Keratin 8 phosphorylation by protein kinase C delta regulates shear stress-mediated disassembly of keratin intermediate filaments in alveolar epithelial cells.
Phosphorylation of keratin intermediate filaments (IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C delta-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC delta and that the PKC delta peptide antagonist, delta V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.
Division of Pulmonary and Critical Care Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA. kridge@northwestern.edu
Karen M
KM
Ridge
Laura
L
Linz
Frederick W
FW
Flitney
Edward R
ER
Kuczmarski
Ying-Hao
YH
Chou
M Bishr
MB
Omary
Jacob Iasha
JI
Sznajder
Robert D
RD
Goldman
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology*; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D007381:Intermediate Filament Proteins / Q000737:chemistry; D007382:Intermediate Filaments / Q000378:metabolism; D053533:Keratin-8; D007633:Keratins / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051745:Protein Kinase C-delta; D021381:Protein Transport; D011650:Pulmonary Alveoli / Q000166:cytology*; D051381:Rats; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013314:Stress, Mechanical; D013997:Time Factors
D004791:Enzyme Inhibitors; D007211:Indoles; D007381:Intermediate Filament Proteins; C426203:KRT8 protein, human; D053533:Keratin-8; D008301:Maleimides; D010455:Peptides; D010768:Phosphoserine; D012694:Serine; D007633:Keratins; D000255:Adenosine Triphosphate; C497260:Prkcd protein, rat; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; C088060:bisindolylmaleimide