necessary for full activation of the DRP-1, as long as the CaM regulatory domain is present [12]. DRP-1 is a cytoplasmic protein, and upon ectopic expression it induces autophagy, and caspase- independent autophagic cell death [8]. TNF-a induces both dephosphorylation and dimerization of DRP-1 and a functional interaction between DRP-1 and DAPk has been proposed as well [8,12,13,14]. The third member of the DAPk family is ZIP kinase, a 55 kDa, Ser/Thr kinase. ZIPk-induced cell death can involve both caspase dependent and independent pathways, the former being mito- chondrial dependent [15]. Unlike DAPk and DRP-1, ZIPk is not regulated by Ca+2/CaM. It contains a leucine zipper like domain at the C-terminus, needed for homo-oligomerization that is critical for its death promoting effects. ZIPk also contains a nuclear localization signals (NLS) and is localized both in the nucleus and the cytoplasm. Quite surprisingly it was recently found that the murine orthologs of ZIPk underwent a unique type of sequence divergence compared to other vertebrate species. As a conse- quence they are localized exclusively to the nucleus and also acquired several additional changes to compensate for their divergence [16]. A non-PCD function of ZIPk was observed in smooth muscle cells, where ZIPk-dependent phosphorylation of MLC led to Ca+2 sensitization and smooth muscle contraction. This was attributed to direct phosphorylation of MLC as well as inactivation of Smooth Muscle Myosin Phosphatase (SMMP-1M), through phosphorylation of the phosphatase’s myosin binding subunit, and phosphorylation of its inhibitor protein CPI17 [17,18]. Both DRP-1 and ZIPk genes are only present in vertebrates. Previously it has been shown in our lab that there is a physical and functional cross talk between ZIPk and DAPk. DAPk is able to trans-phosphorylate ZIPk on six distinct sites in the extra-catalytic domain, thus increasing the cytoplasmic localization of ZIPk and the homo-trimerization towards a more potent cell death inducer. Accordingly, co-expression of both kinases causes a synergistic effect in promoting the membrae blebbing phenotype [15,19,20,21,22,23]. These data, together with the epistatic relationship mentioned above, imply that the DAPk family may have a signaling capacity greater than the sum of signaling attributed to its individual members, perhaps even creating a cell death inducing kinase-kinase cascade. In this work we illustrate an additional level of complexity in which transcripts derived from the genomic locus of DRP-1 can undergo alternative splicing to give rise to a new kinase isoform, found to be expressed in embryonic stem cells and brain tissues. The alternatively spliced exon is homologous and highly similar to the C-terminus of ZIPk, thus generating a novel DRP-1 kinase isoform which shares functional characteristics with ZIPk. The DRP-1 gene organization, and its potential for alternate isoforms, is conserved in all known DRP-1 loci. Together with known sequences of other DAP kinases this provides an evolutionary model for the expansion and evolution of these kinases within vertebrates, and suggests that the DAP kinases sequence diversion is accompanied by retained sequence features. Results The genomic locus of DRP-1 contains an alternative exon Analyzing genomic loci of DRP-1 (DAPk2) we identified a previously unknown putative exon. The exon is found in all vertebrate DRP-1 gene loci that have been sequenced (Figure S1), is well conserved, and codes for 165–220 amino acids. This region is significantly similar to only one protein in the current sequence databases, to the extra catalytic domain of ZIPk (DAPK3), which DRP-1b - A Novel ZIPk-Like Isoform is also encoded by a single exon. The new DRP-1 exon is located between its previously known exons 8 and 9, immediately downstream of the catalytic domain encoding exons, and upstream of the two exons coding for DRP-1 regulatory extra catalytic domain (Figure 1B). All DRP-1 loci, from fish to mammals, include a tightly conserved splice acceptor sequence at the 39 end of the upstream intron (Figure 1C), and a stop codon in a well conserved position. Sequence analysis of the coding capacity of the exon shows its conservation pattern is typical to protein coding regions and includes numerous codons undergoing purifying selection (data not shown). Thus DRP-1 has the potential to encode another isoform to its previously known product, where an alternative splicing event will replace the known CaM regulatory and dimerization domains with a ZIPk-like extra catalytic domain. The human DRP-1 exon we identified is 202 amino acids long. Residues 5–165 of this exon have 42% identity (and 65% similarity) to residues 289–444 of human ZIPk (Figs. 1B and 2). The C-terminal 37 amino acids of the exon are Ser/Thr rich, with no significant similarity to any known protein in the current sequence databases. Thus, in case of an alternative splicing event at the locus of DRP-1, the predicted translated protein would be very similar to ZIPk- e.g., in humans 79% identity in the catalytic domain and 42% identity in the extra catalytic domain (Fig. 1B). Unlike DRP-1, this protein is not expected to be regulated by calcium, as it lacks its Ca+2/CaM binding domain. Detailed analysis of available sequence data identified transcripts of the new isoform in pig, cow, chicken and several fish (Table S1). We termed the alternative spliced isoform DRP-1b. Sequence analysis of DRP-1b alternative exon To further study the features of the DRP-1 new exon, we aligned all the protein sequences we found for it and compared the alignment to a similar alignment of the ZIPk extra catalytic domain (Figure 2). The two regions are very similar and can be confidently aligned across their N-terminal 80%. The most conserved region is at human ZIPk positions 297–332. ZIPk contains at this region several sites that are phosphorylated by DAPk and several autophosphorylation sites, shown to be important for full activation of the protein [21,24]. Most of these sites are conserved in DRP-1b (Figure 2) and may undergo similar regulation. Another conserved region corresponds to the Leucine zipper-like motif of ZIPk, at position 433–447 of DRP-1b, especially due to the presence of hydrophobic amino acids at the key positions 433/440/447 of the heptameric repeat that creates the zipper itself [25] (Figure S2). Sequence prediction for coiled coil domains showed both these DRP-1b and ZIPk regions to most probably adopt this structure, as expected for Leucine Zipper type dimerization regions (data not shown). It is interesting to note that unlike ZIPk, DRP-1b is conserved in murines, and did not undergo the murine-specific divergence characteristic of murine ZIPk which we have previously described [16]. Thus, while the extra catalytic domain of mouse ZIPk shows only 81% similarity to that of human ZIPk, mouse alternative exon DRP-1b is 92% similar to its human ortholog, This suggests that DRP-1b has a distinct, separate role from ZIPk, and thus was not under the same evolutionary pressure which led ZIPk to diverge from the common consensus in murines. mRNA and protein expression of DRP-1b Database searches identified a few DRP-1b expressed sequence tags (ESTs) suggesting that this isoform may be expressed in some settings (Table S1). Yet the low number of DRP-1b ESTs stands in contrast to the numerous ESTs of DAPk, DRP-1 and ZIPk PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e17344