Death-Associated Protein Kinase 2 Is a New Calcium/Calmodulin-Dependent Protein Kinase That Signals Apoptosis Through Its Catalytic Activity

Oncogene (1999) 18, 3471 ± 3480 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Death-associated protein kinase 2 is a new calcium/calmodulin-dependent protein kinase that signals apoptosis through its catalytic activity

Taro Kawai1,3, Fumiko Nomura1,3, Katsuaki Hoshino1,3, Neal G Copeland2, Debra J Gilbert2, Nancy A Jenkins2 and Shizuo Akira*,1,3

1Department of Biochemistry, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan; 2Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland, MD 21702, USA; 3CREST of Japan Science and Technology Corporation, Japan

We have identi®ed and characterized a new calcium/ gene ced-3 (Yuan et al., 1993; Nagata, 1997; calmodulin (Ca2+/CaM) dependent protein kinase termed Thornberry and Lazebnik, 1998). Mutant mice lacking death-associated protein kinase 2 (DAPK2) that contains the caspase family member revealed its essential role an N-terminal protein kinase domain followed by a for execution of apoptosis in vivo (Kuida et al., 1996, conserved CaM-binding domain with signi®cant homo- 1998; Hakem et al., 1998). In addition to the activation logies to those of DAP kinase, a protein kinase involved of caspases, apoptosis is known to be mediated by in apoptosis. DAPK2 mRNA is expressed abundantly in activation of c-Myc, p53 and protein kinases (Askew et heart, lung and skeletal muscle. The mapping results al., 1991; Evan et al., 1992; Lowe et al., 1993; indicated that DAPK2 is located in the central region of Anderson, 1997). Evidence has been presented that mouse chromosome 9. In vitro kinase assay revealed that certain protein kinases have an ability to induce DAPK2 is autophosphorylated and phosphorylates apoptosis, or are activated by various apoptotic myosin light chain (MLC) as an exogenous substrate. stimuli. Activation of cyclin dependent-protein kinase DAPK2 binds directly to CaM and is activated in a (CDK), Cdc2 kinase was correlated with apoptosis of Ca2+/CaM-dependent manner. A constitutively active lymphoma cells induced by granzyme B (Shi et al., DAPK2 mutant is generated by removal of the CaM- 1994). An additional CDK, p58 PITSRLE induced binding domain (DCaM). Treatment of agonists that apoptosis upon overexpression in Chinese hamster elevate intracellular Ca2+-concentration led to the ovary cells and was activated by Fas-stimulation activation of DAPK2 and transfection studies revealed (Lahti et al., 1995). FAST and RICK (also known as that DAPK2 is localized in the cytoplasm. Overexpres- RIP2) kinases are also implicated in Fas-mediated sion of DAPK2, but not the kinase negative mutant, apoptosis (Tian et al., 1995; Inohara et al., 1998; signi®cantly induced the morphological changes char- McCarthy et al., 1998). Various apoptotic stimuli such acteristic of apoptosis. These results indicate that as Fas, TNF-a, ceramide and stresses induce activation DAPK2 is an additional member of DAP kinase family of c-Jun N-terminal kinase (JNK), also known as stress involved in apoptotic signaling. activated protein kinase (SAPK) (Hibi et al., 1993; Derijard et al., 1994; Verheij et al., 1996; Xia et al., Keywords: DAP kinase family; calcium/calmodulin- 1995). This suggests the involvement of the JNK dependent kinase; apoptosis pathway in the apoptotic signaling. Indeed, upstream kinases of JNKs, MEKK1 and ASK1, and downstream target c-Jun were reported to induce apoptosis of target cells (Ichijo et al., 1997; Lassignal Johnson et Introduction al., 1996; Cardone et al., 1997; Widmann et al., 1998; Bossy-Wetzel et al., 1997). Activation of the calcium/ Apoptosis, an essential mechanism for normal devel- calmodulin (Ca2+/CaM) dependent protein kinase opment and maintainence of tissue homeostasis, can be (CaMK) family was also shown to mediate apoptosis induced