TGF-ALPHA-INDUCED BREAKDOWN OF STRESS FIBERS AND DEGRADATION OF TROPOMYOSIN IN NRK CELLS IS BLOCKED BY A PROTEASOME INHIBITOR

Treatment of NRK cells with TGF-alpha in the presence of serum initiat es disassembly of cytoskeletal stress fibers and suppresses the synthe sis of tropomyosin isoforms (TMs) 1, 2, and 3 but not TMs 4 and 5 (Coo per et al., Cancer Res. 47, 4493-4500, 1987). In order to determine ho w the loss of tropomyosin is induced and what role it plays in cytoske letal disruption, the turnover of tropomyosin was studied in the prese nce of the transforming growth factor and protease inhibitors. Cells w ere pulse-labeled with [S-35]methionine and chased in the absence or t he presence of the growth factor. It was found that TMs 1, 2, and 3 ar e degraded at about twice the rate of TMs 4 and 5 in control cells and that the rate of degradation of TMs 1-3 is accelerated by the growth factors. Degradation of TMs in control and growth factor-treated cells is blocked by a membrane-permeable inhibitor of cysteine proteases (L LnL) that acts upon calpains and proteasomes, and the cells maintain a flattened shape with a normal complement of stress fibers. Applicatio n of inhibitors that block calpains but not proteasomes does not block TM degradation. Treatments (suspension culture or cytochalasin B) tha t disrupt stress fibers without application of the growth factors also accelerate TM degradation, suggesting that acceleration of TM degrada tion is a consequence of its release from stress fibers during their b reakdown. The normally more rapid turnover of the TM isoforms 1-3 that are lost in the phenotypically transformed cells could serve to facil itate the cytoskeletal reorganization that follows the activation of s ignal transduction pathways by the transforming growth factors observe d in this study or during other rearrangements of the cytoskeleton suc h as occur during cell migration or mitosis. (C) 1997 Academic Press.