Original Article

Subject Category: Experimental Medicine

Citation: Cell Death and Disease (2010) 1, e102; doi:10.1038/cddis.2010.80
Published online 18 November 2010

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

Edited by R Mantovani

T Karasawa1, Q Wang1, L L David2 and P S Steyger1

  1. 1Oregon Hearing Research Center, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, USA
  2. 2Proteomics Shared Resource, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, USA

Correspondence: T Karasawa, Oregon Hearing Research Center, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. Tel: 503 494 2373; Fax: 503 494 5656; E-mail: karasawa@ohsu.edu

Received 15 June 2010; Revised 27 September 2010; Accepted 14 October 2010.

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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 63kDa (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 1h. 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.

Keywords:

aminoglycosides; gentamicin; CLIMP-63; CKAP4; cytotoxicity; 14-3-3

Abbreviations:

GBPs, gentamicin-binding proteins; CLIMP-63, cytoskeleton-linking membrane protein of 63kDa; ER, endoplasmic reticulum; DTT, dithiothreitol; GTTR, gentamicin-Texas Red; MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling