| Keratin intermediate filaments (IFs) have traditionally been understood as a mechanical scaffold, enabling epithelial cells and tissues to withstand various types of stress. However, recent evidence suggests that keratins could also play a role in the control of cellular events by modulating the activity of keratin-binding proteins. To investigate a potential role for keratin IFs in the regulation of cell growth, I set out to identify keratin-binding proteins and characterize their potential functions, thereby establishing a connection between keratin cytoskeleton and growth signaling networks.; In Chapter 1, I show that keratin 17 (K17), a wound-inducible keratin, regulates the Akt/mTOR signaling pathway through binding to the signaling adaptor protein 14-3-3sigma. First, K17 null keratinocytes in both wounded embryonic tissues and primary culture are smaller in size than control cells. This hypertrophic response results from the hypoactivation of the Akt/mTOR pathway, leading to translational depression. I performed a screen for K17-binding proteins by using a chemical crosslinker and identified 14-3-3sigma, a known positive regulator of mTOR. Detailed functional studies revealed that K17 promotes the serum-dependent relocalization of 14-3-3 from the nucleus to the cytoplasm, thereby leading to the full activation of the Akt/mTOR pathway and translation.; Interactions between cytoskeletal networks and the translation apparatus have been recognized for many years, but the physiological significance remains unclear. In Chapter 2, I show that keratin but not vimentin intermediate filaments directly bind to eEF1Bgamma (gamma subunit of eukaryotic elongation factor 1-B), which is essential for peptide chain elongation. I identified a unique role for eEF1Bgamma in the organization of keratin bundles in vitro and in cell culture. In epithelial cells, overexpression of the keratin-binding domain of eEF1Bgamma not only dissociates endogenous eEF1Bgamma from keratin filaments, but also depresses the total rate of protein synthesis by ∼15--20%, suggesting a potential role for keratin cytoskeleton in the control of translation.; In conclusion, from my thesis studies, keratin intermediate filaments are shown to dynamically orchestrate the cellular growth response by functionally associating with signaling networks and the translational machinery. |
