Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis.
We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.
de Graauw
Marjo
M
Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.
Tijdens
Ine
I
Cramer
Rainer
R
Corless
Steve
S
Timms
John F
JF
van de Water
Bob
B
D000818:Animals; D017209:Apoptosis; D002448:Cell Adhesion; D003545:Cysteine / Q000031:analogs & derivatives / Q000633:toxicity; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology*; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006360:Heat-Shock Proteins / Q000502:physiology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007668:Kidney / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D040901:Proteomics; D013552:Swine; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D006360:Heat-Shock Proteins; C039961:S-(1,2-dichlorovinyl)cysteine; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D003545:Cysteine