Figure 3. mRNA and protein expression of DRP-1b. A. DRP-1b and DRP-1 mRNA fragments were amplified by PCR, using total embryo mouse cDNA from the indicated days as template, followed by ethidium bromide gel detection. B. Western blot detection of DRP-1b and DRP-1 protein levels in brain tissues of mice and human embryonic stem cells, using anti N’-DRP-1 antibody. E12- embryonic day 12, P5 - postnatal day 5, P42 – postnatal day 42 (adult mouse). doi:10.1371/journal.pone.0017344.g003 DAP kinase family of proteins induce membrane blebbing by phosphorylating the regulatory light chain of myosin II, MLC [1]. To verify that DRP-1b retains this ability, we performed an in vitro kinase assay, using MLC as substrate. As shown in Figure 4D, DRP-1b can phosphorylate MLC on serine 19 to a level comparable to ZIPk and DRP-1. Transmission electron microscopy (TEM) studies were next performed to find out whether DRP-1b induces the accumulation of autophagosomes like the other members of the DAPk family. It was found that double membrane vesicles characteristic of autophagosomes were clearly evident upon DRP-1b transfection; the autophagosomes were detected at high number within the membrane blebs (Figure 5A) and the cell body (Figure 5B). This is consistent with the autophagic phenotype induced by DRP-1 and ZIPk ([8,21], and Kimchi et al., unpublished data). Western blot analysis revealed that the ectopic expression of each of the three kinases induced the conversion of LC3-I to the lipidated LC3-II form which is a marker of autophagy activation (Figure S3) while none of them activated caspases, a marker of apoptosis (data not shown). Altogether, the ectopic expression experiments indicate that DRP-1b shares some biochemical and cellular properties with both DRP-1 and ZIPk, including MLC phosphorylation, mem- brane blebbing and autophagy. DRP-1b - A Novel ZIPk-Like Isoform DRP-1b and ZIPk but not DRP-1 share a common interacting protein Since DRP-1b shows high similarity to ZIPk, we next examined whether it retains some of the ZIPk unique characteristics. The high degree of conservation of the leucine zipper-like motif of ZIPk in DRP-1b led us to examine whether both proteins can interact with the same partners through this structural domain. Activating transcription factor 4 (ATF4) was previously shown to bind ZIPk through its leucine zipper [25] and was thus selected for this study. To this end, we performed a co-immunoprecipitation experi- ment to examine whether the ectopically expressed DRP-1b and ATF4 proteins interact with each other. As shown in Figure 6, both ZIPk and DRP-1b were able to pull down ATF4, while DRP- 1 could not. Thus, DRP-1b shares at least one interacting protein with ZIPk, a function gained by the alternative splicing which does not exist in the canonical DRP-1 isoform. A leucine zipper mutant of DRP-1b, in which three key hydrophobic amino acids (at the d position of the heptamer in Figure S2) were substituted to alanines, displayed a reduced ability to pull down ATF4 (Figure S4). DRP-1 and ZIPk evolved from DAPk at the emergence of jawed vertebrates To find out the relation between the different DAPK family members we calculated a phylogenetic dendogram from an alignment of the kinase catalytic domains from DAPk, DRP-1 and ZIPk. To determine the root of the dendogram and its time dimension we included DRAK proteins that are more distant from DAPk, DRP-1 and ZIPk proteins than the distance in between these members [1,26]. (Figure 7) The resulting dendogram clearly separates the DAPk, DRP-1 and ZIPk proteins from each other, and within each cluster most sequences are grouped according to accepted taxonomic relations of the species they are found in. DAPk proteins are most diverse, confirming their identification in both invertebrates and verte- brates. DRP-1 and ZIPk clusters appear on the dendogram next to the vertebrate DAPk cluster. The closest cluster to these three vertebrate clusters is a clearly separated (bootstrap value of 95/ 100) and earlier branching cluster with two lamprey sequences. The sequences from this jawless vertebrate have a DAPk kinase domain (but their extra catalytic domain is yet undetermined), and their dendogram position is in between the jawed vertebrate DAPk proteins and the DAPk proteins of other invertebrates, simpler chordates (i.e, Amphioxus), and urochordates (i.e, Ciona). DRP-1 and ZIPk clusters each include a sequence from a shark species. The Elephant shark (Callorhinchus milii) includes at least five DAPk genes but their publicly available sequences are partial, highly fragmented, and most are too short to include in phylogenetic dendograms (SP, data not shown). Ciona species belong to a basal urochordate sub phyla that diverged before the emergence of vertebrates [27]. Two Ciona species C.intestinalis and C.savignyi include a DAPk protein with an extra catalytic domain different from those of DAPk, DRP-1 and ZIPk. These proteins are clearly placed within the DAPk cluster on the dendogram, showing them to be a novel ‘‘offshoot’’ of these proteins. Other interesting DAPk sequences appear in the insects Honeybee (Apis mellifera), Jewel wasp (Nasonia vitripennis), ants (Camponotus floridanus, Harpegnathos saltator, Atta cephalotes), and Red flour beetle (Tribolium castaneum). No DAPk sequences were identified in any of the Drosophila and mosquito genomes sequenced so far, but probable DAPk sequences are found in other arthropods including insects, arachnids, and crustaceans (Table S1). It thus seems most likely that the DAPk gene was lost in a dipteran progenitor of mosquitos and flies. Partial sequences of PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e17344