Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation

Abstract

The Bcl-2 oncoprotein is a key regulator of apoptosis and the Bag-1 protein interacts with Bcl-2 and cooperates with Bcl-2 to suppress apoptosis. The human Bag-1 cDNA is essentially identical with a previously described cDNA encoding RAP46, which interacts with activated steroid hormone receptors. However, there is considerable confusion over the structure of Bag-1/RAP46 proteins and their relationship to endogenous Bag-1 proteins. Here we have characterized Bag-1 expression in mammalian cells. We demonstrate that, in addition to the previously identified 32 kDa murine and 36 kDa human Bag-1 proteins, cells express a second 50 kDa Bag-1 isoform. In some murine cell lines p50 is expressed at the same level as p32 Bag-1, and p50 and p32 Bag-1 proteins have distinct subcellular localizations, suggesting that they are functionally distinct. The published mouse Bag-1 cDNA is partial, and sequencing of additional murine Bag-1 RNA 5' sequences demonstrated that human and murine Bag-1 cDNAs contain longer open reading frames than originally suspected. We determined which open reading frames gave rise to the Bag-1 isoforms in human cells. Surprisingly, translation of neither protein initiated at the first in-frame methionine, and cells do not express Bag-1/RAP46 proteins with the previously proposed structures; p50 Bag-1 initiates at an upstream CUG codon, whereas p36 Bag-1 initiates at a downstream AUG codon. Therefore, cells express two differently localized Bag-1 isoforms generated by alternative translation initiation, and Bag-1 proteins may play a dual role in regulating apoptosis and steroid hormone-dependent transcription.

BAG-1 overexpression attenuates luminal apoptosis in MCF-10A mammary epithelial cells through enhanced RAF-1 activation

“…The unique amino terminus of BAG-1 mediates the intracellular localization of the isoforms with BAG-1L containing a nuclear localization signal consistent with its predominantly nuclear expression, and BAG-1S and BAG-1M (both lacking the nuclear localization signal) located in the cytoplasm and cytoplasm/nucleus, respectively (Packham et al, 1997;Takayama et al, 1998). This differential localization may also dictate functional differences between the isoforms.…”

BAG-1 accelerates cell motility of human gastric cancer cells

“…The most abundant form of the BAG-1 protein is predicted to be 230 and 219 amino acids in length in human and mice, respectively, but longer isoforms of BAG-1 (known as BAG-1M/RAP46 and BAG-1L) have been attributed to alternative translation site initiation form of common mRNAs (Packham et al, 1997;Takayama et al, 1998). BAG-1 proteins contain a ubiquitin-like domain, the signi®cance of which is presently unknown, and several copies of a EE (A/V/L/ M)T(Q/R/K)(S/T) repeat.…”

BAG-1 expression and function in human cancer

“…We initially identified a potential nuclear localisation signal (NLS) within the unique N-terminal domain of BAG-1L, consistent with the predominantly nuclear localisation of this isoform (Packham et al, 1997;Takayama et al, 1998;Yang et al, 1998;Brimmell et al, 1999). By contrast, BAG-1S and BAG-1M lack this sequence and BAG-1S is largely located in the cytoplasm whilst BAG-1M partitions between the nucleus and cytoplasm (Packham et al, 1997;Takayama et al, 1998;Yang et al, 1998). The nuclear/cytoplasmic distribution of BAG-1 isoforms is regulated under some conditions and it is important to recognise that nuclear BAG-1 immunostaining in cancer cells may not be a reliable measure of BAG-1L expression.…”

“…BAG-1 proteins are expressed as multiple isoforms generated by alternate translation initiation from a single mRNA (Packham et al, 1997;Takayama et al, 1998;Yang et al, 1998). The major human BAG-1 isoform, BAG-1S, initiates at an internal AUG codon, whereas translation of the larger BAG-1L and BAG-1M proteins initiate at upstream CUG and AUG codons, respectively.…”

“…BAG-1 proteins contain different numbers of copies of repeats rich in acidic amino-acids. Human BAG-1L and BAG-1M contain 10 copies whereas BAG-1S contains four (Packham et al, 1997). The function of the repeats remains unclear since this part of the molecule is not essential for suppression of apoptosis, but is required for regulating glucocorticoid receptor (GR)-dependent transcription (Schneikert et al, 1999).…”