New Modularity of DAP-Kinases: Alternative Splicing of the DRP-1 Gene Produces a ZIPk-Like Isoform Yishay Shoval, Hanna Berissi, Adi Kimchi*, Shmuel Pietrokovski* Department of Molecular Biology, Weizmann Institute of Science, Rehovot, Israel Abstract DRP-1 and ZIPk are two members of the Death Associated Protein Ser/Thr Kinase (DAP-kinase) family, which function in different settings of cell death including autophagy. DAP kinases are very similar in their catalytic domains but differ substantially in their extra-catalytic domains. This difference is crucial for the significantly different modes of regulation and function among DAP kinases. Here we report the identification of a novel alternatively spliced kinase isoform of the DRP-1 gene, termed DRP-1b. The alternative splicing event replaces the whole extra catalytic domain of DRP-1 with a single coding exon that is closely related to the sequence of the extra catalytic domain of ZIPk. As a consequence, DRP-1b lacks the calmodulin regulatory domain of DRP-1, and instead contains a leucine zipper-like motif similar to the protein binding region of ZIPk. Several functional assays proved that this new isoform retained the biochemical and cellular properties that are common to DRP-1 and ZIPk, including myosin light chain phosphorylation, and activation of membrane blebbing and autophagy. In addition, DRP-1b also acquired binding to the ATF4 transcription factor, a feature characteristic of ZIPk but not DRP-1. Thus, a splicing event of the DRP-1 produces a ZIPk like isoform. DRP-1b is highly conserved in evolution, present in all known vertebrate DRP-1 loci. We detected the corresponding mRNA and protein in embryonic mouse brains and in human embryonic stem cells thus confirming the in vivo utilization of this isoform. The discovery of module conservation within the DAPk family members illustrates a parsimonious way to increase the functional complexity within protein families. It also provides crucial data for modeling the expansion and evolution of DAP kinase proteins within vertebrates, suggesting that DRP-1 and ZIPk most likely evolved from their ancient ancestor gene DAPk by two gene duplication events that occurred close to the emergence of vertebrates. Citation: Shoval Y, Berissi H, Kimchi A, Pietrokovski S (2011) New Modularity of DAP-Kinases: Alternative Splicing of the DRP-1 Gene Produces a ZIPk-Like Isoform. PLoS ONE 6(2): e17344. doi:10.1371/journal.pone.0017344 Editor: Richard Cordaux, University of Poitiers, France Received September 13, 2010; Accepted January 29, 2011; Published March 8, 2011 Copyright: 2011 Shoval et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Center of Excellence grant from the Flight Attendant Medical Research Institute (FAMRI), by a grant from the European Union FP7 consortium (APO-SYS), and by a grant from the Israel Science Foundation (ISF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Shmuel.Pietrokovski@weizmann.ac.il (SP); Adi.Kimchi@weizmann.ac.il (AK) Introduction The Death Associated Protein Kinase (DAPK) family of proteins is a family of five Ser/Thr kinases which are very similar in their catalytic domain and are involved in programmed cell death (PCD) mechanisms. Three members including DAPk (also called DAPK1), DRP-1, (also called DAPK2), and ZIP-kinase (ZIPk, also called DAPK3), share about 80% identity in their catalytic domains thus creating a sub-family which is in the focus of this work. Two other members, DAPk Related Apoptosis inducing Kinase 1 and 2 (DRAK1 and DRAK2) are more distantly related, sharing only about 50% identity with DAPk [1]; also see Figure 1A). DAPk is a 160 kDa, multi domain Ca+2/Calmodulin (CaM) regulated, Ser/Thr kinase. In addition to the catalytic and the CaM regulatory domains, it possesses several ankyrin repeats, a potential P loop motif, a cytoskeleton binding domain, a death domain and a C-terminal Serine rich region (Figure 1A). Ectopic expression of DAPk (as well as of ZIPk and DRP-1) induces membrane blebbing and cellular rounding through the phosphor- ylation of the regulatory light chain of myosin II (MLC). DAPk is activated by dephosphorylation of a specific site in the CaM regulatory domain and by Ca+2/CaM binding [2]. DAPk is involved in several pathways leading to cell death, including apoptosis, autophagy and anoikis-like cell death. It mediates several types of stress signals induced by IFN-c TNF-a, Fas, TGF- b, ceramides, deprivation of neuronal cells from Netrin-1, and stimulation of NMDA receptors in cerebral ischemia [3,4]. The gene is frequently silenced in cancer by promoter DNA methylation, suggesting that it functions as a tumor suppressor [5]. Moreover, a germline mutation in the human DAPK1 promoter leads to a familial case of Chronic Lymphocytic Leukemia CLL [6]. DAPk may have also other functions, not related to PCD, such as a role in cytokinesis and cell migration [7,8,9,10]. The DAPK1 gene is well conserved in evolution from various invertebrates, such as C. elegans [11], to chordates and mammals. DRP-1 is a 42 kDa cell death-promoting kinase. Like DAPk, it contains a CaM regulatory domain which shares high sequence and functional similarity with that of DAPk, but its C- terminus differs completely from DAPk, possessing a unique 40 amino acid tail at its C terminus necessary for stabilizing the homo-dimerization state of the kinase [12]. Full activation of DRP-1 depends on relieving the inhibitory effects of the CaM regulatory domain by its binding to Ca+2/CaM and by the dephosphorylation of an critical Ser residue in this domain similar to DAPk regulation. In addition, homo-dimerzation is also PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e17344