in response to a variety of stimuli including in some cells. CaMKII is a multifunctional protein cytokines, growth factor withdrawal and stresses kinase that is expressed in various tissues, and is (White, 1996; Steller, 1995). Cells undergoing apopto- activated by apoptotic stimuli including tumor necrosis sis exhibit biochemical and morphological changes factor (TNF) -a and UV-irradiation at doses sucient including membrane blebbing, cell shrinkage, genomic for induction of apoptosis (Wright et al., 1997). DNA fragmentation, elevation of cytoplasmic Ca2+ Furthermore, selective inhibitors of CaMKII signifi- concentration and formation of an apoptotic body. cantly inhibited apoptosis induced by such stimuli. Increasing number of essential molecules that partici- DAP kinase is a member of the CaMK family that pate in these events have been identi®ed. Critical and was originally identi®ed as an essential molecule in widely used molecules that play a central role in the interferon (IFN) -g-induced apoptosis in HeLa cells apoptotic pathway are a caspase family of cystaine (Deiss et al., 1995; Cohen et al., 1997). It was shown proteases that are related to the C. elegans cell death that the reduced expression of DAP kinase mediated by antisense mRNA protected HeLa cells from apoptosis induced by IFN-g, and overexpression of *Correspondence: S Akira, Department of Biochemistry, Hyogo DAP kinase induced apoptosis (Cohen et al., 1997). College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663- 8501, Japan DAP kinase has been reported to function as a Received 2 October 1998; revised 14 January 1999; accepted 14 possible tumor suppressor gene (Kissil et al., 1997; January 1999 Inbal et al., 1997). Recently, ZIP kinase has been Apoptosis inducing kinase TKawaiet al 3472 identi®ed by the yeast two-hybrid screening in which strong similarity to that of DAP kinase. Ectopic the leucine zipper domain of ATF4, a member of expression of ZIP kinase also induced the morpholo- Activating Transcription Factor/ cAMP responsive gical changes of apoptosis in NIH3T3 cells. It was element binding protein (ATF/CREB) family of shown that the catalytic activities of both DAP- and transcriptional factor was used as a bait (Kawai et ZIP-kinases were required for the induction of al., 1998). The catalytic domain of ZIP kinase shares a apoptosis. Recently, we have identi®ed two novel

a c

Figure 1 Amino acid sequences of DAPK2 and alignment with other protein kinases. (a) Amino acid sequences of human and mouse DAPK2. Indicated are the putative kinase domains and the CaM-binding domains. Identical amino acid residues are highlighted with solid boxes. (b) Alignment of the kinase domain of human DAPK2 with those of human DAP kinase (DAPK), human ZIP kinase (ZIPK), human DRAK1 and human DRAK2. Amino acid residues conserved among at least three molecules are highlighted with solid boxes. (c) Alignment of the Ca2+/CaM binding domains between DAPK2 and DAP kinase. Amino acid residues conserved between two molecules are highlighted with solid boxes. (d) Amino acid identities of the catalytic domains of DAP kinase (DAPK), DAPK2, ZIP kinase (ZIPK), DRAK1 and DRAK2. (e) Unrooted phylogenic tree of the kinase domains of the DAP kinase family. The Clustal W program was used to align the kinase domains of the DAP kinase family. The unrooted phylogenic tree was constructed by the DRAWTREE program (Thompson et al., 1994). (f) Schematic representation of DAP kinase family. The known domains are indicated. The C-terminal region of DRAK1 shows homology to that of DRAK2 Apoptosis inducing kinase TKawaiet al 3473 protein kinases termed DAP kinase related apoptosis- domain, it was found that DAPK2 also shared inducing kinase (DRAK)1 and DRAK2, both of homology with DAP kinase but not ZIP kinase, which also show homology to DAP- and ZIP-kinases DRAK1 and DRAK2 just downstream from the in