Figure 6. ZIPk and DRP-1b bind ATF-4, while DRP-1 fails to do so. HEK293T cells were co-transfected with the indicated vectors and harvested 24 h post transfection. Lysates were immunoprecipitated using anti-FLAG antibodies, and protein levels were detected using western blot. doi:10.1371/journal.pone.0017344.g006 similar role for DRP-1b. These possibilities should be further pursued in order to establish them. In any case, this interaction proves that DRP-1b, although possessing the DRP-1 kinase domain, has functions similar to those of ZIPk. DRP-1b has also lost some of the DRP-1 features, like the regulation by Ca+2/CaM. The conservation of DRP-1b and ZIPk and their gene organization across vertebrates, perhaps even from the very emergence of this subphylum, excludes complete redundancy and indicates some essential and specific roles for both kinases. On the one hand the localization of the ZIPk like extra-catalytic exon in the same genetic locus of DRP-1 may allow a coordinated control of both DRP-1 and DRP-1b as opposed to their placement in separate genomic loci. On the other hand, the additional presence of ZIPk on a distinct locus is assumed to have a significant advantage suggesting some unidentified yet functional difference between ZIPk and DRP-1b. The two genes are driven by different promoters, and their extra-catalytic domains are not completely identical (Figure 2). The discovery of a ZIPk-like encoding exon sheds light on the evolution of the entire DAPK family, and enables us to hypothesize on the events leading to the emergence of DRP-1 and ZIPk genes. One parsimonious course of evolutionary events that could have led to the present nature and distribution of DAPk genes is shown in Figure 8. DAP-kinase emerged early in the evolution of metazoa, appearing already in nematodes, flat worms and other bilateral invertebrates as a single DAPk gene in their genomes. Around the development of the jawed vertebrates, the DAPk gene underwent a duplication with one copy losing the extra catalytic coding regions downstream of the CaM regulatory domain and acquiring (from an unknown source) two single coding-exon regions for protein dimerization. A region coding for a leucine-zipper like dimerization domain was inserted between the catalytic domain exons and the CaM regulatory domain, and a much shorter exon coding for a different dimerization domain was inserted downstream of the CaM regulatory domain. We have no data at present to determine if this happened in a single or multiple events. The resulting gene progenitor of DRP-1 and ZIPk then underwent a second gene duplication. One gene lost the Ca+2/ CaM and adjacent dimerization exons, creating the ZIPk progenitor. The second gene kept all exons, but probably expressed either the leucine zipper encoding exon or the 39 Ca+2/CaM and dimerization exons by alternate splicing, thus giving rise to DRP-1 and DRP-1b isoforms. In conclusion, our integrated research approach and analysis of diverse data allowed us to identify a complex case of gene DRP-1b - A Novel ZIPk-Like Isoform evolution and expression. Examining all available DRP-1 loci confirmed the coding nature of the cryptic exon which we found. This, together with subsequent experimental data, transformed, in turn, the absence of human and mouse ESTs for this exon, from a trivial and uninformative observation to a hypothesis for a restricted and potentially interesting expression of a new DRP-1 isoform. Identifying the DRP-1b isoform in the brain of embryonic and young mice excluding the adult phase may have functional implications for our future understanding of why such domain modularity evolved in this family of death –inducing kinases. Materials and Methods Sequences Sequences accession numbers and compositions are detailed in the supplementary material. Multiple sequence alignments and Phylogenetic tree Multiple sequence alignments were generated using the BLAST [30], DIALIGN2 [31], GLAM2 [32], and LAMA [33] programs. Sequence reads were assembled using the CAP3 program [34]. Sequences were aligned and edited analysis with Se-Al (http:// tree.bio.ed.ac.uk/software/seal/) program. Sequence dendograms were calculated based on the multiple alignment of the catalytic domains of DAPk, DRP-1 and ZIPk, and rooted using the catalytic domain of DRAK, using the PHYML v.2.4.4 program [35]. Plasmids Human DRP-1 and ZIPk plasmid were previously described [13,21]. Human DRP-1b exon was amplified through PCR using genomic DNA as template, ligated to DRP-1 catalytic domain coding sequence and finally subcloned into a FLAG-tagged pcDNA3 expression plasmid. HA tagged human ATF4 expression plasmids were kindly provided by Prof. Michael S. Kilberg and by Prof. Fung-Fang Wang. The leucine zipper perturbation of DRP- 1b was created by introducing the substitutions L433A/I440A/ F447A to human DRP-1b, using the PCR site-directed mutagen- esis protocol. mRNA and protein detection DRP-1 and DRP-1b mRNA detection was performed using PCR amplification with cross-exon primers. Mouse embryo cDNA (MD-104, Zygen) of the indicated days was used as template. Anti- DRP-1 (N’ terminus) Rabbit monoclonal antibody (dilution 1:500) (AbCaM, EP1633Y) was used for endogenous DRP-1b protein detection in lysates of the indicated cells or tissues. Mouse brain tissues were kindly provided by Prof. Orly Reiner. Cell culture and transient transfection 293T Human Embryonic Kidney (HEK) cells and HeLa cells were grown in DMEM (Biological Industries) supplemented with 10% fetal bovine serum (Hyclone) and 1% L-Glutamine (GibcoBRL) and a mixture of antibiotics (100 u/ml penicillin and 0.1 mg/ml streptomycin). For transient transfections, 1.26106 (293T) or 0.86106 (HeLa) cells were plated on 9 cm plates 24 hours prior to transfection. Transfections were done by the calcium phosphate method with 10 mg DNA per plate. To assess the membrane blebbing potency of DRP-1b, DRP-1 and ZIPk, 293T cells were transfected with the appropriate plasmid and 1 mg of peGFP expression vector. After 24 hours, green cells were counted and the percent of blebbing cells was calculated. Western PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e17344