Ubiquitin-Proteasome-mediated Degradation of Keratin Intermediate Filaments in Mechanically Stimulated A549 Cells

Abstract

We previously reported that shear stress induces phosphorylation and disassembly of keratin intermediate filaments (IFs). Shear stress also induces a time-and strain-dependent degradation of keratin IFs, and the current study examines the mechanisms involved in degradation of keratin proteins in human A549 cells exposed to 0 -24 h of shear stress (7.5-30 dynes/cm 2 ). Ubiquitin was found to be covalently associated with keratin proteins immunoprecipitated from shear-stressed cells, and pretreatment with the proteasomal inhibitor MG132 prevented the degradation of the keratin IF network. Importantly, phosphorylation of K8 Ser-73 is required for the shear stress-mediated ubiquitination, disassembly, and degradation of the keratin IF network. Immunofluorescence microscopy revealed that shear stress caused the thin array of keratin fibrils observed in control cells to be reorganized into a perinuclear aggregate, known as an aggresome, and that ubiquitin was also associated with this structure. Finally, the E2 enzymes, UbcH5b, -c, and Ubc3, but not E2-25K are required for the shear stress-mediated ubiquitin-proteasomal degradation of keratin proteins. These data suggest that shear stress promotes the disassembly and degradation of the keratin IF network via phosphorylation and the ubiquitin-proteasome pathway.Mechanical stimuli are important modulators of cellular function; this is especially true in the mechanically ventilated lungs of patients with acute lung injury. In these patients, shear stress is created by the cyclic opening and closing of the edematous, surfactant-depleted, collapsed alveoli. It has been previously demonstrated that physical forces, such as shear stress, induce rapid and global changes in the patterns of intermediate filaments (IFs) 2 (1-4).Keratin IFs are the major cytoskeletal component of epithelial cells and are assembled as obligate heteropolymers of type I (K9-K20) and type II (K1-K8) IF proteins (5-7). Lung alveolar epithelial cells (AEC) express primarily K8 and K18 with variable levels of K7 and K19 (1, 8, 9). The prototype structure of all IF proteins, including keratins, consists of a structurally conserved central coiled-coil ␣-helix termed the "rod" that is flanked by non-␣-helical N-terminal "head" and C-terminal "tail" domains (6). Most of the structural heterogeneity of the different keratins resides in their head and tail domains, which contain the sites for several post-translational modifications including phosphorylation and ubiquitination.The ubiquitin-proteasome pathway (UPP) (10) is involved in regulating the cell cycle, signal transduction, differentiation, and stress response. Most of these functions are mediated by the conditional turnover of regulatory and structural proteins (10 -12). The process begins with activation of ubiquitin by the ubiquitin-activating enzyme (E1) (10), followed by transfer of ubiquitin to E2, an ubiquitin-conjugating enzyme. E2 shuttles the ubiquitin molecule to the substrate-specific ubiquitin ligase (E3), which then delivers the ubiquiti...

Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18

“…This seems plausible because phosphorylation on many proteins acts as a signal for ubiquitination-mediated proteasomal degradation (55). Even on K8, shear stress-induced phosphorylation at Ser 73 enables E2 ligases to mediate proteasomal degradation (56). Indeed, loss of phosphorylation at Ser 33 increased the stability of K18, which could not be rescued by increasing O-GlcNAcylation on K18 (Fig.…”

Cytokeratins mediate epithelial innate defense through their antimicrobial properties

“…Turnover of intermediate filaments, and soluble keratin proteins, in epithelial cells is a highly regulated and complex process that is known to involve the ubiquitin-proteasome pathway (UPP) (55,56). It is not yet known whether the UPP contributes to the generation or degradation of KDAMPs.…”

Shear Stress Induced Reorganization of the Keratin Intermediate Filament Network Requires Phosphorylation by Protein Kinase C ζ

“…The alveolar epithelial cells used in this study contain polymers of keratin 8 and 18 (K8/K18; Ridge et al, 2005;Jaitovich et al, 2008;Sivaramakrishnan et al, 2008). KIF proteins are important for maintaining the mechanical integrity of epithelial cells (Fuchs and Cleveland, 1998;Herrmann and Aebi, 2004).…”