Proteomic analysis of alternative protein tyrosine phosphorylation in 1,2-dichlorovinyl-cysteine-induced cytotoxicity in primary cultured rat renal proximal tubular cells.
Toxicant exposure affects the activity of various protein tyrosine kinases. Using phosphotyrosine proteomics, we identified proteins that were differentially phosphorylated before renal cell detachment and apoptosis. Treatment of primary cultured rat proximal tubular epithelial cells with the model nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC) resulted in early reorganization of F-actin stress fibers and formation of lamellipodia, which was followed by cell detachment from the matrix and apoptosis. This was prevented by genistein-mediated inhibition of protein tyrosine kinases and enhanced by inhibition of protein tyrosine phosphatases using vanadate. Phosphotyrosine proteomics revealed that DCVC-induced renal cell apoptosis was preceded by changes in the tyrosine phosphorylation status of a subset of proteins, as identified by matrix-assisted laser desorption ionization/time of flight-mass spectrometry (MS)/MS including actin-related protein 2 (Arp2), cytokeratin 8, t-complex protein 1 (TCP-1), chaperone containing TCP-1, and gelsolin precursor. The major differentially tyrosine-phosphorylated protein was Arp2, whereas phosphorylation of Arp3 was not affected. Arp2 was located in the lamellipodia that were formed before the onset of apoptosis. Because DCVC-induced cell detachment and apoptosis is regulated by tyrosine kinases, we propose that alterations in tyrosine phosphorylation of a subset of proteins, including Arp2, play a role in the regulation of the F-actin reorganization and lamellipodia formation that precede renal cell apoptosis caused by nephrotoxicants.
de Graauw
Marjo
M
Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratoria, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Le Dévédec
Sylvia
S
Tijdens
Ine
I
Smeets
Mirjam B
MB
Deelder
André M
AM
van de Water
Bob
B
D051377:Actin-Related Protein 2 / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002450:Cell Communication / Q000187:drug effects; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives* / Q000633:toxicity; D007687:Kidney Tubules, Proximal / Q000187:drug effects*; D008297:Male; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D040901:Proteomics; D051381:Rats; D017208:Rats, Wistar; D014443:Tyrosine / Q000378:metabolism
D051377:Actin-Related Protein 2; D014443:Tyrosine; C039961:S-(1,2-dichlorovinyl)cysteine; D011505:Protein-Tyrosine Kinases; D003545:Cysteine