Two or three lysine residues in the C terminus of XBP-1u were replaced by arginines to generate XBP-1uKK (K235R, K252R) and XBP-1uKKK (K146R, K235R, K252R) by site-directed mutagenesis (22). recently been observed with a series of novel medicines that inhibit the proteasome, a highly conserved multienzyme complex that destroys proteins covalently revised by ubiquitin (1C5). One such drug, PS-341, induces apoptosis of MM cells and interferes with their interaction with the stromal microenvironment and subsequent production of the OSU-T315 MM survival cytokine IL-6 (6C9). In MM cells, PS-341 decreases levels of several antiapoptotic proteins, resulting in mitochondrial cytochrome launch and activation of caspase-9, jun kinase, and Fas-dependent pathways (9, 10). The molecular switch that initiates these apoptotic cascades, however, has not yet been defined. Both the normal and malignant plasma cells create and secrete abundant Igs. This requires a highly developed endoplasmic reticulum and the production of chaperone proteins that effect appropriate translation and folding. A signaling pathway called the unfolded protein response (UPR), or stress response, ensures that the plasma cells can handle the proper folding of Ig proteins (11). Three signaling pathways responsible for mediating the UPR have been described. Two of them involve the activation of transcription factors XBP-1 and ATF6, whereas the third depends on translational repression mediated by PERK/eIF2.On sensing unfolded proteins, an endoplasmic reticulum (ER) transmembrane endoribonuclease and kinase called IRE1 oligomerizes, is activated by autophosphorylation, and uses its endoribonuclease activity to excise an intron from candida Hac1p or mammalian XBP-1 mRNA, resulting in the conversion of a ARHGEF2 267-aa unspliced XBP-1 protein to a OSU-T315 371-aa spliced XBP-1 protein (12C20). XBP-1 then translocates into the nucleus where it binds to its target sequence in the regulatory regions of the chaperone genes to induce their transcription. We recently reported that XBP-1 is required for the generation of plasma cells (21) and that only the spliced XBP-1 varieties can reconstitute Ig secretion (22). The abundant manifestation of XBP-1 in myelomas suggested a role for it in perpetuating this malignancy (23) and raised the possibility that it was one molecular target of the novel anticancer compounds that target the proteasome. Materials and Methods Western Blot and OSU-T315 PulseCChase Experiments. Cells were lysed in RIPA buffer (50 mM Tris, pH 7.4/150 mM NaCl/1 mM EDTA/1% Triton X-100/1% sodium deoxycholate/0.1% SDS), and lysates were subjected to SDS/PAGE and transferred to Hybond-P membranes (Amersham Biosciences). Blots were exposed by anti-XBP-1 (Santa Cruz Biotechnology), anti-caspase-12 (gift of J. Yuan, Harvard University or college, Boston), and anti-IRE1 (24) antibodies by standard methods. HeLa cells were cotransfected with XBP-1u and His-tagged ubiquitin manifestation plasmids (pMT107, gift of D. Bohmann, EMBL, Heidelberg, Germany) by using Lipofectamine 2000 reagent (Invitrogen). Cell components were purified through Ni-NTA columns as explained (25), and ubiquitinated XBP-1u proteins were revealed by Western blot analysis with anti-XBP-1 antiserum. Degradation rates of XBP-1u and -1s proteins were determined by pulse labeling J558 cells with [35S]Met/[35S]Cys for 1 h and chasing after for the indicated instances. Radiolabeled XBP-1 proteins were immunoprecipitated from total cell components, separated on 10% SDS/PAGE, and exposed by autoradiography. Northern Blot and RT-PCR Analysis. Total RNA was prepared by using TRIzol reagent, electrophoresed on 1.2% agarose/6% formaldehyde gels, and then transferred onto Genescreen Plus membrane (NEN). Hybridizations with 32P-radiolabeled probes were performed as shown (22). Probes for ERdj4 and p58IPK were generated from cDNA excised from EST clones (American Type Tradition Collection) by using appropriate restriction enzymes (ERdj4, IMAGE:1920927; p58IPK, IMAGE:2646147). The percentage of XBP-1u to -1s mRNA was exposed by RT-PCR analysis having a probe arranged spanning the spliced-out region as shown (22). Plasmids and Reporter Assays. Two or three lysine residues in the C terminus of XBP-1u.