Cell Lines Protein BAG-1 and Its Variants in Normal Tissues and Tumor Expression and Location of Hsp70/Hsc-Binding Anti-Apoptotic

BAG-1 is a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hor mone receptors, possibly by virtue of its ability to regulate the Hsp70/ Hsc70 family of molecular chaperones. Two major forms of the human and mouse BAG-1 proteins were detected by immunoblotting. The longer human and mouse BAG-1 proteins (BAG-1L) appear to arise through translation initiation at noncanonical CTG codons located upstream of and in-frame with the usual ATG codon used for production of the originally described BAG-1 protein. Immunoblotting experiments using normal tissues revealed that BAG-1 L is far more restricted in its expres sion and is present at lower levels than the more prevalent BAG-1 protein. Human but not mouse tissues also produce small amounts of an additional isolot m of BAG-1 of intermediate size (BAG-1M) that probably arises through translation initiation at yet another site involving an ATG codon. All three isoforms of human BAG-1 (BAG-1, BAG-1M, and BAG-1L) retained the ability to bind Hsc70. Subcellular fractionation and iniiminofluorescence confocal microscopy studies indicated that BAG-1 L often resides in the nucleus, consistent with the presence of a nuclear localiza tion sequence in the Ml ,-lmuinal unique domain of this protein. In immunohistochemical assays, BAG-1 immunoreactivity was detected in a wide variety of types of cells in normal adult tissues and was localized to either cytosol, nucleus, or both, depending on the particular type of cell. In some cases, cytosolic BAG-1 immunostaining was clearly associated with organelles resembling mitochondria, consistent with the reported interac tion of BAG-1 with Bcl-2 and related proteins. Furthermore, experiments using a green fluorescence protein (GFP)-BAG-l fusion protein demon strated that overexpression of Bcl-2 in cultured cells can cause intracellular redistribution of GFP-BAG-1, producing a membranous pattern typical of Bcl-2 family proteins. The BAG-1 protein was found at high levels in several types of human tumor cell lines among the 67 tested, particularly leukemias, breast, prostate, and colon cancers. In contrast to normal tissues, which only rarely expressed BAG-1L, tumor cell lines commonly contained BAG-1L protein, including most prostate, breast, and leukemia cell lines, suggesting that a change in BAG-1 inKNA trans lation frequently accompanies malignant transformation.