3σ stabilizing the solubilized actin/keratin complex to coordinate cell motility. (A) Protein 14-3-3σ stabilizes formation of the soluble actin/keratin/14-3-3σ complex. Less actin and PKCζ coimmunoprecipitated with keratin 5 in sh-14-3-3σ cell lysate relative to control cells, suggesting that 14-3-3σ is required for stable interaction between soluble actin and keratin 5/17. IB, Western blot; IP, immunoprecipitation. (B) Formation of the soluble actin/keratin/14-3-3σ complex requires PKCζ-dependent phosphorylation. Treatment with a myristoylated PKCζ pseudosubstrate peptide inhibitor (PKCζ PSi) eliminates interactions between 14-3-3σ, actin, and keratin 5/17 (B, i) and concomitantly decreases the pool of soluble actin and keratins (B, ii). Cells were treated with EGF, okadaic acid (OA), PKCζ PSi, or cytochalasin D (CCD) before immunoprecipitation or fractionation into 1% Triton X-100–soluble and –insoluble pools. IB, Western blot; IP, immunoprecipitation. (C) The influence of pharmacological inhibitors on the cytoskeletal architecture. In agreement with B, treating cells with OA leads to rapid solubilization of F-actin and intermediate filaments; conversely, treating cells with PKCζ PSi dramatically promotes filament formation. (D) All cytoskeletal pharmacological inhibitors terminate cell migration, indicating that the correct balance of soluble to insoluble cytoskeleton is necessary for optimal cell migration. Velocities for 25 individual cells were calculated using particle tracking analysis. Error bars represent the SEM, and statistical significance was calculated using a one-way analysis of variance with Bonferroni's multiple comparison posttest. (E) A proposed model by which cytoskeleton dynamic equilibrium and cell motility are integrated by 14-3-3σ; 14-3-3σ maintains a soluble pool of nonfilamentous cytoskeletal nuclei to be assimilated into filaments as needed during motility. Formation of this soluble pool depends on PKCζ and is antagonized by PP2A. Disrupting cytoskeletal homeostasis, either by using pharmacological inhibitors that shift the equilibrium away from the optimal ratio of soluble to filamentous cytoskeleton or by using shRNAs targeting 14-3-3σ, leads to decreased tumor cell motility and invasion.