their catalytic domains (Sanjo et al., 1998). kinase domain (31.4% identity) (Figure 1c). It was Overexpression of DRAKs but not the kinase reported that there is a conserved CaM binding negative mutants also induced the morphological domain in this region of DAP kinase. Therefore, it is changes of apoptosis in NIH3T3 cells. Collectively, suggested that DAPK2 is also regulated by CaM these kinases represent a new family of protein kinase through the corresponding region as in the case of whose catalytic activity is sucient for induction of DAP kinase or other Ca2+/CaM kinases. Unlike DAP apoptosis. However, to date, the precise mechanisms kinase, no ankyrin repeats or death domain was found by which these kinases mediate apoptosis are unclear. in the C-terminal region of DAPK2 (Figure 1f). The C- In this report, we have identi®ed a novel protein terminal region of DAPK2 showed no homology to kinase designated DAP kinase 2 (DAPK2). DAPK2 any other proteins or other known domains. The contains an N-terminal protein kinase domain followed structure of the DAP kinase family member is shown by a region homologous to the Ca2+/CaM binding in Figure 1f. domain, both of which share homology with those of DAP kinase. Overexpression of DAPK2, but not the Tissue distribution of human DAPK2 mRNA kinase negative mutant induced the morphological changes of apoptosis in 293 and NIH3T3 cells. These We carried out the multiple tissue Northern blot to results indicate that DAPK2 is a new member of DAP investigate the tissue distribution of human DAPK2. kinase family involved in apoptosis. As shown in Figure 2, three major transcripts approximately the size of 6.0, 3.2 and 2.0 kb were detected in all tissues tested with strong hybridizing signals in heart, lung and skeletal muscle. The shortest Results transcript detected at 2.0 kb matched the obtained cDNA clone. The other transcripts were thought to be cDNA cloning and structure of human and mouse isoforms generated by alternative splicing or the DAPK2 transcripts encoding highly homologous proteins. To obtain cDNAs with signi®cant similarity to DAP kinase, we searched for EST clones in the DDBJ, In vitro kinase activity of DAPK2 EMBL and GenBank databases with the nucleotide sequence of the catalytic domain of human DAP To investigate the biochemical function and the kinase as the query. A search using the BLAST regulation of DAPK2, we carried out in vitro kinase program revealed that two EST clones with the accession numbers AA858002 (human) and W82116 (mouse) showed a high degree of homology to the catalytic domain of DAP kinase. On the basis of the reported sequences of these clones, we performed the RACE protocols with the corresponding primers to isolate full length cDNAs from human skeletal muscle and mouse lung cDNA libraries. Considering the sequence similarity, we designated this novel molecule DAP kinase 2 (DAPK2). Predicted amino acid sequences of both human and mouse DAPK2 are shown in Figure 1a. Both DAPK2 contain an open reading frame of 1110 bp and were predicted to encode a protein of 370 amino acid residues with a calculated molecular weight of 42.897 kDa (human) and 42.768 kDa (mouse). The N-terminal end of DAPK2 demonstrated a signi®cant homology to the catalytic domain of DAP kinase that began with amino acid 23 and extended to 285, and shared 80.2% sequence identity in the protein kinase domain (Figure 1b). Recently, we have identi®ed ZIP kinase, DRAK1 and DRAK2 that also show signi®cant sequence similarities to DAP kinase in the catalytic domains (Kawai et al., 1998; Sanjo et al., 1998). Additionally, the protein kinase domain of DAPK2 showed homology to ZIP kinase, DRAK1 and DRAK2 with 80.6, 46.4 and 48.3% identities, respectively (Figure 1b and d). Based on the sequence similarity and their biological functions, we propose that these new kinases represent a new family, which we designate