Abstract
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.
Footnotes
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This work was supported by grants from The Netherlands Organization for Scientific Research (Grants 902-21-229 and 911-02-022). B.v.d.W. was supported by a fellowship of the Royal Netherlands Academy for Arts and Sciences.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.117689.
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ABBREVIATIONS: RPTE, renal proximal tubular epithelial; ECM, extracellular matrix; pTyr, protein tyrosine phosphorylation; PY, phosphotyrosine; DCVC, 1,2-dichloro-vinyl-l-cysteine; FA, focal adhesion; FAK, focal adhesion kinase; AJ, adherens junctions; PTK, protein tyrosine kinase, 2D, two-dimensional; MALDI, matrix-assisted laser desorption ionization; Van, orthovanadate; Gen, genistein; PAGE, polyacrylamide gel electrophoresis; Arp2, actin-related protein 2; PBS, phosphate-buffered saline; G6PDH, glucose-6-phosphate dehydrogenase; EGF, epidermal growth factor; DPPD, N,N′-diphenyl-p-phenylenediamine; LDH, lactate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin; z, benzyloxycarbonyl; fmk, fluoromethyl ketone; MS, mass spectrometry; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; AG1714, 4-nitrobenzylidene malonitrile; IPG, immobilized pH gradient; HSC70, heat shock cognate protein 70.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received November 28, 2006.
- Accepted April 17, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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