DRP-1b - A Novel ZIPk-Like Isoform Figure 4. Ectopically expressed DRP-1b induces MLC phosphorylation and membrane blebbing in cells. A. DRP-1b ectopic expression induces membrane blebbing. HEK293T cells were co-transfected with FLAG DRP-1b and GFP expression vectors, and examined under fluorescent microscope after 24 h. White arrows- cells exhibiting membrane blebs. B. Quantification of the blebbing inducing ability of ZIPk, DRP-1 and DRP-1b. Note that the number of blebbed cells in cells transfested with control plasmids is below detection levels C. Western blot detection of the kinases, (detection was done with anti-FLAG Abs, running the samples in the same gels and the same exposure time of the blots) indicating comparable expression levels. D. ZIPk, DRP-1b and DRP-1 phosphorylate myosin light chain (MLC). FLAG tagged kinases were expressed in HEK293T, immunoprecipitated using anti-FLAG antibodies and eluted from beads. His-tagged MLC was purified from bacteria, and used as a substrate in an in vitro kinase assay. MLC phosphorylation was detected using an antibody against phospho-serine 19 on MLC. doi:10.1371/journal.pone.0017344.g004 DAPK-like kinase domains from cnidaria, basal metazoa with radial symmetry, could be found and cluster with the invertebrate DAPk sequences. However, they are not all clustered together and their exact nature would be better understood once their full sequences will be available. Fish DRP-1 genes Fish exhibit a unique composition of DAP kinases. Two DRP-1 gene loci were found by us in each of six different teleost fish species where relatively large genomic and EST sequence data is available. These two DRP-1 sub-type groups cluster together (Figure 8) and are the probable result of the whole genome duplication that occurred after the divergence of ray-finned fish [28]. One fish DRP-1 sub type has the same genomic structure as the DRP-1 gene in other vertebrates, i.e., including 39 exons coding for both the previously known DRP-1 and ZIPk-like extra- catalytic domains. The second fish DRP-1 sub type gene only has the exon for the ZIPk-like domain, missing the previously known DRP-1 39 exons (Figure 1A). In zebrafish there is another change - its first DRP-1 sub type gene does not have a C-terminal ZIPk-like exon. Thus, the two zebrafish DRP-1 genes each have a different extra-catalytic domain. It is possible that in the teleost fish, DRP-1 gene has evolved, or is even yet evolving, into two distinct genes. The function, or at least coding capacity, of these duplicated DRP-1 genes could be equivalent to the different transcripts of the single DRP-1 gene of other vertebrates. It is also interesting that we found no evidence in fish for DAPk and ZIPk gene duplicates. Perhaps the two DRP-1 gene duplicates were retained after the fish whole genome duplication because of the distinct alternative messages possible from their progenitor gene. Discussion Here we report the discovery of a novel member of the DAP kinase family DRP-1b, highly conserved from fish to mammals, which is generated by alternative splicing event of the DRP-1 gene. The uniqueness of this isoform is in its close resemblance to ZIPk, another member of the DAP kinases family, due to an interesting modular organization discovered here. The resulting modular cross similarity within the DAP kinase proteins could allow for intricate control of their function and is unique, to the best of our knowledge. We show that this alternative splicing takes place in mouse embryonic tissues and that the product of the new isoform is expressed in the embryonic brains of mice and human embryonic stem cells. We further show that DRP-1b is an active kinase, able to phosphorylate MLC on serine 19 and induce membrane blebbing, and autophagic vesicle formation. Further studies will need to be conducted in order to determine the physiological roles of DRP-1b and the specific differences between this kinase and the other DAP kinases, that appear to have kept the relative complicated DAP kinase genes arrangement throughout verte- brate evolution. It is interesting to note that a recent paper by Tang et al. shows that ZIPk plays a role in induction of autophagy by phosphorylating the ULK1 protein [29]. It should be examined if DRP-1b can perform the same function. The interaction of DRP-1b and ATF-4 has unclear functional implications, as the function of the interaction of ZIPk and ATF-4 is not known either. It is possible that these interactions sequester ATF-4 from the nucleus and its genomic targets, thus halting the induction of pro-survival genes. On the other hand, it has been suggested that ATF-4 may block the pro-death activities of ZIPk by preventing its homo dimerization [25], and it may play a Figure 5. Ectopic expression of DRP-1b induces the accumulation of autophagic vesicles. HEK293T cells were transfected with DRP-1b expression vector, fixed 24 h after transfection and examined using Transmission Electron Microscopy (TEM). A. a cell undergoing membrane blebbing; B. larger magnification of induced vesicles. Arrowheads indicate double membrane, autophagic vesicles. doi:10.1371/journal.pone.0017344.g005 PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e17344