Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation.
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.
Packham
G
G
Ludwig Institute for Cancer Research, Imperial College School of Medicine at St Mary's, London W2 1PG, U.K.
Brimmell
M
M
Cleveland
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D002458:Cell Fractionation; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014407:Tumor Cells, Cultured
C091732:BCL2-associated athanogene 1 protein; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D014157:Transcription Factors