Shear stress induced reorganization of the keratin intermediate filament network requires phosphorylation by protein kinase C zeta.

Keratin intermediate filaments (KIFs) form a fibrous polymer network that helps epithelial cells withstand external mechanical forces. Recently, we established a correlation between the structure of the KIF network and its local mechanical properties in alveolar epithelial cells. Shear stress applied across the cell surface resulted in the structural remodeling of KIF and a substantial increase in the elastic modulus of the network. This study examines the mechanosignaling that regulates the structural remodeling of the KIF network. We report that the shear stress-mediated remodeling of the KIF network is facilitated by a twofold increase in the dynamic exchange rate of KIF subunits, which is regulated in a PKC zeta and 14-3-3-dependent manner. PKC zeta phosphorylates K18pSer33, and this is required for the structural reorganization because the KIF network in A549 cells transfected with a dominant negative PKC zeta, or expressing the K18Ser33Ala mutation, is unchanged. Blocking the shear stress-mediated reorganization results in reduced cellular viability and increased apoptotic levels. These data suggest that shear stress mediates the phosphorylation of K18pSer33, which is required for the reorganization of the KIF network, resulting in changes in mechanical properties of the cell that help maintain the integrity of alveolar epithelial cells.

[1]  J. Sznajder,et al.  Cyclic stretch activates ERK1/2 via G proteins and EGFR in alveolar epithelial cells. , 2002, American journal of physiology. Lung cellular and molecular physiology.

[2]  M. Omary,et al.  "Heads and tails" of intermediate filament phosphorylation: multiple sites and functional insights. , 2006, Trends in biochemical sciences.

[3]  Sivaraj Sivaramakrishnan,et al.  Micromechanical properties of keratin intermediate filament networks , 2008, Proceedings of the National Academy of Sciences.

[4]  M. Omary,et al.  Implications of intermediate filament protein phosphorylation , 1996, Cancer and Metastasis Reviews.

[5]  R. Vallee,et al.  A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization , 2002, The Journal of cell biology.

[6]  M. Omary,et al.  Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Sznajder,et al.  Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta. , 2002, Molecular biology of the cell.

[8]  M. Omary,et al.  Intermediate filament proteins and their associated diseases. , 2004, The New England journal of medicine.

[9]  M. Omary,et al.  The Intermediate Filament Protein Keratin 8 Is a Novel Cytoplasmic Substrate for c-Jun N-terminal Kinase* , 2002, The Journal of Biological Chemistry.

[10]  Kay C Dee,et al.  Mechanisms of surface-tension-induced epithelial cell damage in a model of pulmonary airway reopening. , 2003, Journal of applied physiology.

[11]  R. Goldman,et al.  Rapid displacement of vimentin intermediate filaments in living endothelial cells exposed to flow. , 2000, Circulation research.

[12]  J. Sznajder,et al.  Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-ε and -δ , 2002 .

[13]  M. Omary,et al.  Keratin modifications and solubility properties in epithelial cells and in vitro. , 1998, Sub-cellular biochemistry.

[14]  J. Sznajder,et al.  LUNG INJURY INDUCED BY MECHANICAL VENTILATION , 1998 .

[15]  E. Fuchs,et al.  A structural scaffolding of intermediate filaments in health and disease. , 1998, Science.

[16]  P. Coulombe,et al.  Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm. , 2007, Genes & development.

[17]  A. Ciechanover,et al.  Ubiquitin-Proteasome-mediated Degradation of Keratin Intermediate Filaments in Mechanically Stimulated A549 Cells* , 2008, Journal of Biological Chemistry.

[18]  Jesse D. Martinez,et al.  Reduction of 14-3-3 Proteins Correlates with Increased Sensitivity to Killing of Human Lung Cancer Cells by Ionizing Radiation , 2003, Radiation research.

[19]  Ueli Aebi,et al.  Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular Scaffolds. , 2003, Annual review of biochemistry.

[20]  Robert D Goldman,et al.  Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments , 2004, Journal of Cell Science.

[21]  D. Terrian,et al.  Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function , 1996, The Journal of cell biology.

[22]  Robert D Goldman,et al.  Keratin 8 Phosphorylation by Protein Kinase C δ Regulates Shear Stress-mediated Disassembly of Keratin Intermediate Filaments in Alveolar Epithelial Cells* , 2005, Journal of Biological Chemistry.

[23]  M. Omary,et al.  Phosphorylation of human keratin 18 serine 33 regulates binding to 14‐3‐3 proteins , 1998, The EMBO journal.

[24]  M. Omary,et al.  Keratin 8 Phosphorylation by p38 Kinase Regulates Cellular Keratin Filament Reorganization , 2002, The Journal of Biological Chemistry.

[25]  J. Eriksson,et al.  Intermediate filament dynamics. , 1992, Current opinion in cell biology.

[26]  M. Liu,et al.  Mechanical force-induced signal transduction in lung cells. , 1999, The American journal of physiology.

[27]  M. Omary,et al.  Identification of the major physiologic phosphorylation site of human keratin 18: potential kinases and a role in filament reorganization , 1994, The Journal of cell biology.

[28]  Ueli Aebi,et al.  Intermediate filaments: from cell architecture to nanomechanics , 2007, Nature Reviews Molecular Cell Biology.

[29]  J. Eriksson,et al.  Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments. , 1997, Journal of cell science.

[30]  M. Omary,et al.  Phosphorylation of Human Keratin 8 in Vivo at Conserved Head Domain Serine 23 and at Epidermal Growth Factor-stimulated Tail Domain Serine 431* , 1997, The Journal of Biological Chemistry.

[31]  I. Weinstein,et al.  Response Element Serum Fos Kinase C in Activation of the C- Novel Roles of Specific Isoforms of Protein , 1999 .

[32]  J. Sznajder,et al.  Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta. , 2003, The Journal of clinical investigation.

[33]  D. Morrison,et al.  Unlocking the code of 14-3-3 , 2004, Journal of Cell Science.

[34]  M. Omary,et al.  Raf-1 activation disrupts its binding to keratins during cell stress , 2004, The Journal of cell biology.

[35]  Robert D. Goldman,et al.  Rapid Movements of Vimentin on Microtubule Tracks: Kinesin-dependent Assembly of Intermediate Filament Networks , 1998, The Journal of cell biology.

[36]  M. Shoji,et al.  Immunocytochemical evidence for phorbol ester-induced protein kinase C translocation in HL60 cells. , 1986, Biochemical and biophysical research communications.

[37]  Richard J. Goss,et al.  10 – Heads and Tails , 1969 .

[38]  Kathleen J Green,et al.  Intermediate filament assembly: dynamics to disease. , 2008, Trends in cell biology.

[39]  R. Goldman,et al.  Intermediate filaments: versatile building blocks of cell structure. , 2008, Current opinion in cell biology.

[40]  M. Omary,et al.  14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor , 1996, The Journal of cell biology.

[41]  J. Sznajder,et al.  Ventilator-associated lung injury decreases lung ability to clear edema and downregulates alveolar epithelial cell Na,K-adenosine triphosphatase function. , 1999, Chest.

[42]  F. W. Flitney,et al.  Insights into the Dynamic Properties of Keratin Intermediate Filaments in Living Epithelial Cells , 2001, The Journal of cell biology.