as the DAP kinase Figure 2 Tissue distribution of DAPK2. Full length cDNA of family. The amino acid identities and dendrogram of human DAPK2 was used to probe human Multiple Tissue the catalytic domains of the DAP kinase family were Northern blot containing 2 mg of poly (A)+ RNA isolated from shown in Figure 1d and e. In addition to the catalytic various tissues Apoptosis inducing kinase TKawaiet al 3474 assay for DAPK2. To express DAPK2 into mamma- binding domain. These results are in accord with the lian cells, the N-terminal Flag epitope-tagged wild type case of DAP kinase, as well as other Ca2+/CaM protein (WT) DAPK2 was constructed into the pEF-BOS kinases (Cohen et al., 1997). expression vector. We also prepared two versions of the mutant DAPK2; a mutant in which the lysine DAPK2 is a Ca2+/CaM-dependent protein kinase residue at amino acid 52 that is predicted to the ATP- binding site was mutated to alanine (Flag-DAPK2 The region just downstream to the catalytic domain K52A) and a mutant missing the region corresponding shares homology to the CaM-binding domain of DAP to the CaM-binding domain of DAP kinase (aa 299 ± kinase (Figure 1c), suggesting the regulation of 330) (Flag-DAPK2 DCaM). Human embryonic kidney DAPK2 by Ca2+/CaM. The eect of Ca2+/CaM on 293 cells were transiently transfected with Flag-DAPK2 the kinase activity of DAPK2 was examined by in vitro WT, K52A and DCaM. Cell lysates were prepared and kinase assay. The Flag-DAPK2 WT was introduced immunoprecipitated with anti-Flag M2 monoclonal antibody. Kinase activity in the immunoprecipitant was measured by in vitro kinase assay. As shown in Figure 3, the phosphorylation of Flag-DAPK2 WT a protein, but not K52A mutant, was detected at 42 kDa as expected, indicating that DAPK2 acts as a protein kinase and the band found in WT is a result from autophosphorylation of DAPK2. The fact that the catalytic domain of DAPK2 shares a signi®cant homology to that of DAP kinase allowed us to investigate whether myosin light chain (MLC) is a potential exogeneous substrate because DAPK2 and DAP kinase showed homology to MLCK and DAP kinase was reported to substrate MLC in vitro (Cohen et al., 1997). As shown in Figure 3, DAPK2 WT phosphorylated the MLC in vitro. In contrast, DAPK2 K52A mutant failed to phosphorylate MLC. The kinase activity of DAPK2 DCaM was also con®rmed by the in vitro kinase assay. The level of MLC- phosphorylation was increased when compared to WT. Furthermore, the autophosphorylation of the DCaM mutant was decreased when compared with that obtained in the WT transfection. It is possible that DAPK2 may be negatively regulated by phosphorylation at the residue(s) present in the predicted CaM-

Figure 4 DAPK2 activation is Ca2+/CaM dependent. (a) Activation of DAPK2 by Ca2+/CaM. 293 cells were transiently transfected with the indicated plasmids. After 24 h, cells lysates were prepared and immunoprecipitated with anti-Flag antibody. In vitro kinase reaction was carried out in the buer containing EGTA/CaM (7) or CaCl2/CaM (+) in the presence of 3.0 mgof MLC. Proteins were dissolved by SDS ± PAGE and visualized by autoradiography (upper panel). The levels of protein expression Figure 3 In vitro kinase activity of DAPK2. Human 293 cells were monitored by Western blot analysis of the same lysates using were transiently transfected with the indicated plasmid. After anti-Flag antibody (lower panel). (b) Recombinant CaM binds to 24 h, cell lysates were prepared and immunoprecipitated with DAPK2. COS-7 cells were transiently transfected with the anti-Flag antibody. In vitro kinase reaction was carried out in the indicated plasmids. The whole cell lysates were dissolved by presence of 3.0 mg of MLC as a substrate. Proteins were dissolved SDS ± PAGE, transferred onto nitrocellulose membrane and by SDS ± PAGE and visualized by autoradiography (upper panel). reacted with recombinant CaM