Inhibition of 20 S and 26 S Proteasome Activity by Lithium Chloride

Anna M Rice,Alan C Sartorelli

doi:10.1074/jbc.m106583200

Anna M Rice, Alan C Sartorelli

Open Access

https://doi.org/10.1074/jbc.m106583200

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Abstract

Lithium affects several enzymatic activities, however, the molecular mechanisms of lithium actions are not fully understood. We previously showed that LiCl interacts synergistically with all-trans-retinoic acid to promote terminal differentiation of WEHI-3B D(+) cells, a phenomenon accompanied by the recovery of the retinoid-induced loss of retinoic acid receptor alpha protein pools. Here, we demonstrate the effects of LiCl on proteasome-dependent degradation of retinoic acid receptor alpha proteins. LiCl alone, or in combination with all-trans-retinoic acid, increased cellular levels of ubiquitinated retinoic acid receptor alpha and markedly reduced chymotryptic-like activity of WEHI-3B D(+) 20 S and 26 S proteasome enzymes. Neither KCl nor all-trans-retinoic acid affected enzyme activity, whereas NaCl produced a modest reduction at relatively high concentrations. In addition, LiCl inhibited 20 S proteasome chymotryptic-like activity from rabbits but had no effect on tryptic-like activity of the 26 S proteasome. This effect has significant consequences in stabilizing the retinoic acid receptor alpha protein levels that are necessary to promote continued differentiation of leukemia cells in response to all-trans-retinoic acid. In support of this concept, combination of proteasome inhibitors beta-clastolactacystin or benzyloxycarbonyl-Leu-Leu-Phe with all-trans-retinoic acid increased differentiation of WEHI-3B D(+) cells in a manner that was analogous to the combination of LiCl and all-trans-retinoic acid.

Highlights

Lithium affects several enzymatic activities, the molecular mechanisms of lithium actions are not fully understood
We previously showed that LiCl interacts synergistically with all-trans-retinoic acid to promote terminal differentiation of WEHI-3B D؉ cells, a phenomenon accompanied by the recovery of the retinoid-induced loss of retinoic acid receptor ␣ protein pools
Inhibition of Proteasome-dependent RAR␣ Degradation by LiCl icance of this action is seen in the differentiation of WEHI-3B Dϩ leukemia cells, where LiCl prevents the degradation of RAR␣ protein pools produced by all-trans-retinoic acid (ATRA) that are critical to promoting retinoid-induced leukemia cell differentiation [8]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Differentiation—WEHI-3B Dϩ cells were cultured in suspension in McCoy’s 5A-modified medium supplemented with 15% fetal calf serum. Immunoprecipitation and Western Blotting—RAR␣ was immunoprecipitated from 100 ␮g of cell lysates prepared in radioimmune precipitation buffer with 2 ␮g of anti-RAR␣ antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 12 h followed by incubation with Protein A/G Plus-agarose beads for 2 h. Protein Purification and Activity Analysis—Proteasomes were partially purified from 2 ϫ 108 WEHI-3B Dϩ cells by homogenization in 50 mM Tris-HCl, 5 mM MgCl2, 2 mM ATP, 0.25 M sucrose, pH 7.4, in a Dounce homogenizer followed by vortexing with glass beads [29]. RAR␣-labeling reaction (4.5 ␮l) was incubated at 37 °C in degradation buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM dithiothreitol, 2 mM ATP, 10 mM creatine phosphokinase, 10 mM creatine phosphate, 5 ␮M ubiquitin) for 30 min in the presence or absence of additional 26 S proteasomes (5 ␮g) partially purified from. Degradation reactions were subjected to 10% SDSpolyacrylamide gel electrophoresis followed by phosphorimaging analysis of radiolabeled RAR␣ bands

RESULTS

26 S proteasome

DISCUSSION