Bortezomib rapidly suppresses ubiquitin thiolesterification to ubiquitin-conjugating enzymes and inhibits ubiquitination of histones and type I inositol 1,4,5-trisphosphate receptor Qun Xu, Michelle Farah, Jack M. Webster, and Richard J.H. Wojcikiewicz Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York Abstract The proteasome inhibitor bortezomib is an emerging anticancer agent. Although the proteasome is clearly its locus of action, the early biochemical consequences of bortezomib treatment are poorly defined. Here, we show in cultured cells that bortezomib and other proteasome inhibitors rapidly inhibit free ubiquitin levels and ubiquitin thiolesterification to ubiquitin-conjugating enzymes. Inhi- bition of thiolesterification correlated with a reduction in the ubiquitination of certain substrates, exemplified by a dramatic decline in histone monoubiquitination and a decrease in the rate of inositol 1,4,5-trisphosphate recep- tor polyubiquitination. Thus, in addition to the expected effect of blocking the degradation of polyubiquitinated substrates, bortezomib can also inhibit ubiquitination. The effect of bortezomib on histone monoubiquitination may contribute to its therapeutic actions. [Mol Cancer Ther 2004;3(10):1263 – 9] Introduction Proteasome inhibitors are currently emerging as therapeu- tic agents, with known efficacy against various cancers and inflammatory diseases (1, 2). Bortezomib (Velcade, PS-341) was recently shown in phase II trials to be effective against multiple myeloma (3) and is the first proteasome inhibitor to be approved by the Food and Drug Administration. The proteasome is a multicatalytic protease that degrades a range of crucial cellular proteins, including regulators of the cell cycle and elements of signaling pathways (1, 2, 4). Proteins are directed to the proteasome by virtue of being polyubiquitinated (4). Polyubiquitination is achieved through the hierarchical action of three enzymes, termed ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). Whereas there is only one E1, there are many E2s (also often termed Ubcs) and E3s (4, 5). First, free ubiquitin becomes conjugated via its COOH terminus to a cysteine residue of E1 through a thiolester bond. Second, this ‘‘activated’’ ubiquitin is transferred and linked, again through a thiolester bond, to the active site cysteine residue of an E2, which may already be associated with an E3. Third, the ubiquitin is coupled to the q-amino group of a lysine residue in the substrate through an isopeptide bond; this transfer is facilitated by the E3 that juxtaposes the E2 and the substrate. A polyubiquitin chain can then be formed by multiple rounds of ubiquitination; the COOH terminus of incoming ubiquitin residues are linked via an isopeptide bond to a lysine residue of the already attached ubiquitin. Finally, the polyubiquitinated protein is recognized by the proteasome and is degraded, with accompanying disas- sembly of the ubiquitin chain and recycling of ubiquitin monomers (4). Additional recent work indicates that ubiquitin conjugation also has other functions that do not involve proteasomal targeting (4), for example, in control- ling the trafficking of cell surface receptors (6) and in regulating histones (7, 8), and that in these contexts monoubiquitination, rather than polyubiquitination, is often the regulatory event (6 – 8). To date, most studies on the mechanism of bortezomib action have focused on changes in gene expression and the levels and activity of relevant proteins (1, 2, 9 – 12). The events that lead to these changes remain unclear, however, because the immediate consequences of proteasome inhibition by bortezomib have not been examined in detail. Whether the biological effects of bortezomib result solely from the stabilization of key proteasomal substrates (e.g., transcription factors) in their polyubiquitinated form, or whether there are other con- sequences of proteasome inhibition, is presently unclear, and an answer to this question would both lead to a better understanding of the effects of bortezomib and stimulate the development of more specific and effective drugs. Thus, we examined the acute biochemical effects of bortezomib and show that it rapidly inhibits free ubiquitin levels and the thiolesterification of ubiquitin to E2s. This correlated with a decrease in the ubiquitination of certain substrates, showing that proteasome inhibition has acute consequences beyond the stabilization of polyubiquitinated proteins. Received 5/12/04; revised 7/16/04; accepted 8/13/04. Grant support: NIH grant 5RO1DK49194 and Pharmaceutical Research and Manufacturers of America Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: J.M. Webster is currently at Wadsworth Center, New York State Department of Health, Division of Genetic Disorders, 120 New Scotland Avenue, Albany, NY 12208. Requests for reprints: Richard J.H. Wojcikiewicz, Department of Pharmacology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210-2339. Phone: 315-464-7956; Fax: 315-464-8014. E-mail: wojcikir@upstate.edu Copyright C 2004 American Association for Cancer Research. Molecular Cancer Therapeutics 1263 Mol Cancer Ther 2004;3(10). October 2004 Downloaded from http://aacrjournals.org/mct/article-pdf/3/10/1263/1867052/1263-1269.pdf by guest on 29 July 2023