centrally located ubiquitin-like domain that forms a link for Hsc/Hsp70 to the proteasome and a conserved C-terminal Hsp70 binding domain that binds to Hsp70/Hsc70 and functions as a nucleotide exchange factor. Bag-1 has also been shown to regulate endoplasmic reticulum stress-induced apoptosis and to bind GADD34, a component of the ER stress but details of its action are not known. In this communication we show that Bag-1 binds to GRP78/ BiP through a peptide overlapping its ubiquitin-like domain. We further show that the GRP78/BiP binding peptide of Bag-1 
inhibits the action of GRP78/BiP and interferes with the UPR leading to the induction of apoptosis. We have narrowed down this peptide and identified a core motif of seven amino acids that appears essential for binding to GRP78/BiP and for the negative regulation of prostate tumor cell growth. This core sequence could be the starting point of future therapeutics directed towards the inhibition of GRP78/BiP action and of the UPR. the empty expression vector pcDNA3.1-HA. pcDNA3.1 Bag1DC47 was cloned into the Bam HI-Xho I sites of pcDNA3.1-HA vector after PCR amplification using pcDNA3.1-HA Bag-1 as template. For GSTpull-down experiments, pGEX4T.1-Bag-1 68mer sequence, pGEX4T.1-N-terminal peptide, pGEX4T.1-C-terminal peptide, pGEX4T.1 Bag-1D Ubi, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22212565 pGEX4T.1-19mer and pGEX4T.119mer mutants were created by fusing PCR amplification products of the respective Bag-1L sequence in frame with GST in the vector pGEX4T.1. GST plasmids encoding for GST-fused GRP78, GRP78-ATPase, GRP78-SBD and Bag-1 isoforms were generated by PCR and cloned into pGEX4T.1 after BamHI-XhoI digestion. The plasmid pCMV6-GRP78 was purchased from OriGene Technologies. Cell Transfection, siRNA Knock-down and Western Blotting Unless otherwise stated, stable transfection was carried out in 22Rv.1 or BPH-1 cells with 10 mg of plasmid DNA using FuGene 6TM Stably transfected 22Rv.1 cells were selected with 0.8 mg/ml while BPH-1 cell clones were selected with 1.2 mg/ml G418. LNCaP, PC3, DU145, PNT2 and RWPE-1 cell clones were selected with 1 mg/ml G418. Specific downregulation of GRP78 expression was achieved by transfecting siRNA-duplexes twice in 72 h using HiPerfectTM. siRNA against GFP was used as a control. Western blot analysis was performed as previously described. Materials and Methods Cell Culture Human benign prostatic hyperplasia cell line BPH-1 was cultured in Dulbeccos modified Eagles medium supplemented with glutamine. PC3 and DU145 cells were also cultured in DMEM but without glutamine 22Rv.1, LNCaP and PNT-2 cells were cultured in RPMI 1640. All the above culture media were supplemented with 10% fetal bovine serum. RWPE-1 cells were cultured in keratinocyte serum free medium. All the culture media were kept at 37uC in an atmosphere of 5% CO2. Co-immunoprecipitation 22Rv.1 cells were used for interaction studies of SB-705498 chemical information endogenous Bag-1 and GRP78/BiP, while for the in vivo interaction of the Bag1 peptide and GRP78/BiP, HEK-293 cells were transfected with a plasmid encoding HA-tagged Bag-1 peptide. Twenty-four hours prior to the experiment, the cells in both cases were washed with phosphate buffered saline and treated with 20 mM Dithiobis in PBS for 30 min at room temperature. The reaction was stopped with 20 mM Tris and the cells were lysed in 50 mM Tris-HCl pH 7.4, 120 mM NaCl, 1 mM EDTA, 0.4% NP40 and 1% protease inhibitor cocktail. Immunoprecipitation of GRP78/BiP was carried out overnight at 4uC with anti-GRP78 anti
