Article

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Oregon Hearing Research Center, Oregon Health & Science University, Portland, 97239, USA.
Cell Death & Disease (Impact Factor: 5.18). 11/2010; 1(11):e102. DOI: 10.1038/cddis.2010.80
Source: PubMed

ABSTRACT Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin-agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63.

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