in the presence of Ca2+ (upper The levels of protein expression were monitored by Western blot panel). The levels of protein expression were monitored by analysis of the same lysates with anti-Flag antibody (lower panel). Western blot analysis of the same lysates using anti-Flag antibody Auto; autophosphorylation, MLC; MLC phosphorylation (lower panel) Apoptosis inducing kinase TKawaiet al 3475 into 293 cells. The cell lysates were immunoprecipitated well-known reagents that elevate the intracellular Ca2+ with anti-Flag antibody and the kinase activity was concentration, for 5 or 10 min followed by measure- subsequently measured in the absence or presence of ment of the activity of DAPK2 by in vitro kinase assay Ca2+/CaM using MLC as the substrate. Both auto- with MLC as the substrate. We also stimulated the and MLC-phosphorylations were signi®cantly in- cells with 10 mM of Thapsigargin, a reagent that creased by adding Ca2+/CaM (Figure 4a upper produces an intracellular calcium signal by inhibiting panel). The lower panel in Figure 4 shows the endoplasmic reticular Ca2+-ATPase. As shown in amounts of proteins expressed by Western blot Figure 5, treatment with A23187, Ionomycin or analysis of the same lysates with anti-Flag antibody. Thapsigargin resulted in increased phosphorylation of To rule out the possibility that DAPK2 is regulated MLC in the Flag-DAPK2 WT transfected cells. In by other Ca2+/CaM-regulatory molecules through contrast, both auto- and MLC-phosphorylations were indirect mechanisms, we tested the ability of recombi- not observed in cells expressing DAPK2 K52A. nant CaM to bind directly to DAPK2. COS-7 cells Furthermore, activity of DAPK2 DCaM showed no were transiently transfected with Flag-DAPK2 WT and change after treatment with these reagents, indicating DCaM. The whole cell lysates were dissolved by SDS ± that the predicted CaM-binding domain is responsible PAGE and transferred onto a nitrocellulose membrane. for Ca2+ induced-activation of DAPK2 and suggesting The membrane was incubated with recombinant-CaM that Ca2+/CaM directly activates DAPK2 through the in the presence of Ca2+. As shown in Figure 4b (upper relief of intrinsic auto-inhibition domain present in the panel), the WT protein but not the DCaM reacted with CaM-binding domain. the recombinant CaM, indicating that DAPK2 binds directly to CaM through the predicted CaM-binding Cellular localization of Flag-DAPK2 domain. The protein expression was monitored by Western blot analysis of the same lysates with anti- Previously, DAP kinase was reported to be associated Flag antibody (Figure 4b lower panel). with cytoskeleton, whereas ZIP kinase, DRAK1 and DRAK2 were localized to the nuclei (Cohen et al., 1997; Kawai et al., 1998; Sanjo et al., 1998). We Calcium signals activate DAPK2 investigated the subcellular localization of DAPK2. The presence of the Ca2+/CaM-binding domain The expression plasmid for Flag-DAPK2 WT was suggests the regulation of DAPK2 activity by transiently transfected into COS-7 cells. Twenty-four intracellular Ca2+-concentration. We investigated the hours after transfection, the subcellular localization eect of Ca2+ on activation of DAPK2. Human 293 was determined by indirect-immunostaining with anti- cells were transiently transfected with Flag-DAPK2 Flag antibody. It was found that the Flag-DAPK2 WT WT, K52A or DCaM. Twenty-four hours after protein localized exclusively in the cytoplasm (Figure transfection, the cells were treated with 10 mM of 6). Ca2+ -ionophore (A23187) and 1.0 mM of Ionomycin, Overexpression of DAPK2 induced the morphological changes of apoptosis Previously, overexpression of DAP kinase, ZIP kinase, DRAK1 or DRAK2 was reported to induce the

