Evolutionarily conserved Bok proteins in the Bcl‐2 family (2000)

Apoptosis is under tight control of rather complex regulatory circuits in which proteins of the Bcl-2 family act as a critical life–death decision point. The Bcl-2 family consists of a growing number of proteins [1]. In Caenorhabditis elegans there is only one member, ced-9, existing. In mammalian cells the regulation of apoptotic process by the Bcl-2 family appears to be very complex. More than 10 members of the Bcl-2-related proteins have been identified so far. Previously, we cloned a human Bok gene (Hbok) from a human infant brain library (GenBank accession number AF174487). By search of the expressed sequence tags (EST) database/Drosophila genome sequence database using the amino acid sequences of HBcl-xL and HBok, and by use of polymerase chain reaction (PCR), we further identified two Drosophila and a chicken Bok homologue (Fig. 1). Briefly, a Blast Search of the GenBank database ESTs was performed and a partial nucleotide sequence of a Drosophila cDNA was identified. Subsequently, 5′-RACE PCR was performed on mRNA of Drosophila Schneider L2 (S2) cells to amplify the 5′ sequences, which were then to be compared to genomic sequences from Flybase. This resulted in obtaining the entire coding region containing a 978 nt open reading frame (Fig. 1A). The predicted amino acid sequence showed 35% identity to the human Bok protein, but about 25% identity to other Bcl-2-related proteins. This gene was then named Dbok. Dbok is identical to Drob-1 recently published by Igaki et al. [2], but longer than Debcl from Colussi et al. [3]. Alignment of cDNA sequence along with genomic sequences from the Drosophila genome sequence database revealed that the genomic sequence of Dbok contains three exons. This result and a further search of the Drosophila genomic sequence database recovered a sequence which represents another distant Bcl-2-related gene, named Dbx (Fig. 1A). In addition, we identified a chicken Bok from three chicken cDNA clones, which contain partial nucleotide sequences. Sequence analysis resulted in identification of a 1158 nt full-length cDNA which contains a 641 nt coding sequence. Its amino acid sequence shares 81% identity with that of human Bok (Fig. 1A). The analysis of Bok proteins with other members in the Bcl-2 family revealed that they contain all four different BH domains, BH1, BH2, BH3 and BH4 (Fig. 1A). But both Drosophila homologues contain two BH4 domains, whereas chicken and human Bok contain only one BH4 domain (Fig. 1B). The alignment shows that a highly conserved stretch of amino acids, ITWGK, is present in the BH1 domain. It differs in this region from other Bcl-2 family members and is a characteristic for this sub-group in the Bcl-2 family (Fig. 1A). Dbok has a long N-terminus with an additional BH4 domain as compared to other Bcl-2 related proteins. The topology prediction of the membrane regions of Dbok showed that addition to a candidate membrane-spanning segment of amino acids 221–241 (BH1 domain) and a putative hydrophobic membrane anchor between amino acid 290 and 310, both of which have been shown to have important features, the N-terminal sequence also contains a transmembrane segment within amino acids 14–34 [4]. This region is not present in vertebrates. To explore the structure and function relationship of Dbok, we have used an apoptosis assay, in which apoptotic REF52 cells round up at an early apoptotic stage and detach from the surface of the dish [5]. The DNA encoding full-length Dbok was cloned into the pcDNA3 expression vector. The constructs were transfected into REF52 rat fibroblasts together with a vector (pEGFP-N1) encoding the green fluorescent protein (GFP). GFP served as marker for successfully transfected cells. 15 h after transfection, apoptotic REF52 cells were counted under a fluorescence microscope [5]. As shown in Fig. 1C, 36% of cells transfected with a plasmid carrying the full-length Dbok cDNA became apoptotic, whereas the expression of Bax induced 38% of apoptosis. Furthermore most of the Dbok-transfected cells could be rescued by co-expression of Bcl-xL or BFL-1. The N-terminal-truncated Dbok construct, Dbok-N, in which the first BH4 domain has been deleted, did not lose the apoptotic activity in REF52 cells, indicating that the N-terminal region is not necessary for its apoptotic function. One possible role of this region is not directly involved in the cell killing but rather in mediating the conformation of the protein through apoptosis stimuli. However, the C-terminal-truncated construct, Dbok-C, which lacks a putative transmembrane domain and the Dbok-BH3, which is a BH3 domain deletion construct, lost their killing activity. These results are consistent with the previous observations that mutants of the Bcl-2-related proteins lacking the transmembrane domain in the C-terminus are functionally ineffective. The loss of function of the C-terminal mutant indicated that the localization of Dbok is important for its function. Thus, Dbok is a pro-apoptotic protein which functions through the BH3 domain. The C-terminal sequence and the BH3 domain of Dbok are indispensable for the pro-apoptotic function but the N-terminal sequence is not required for its apoptotic potential. At this stage we do not know the function of Dbx yet. The amino acid sequence of Dbx is very similar to that of Dbok and Dbx also contains the BH4 and BH3 domains. Most interestingly a proline residue is present in the middle of the BH3 domain, which may well change conformationally the local helical structure of the BH3 domain based on the structural analysis of the Bcl-xL and Bfl-1 [5]. Further experiments are necessary to determine the function of this protein. Like Bok proteins from Drosophila, rat and human, chicken Bok may well function as a pro-apoptotic protein. Most recently, two Bcl-2 homology proteins have been isolated from two different species of sponges [6]. Here we report the identification of three Bok protein genes from Drosophila, human and chicken. These results indicate that the apoptosis pathway is evolutionarily conserved even from sponges to human and the complexity of regulation of apoptosis fits to the evolutionary relationships among animals. H.Z. is supported by The Basler Cancer league (Krebsliga Beider Basel, Grant 8/99). We are grateful to Dr. A. Strasser (Novartis Pharma Research) for providing the SL2 cells and H. He (Institute of Zoology/Basel University) for mRNA from multi-stages of Drosophila.