Figure 5 Ca2+ treatment induces activation of DAPK2. Human 293 cells were transiently transfected with the Flag-DAPK2 WT, K52A or DCaM. After 24 h, cells were stimulated with 10 mM Figure 6 Subcellular localization of Flag-DAPK2 WT. Expres- 2+ Ca -ionophore, 1.0 mM Ionomycin or 10 mM Thapsigargin for sion plasmid for Flag-DAPK2 WT was transiently transfected the indicated periods of time. Cell lysates were immunoprecipi- into COS-7 cells. The cellular localization of the Flag-DAPK2 tated and the kinase activity was examined by in vitro kinase protein was determined by indirect immunostaining with anti- assay using MLC as the substrate. The auto- and the MLC- Flag antibody. The nuclei were simultaneously visualized by phosphorylations are indicated by the arrow and arrowhead, DAPI staining. The nuclei of the transfected cells are indicated by respectively the arrows Apoptosis inducing kinase TKawaiet al 3476 morphological changes of apoptosis in mammalian BOS LacZ) which was used as an indicator. After 36 h, cells (Cohen et al., 1997; Kawai et al., 1998; Sanjo et the cells were ®xed and stained with a 5-bromo-4- al., 1998). We investigated the ability of DAPK2 to chloro-3-indolyl b-D-galactopyranoside (X-Gal) solu- induce apoptosis upon overexpression as in the case of tion to visualize the transfected cells. As shown in these kinases. Human 293 cells were transiently co- Figure 7a, blue cells transfected with DAPK2 DCaM transfected with Flag-tagged DAPK2 WT, K52A and showed the morphological changes typical of apoptosis DCaM along with the LacZ expression vector (pEF- characterized by membrane blebbing and shrunken cell

Figure 7 Apoptosis induced by DAPK2. (a) Morphology of cells transfected with DAPK2 DCaM or K52A. Human 293 or mouse NIH3T3 cells were transiently co-transfected with the expression vectors as indicated together with the LacZ expression vector. Thirty-six hours later, cells were stained with a X-Gal solution. Blue stained cells exhibiting the apoptotic phenotypes characterized by shrunken cell size and membrane blebbing are indicated by arrows. (b) Quantitative representation of the DAPK2 induced apoptosis. The data is the percentage of blue stained cells with apoptotic phenotype relative to the total number of blue cells counted from three independent experiments. (c) Nuclear morphology of cells expressing DAPK2 DCaM. NIH3T3 cells were transiently transfected with Flag-DAPK2 DCaM. Thirty-six hours later, cells were ®xed and doubly stained with anti-Flag antibody and DAPI to identify the nuclei. It was noted that the nuclei of cells transfected with Flag-DAPK2 was signi®cantly condensed as indicated by the arrow Apoptosis inducing kinase TKawaiet al 3477 size (54.7%). In contrast, overexpression of the kinase phenylinodole (DAPI) as an indicator of the nuclei. As negative mutant (K52A) resulted in normal morphol- shown in Figure 7c, the nuclei signi®cantly condensed ogy (2.3%) at approximately the same level as the in cells expressing DCaM. These results indicate that vector transfected (1.0%) (Figure 7b). The ratio of the the apoptotic changes also occurred in the nuclei, that apoptotic cells transfected with DAPK2 WT was DAPK2 is a potent mediator of apoptosis and the slightly decreased as compared with the DCaM mutant induction of apoptosis was correlated to the kinase (28.3%). We also examined the ability of DAPK2 to activity of DAPK2. induce apoptosis in NIH3T3 cells. As in the case of 293 cells, the morphological changes of apoptosis were DAPK2 maps in the central region of mouse induced in NIH3T3 cells expressing DAPK2 WT chromosome 9 (27.7%) and DCaM (60.3%) but not K52A (3.0%) and the vector transfected (1.0%) (Figure 7a and b). The mouse chromosomal location of DAPK2 was The phenotypes and the ratio of the apoptotic cells determined by interspeci®c backcross analysis using were similar comparing NIH3T3 and 293 cells. progeny derived from matings of [(C57BL/6J x Mus To further investigate, we examined whether the spretus) F1 X C57BL/6J] mice. This interspeci®c nuclei condensed upon overexpression of DAPK2. backcross mapping panel has been typed for over NIH3T3 cells were transiently transfected with Flag- 2700 loci that are well distributed among all the DAPK2 DCaM. After 36 h, cells were ®xed and doubly autosomes as well as the X chromosome (Copeland stained with anti-Flag antibody and 4,6-diamidino-2- and Jenkins, 1991). C57BL/6J and M. spretus DNAs were digested with several enzymes and analysed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using a mouse DAPK2 cDNA probe. The 9.1 kb HindIII M. spretus RFLP (see Materials and methods) was used to follow the segregation of the DAPK2 locus in backcross mice. The mapping results indicated that DAPK2 is located in the central region of mouse chromosome 9 linked to Atm, Csk, and Rora. Although 152 mice were analysed for every marker and are shown in the segregation analysis (Figure 8), up to 181 mice were typed for some pairs of markers. Each locus was analysed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analysed for each pair of loci and the most likely gene order are: centromere-Atm- 2/181-Csk-6/158-DAPK2-2/ 164-Rora. The recombination frequencies [expressed as genetic distances in centiMorgans (cM)+the standard error] are-Atm-1.1+/70.8-Csk-3.8+/71.5-DAPK2- 1.2+/70.9-Rora.

Discussion

Protein kinases are known to regulate various cellular events such as cell growth, dierentiation and apoptosis. In this report, we have identi®ed DAPK2 Figure 8 DAPK2 maps in the central region of mouse as a new Ca2+ /CaM dependent protein kinase that is chromosome 9. DAPK2 was placed on mouse chromosome 9 by involved in apoptotic signaling. DAPK2 contains the interspeci®c backcross analysis. The segregation patterns of N-terminal protein kinase domain with signi®cant DAPK2 and ¯anking genes in 152 backcross animals that were typed for all loci are shown at the top of the ®gure. For homology to those of DAP kinase, ZIP kinase, individual pairs of loci, more than 152 animals were typed (see DRAK1 and DRAK2. Just downstream to the text). Each column represents the chromosome identi®ed in the catalytic domain, DAPK2 contains the conserved backcross progeny that was inherited from the (C57BL/6J x M. domain sharing homology to the CaM-binding spretus) F1 parent. The shaded boxes represent the presence of a C57BL/6J allele and white boxes represent the presence of a M. domain of DAP kinase as well as to other CaM spretus allele. The number of ospring inheriting each type of kinases. We demonstrated here that DAPK2 has an chromosome is listed at the bottom of each column. A partial intrinsic kinase activity that phosphorylates both itself chromosome 9 linkage map showing the location of DAPK2 in and MLC as the in vitro substrate. DAPK2 was relation to linked genes is shown at the bottom of the ®gure. activated by Ca2+/CaM through direct association and Recombination distances between loci in centimorgans are shown to the left of the chromosome and the positions of loci in human by treatment of cells with three agonists that elevate chromosomes, where known, are shown to the right. References intracellular Ca2+. These activities were completely for the human map positions of loci cited in this study can be abolished by a point mutation in which the conserved obtained from GDB (Genome Data Base), a computerized lysine residue at 52nd was changed to alanine. The database of human linkage information maintained by The William H Welch Medical Library of The Johns Hopkins substitution of this conserved residue is known to University (Baltimore, MD, USA) block ATP-binding, resulting in a kinase negative Apoptosis inducing kinase TKawaiet al 3478 mutant in a number of kinases including DAP kinase, homodimerization of DAPK2 was not detected by ZIP kinase, DRAK1 and DRAK2. Removal the CaM- the yeast two-hybrid system (Kawai and Akira, binding domain resulted in a constitutively active form unpublished observation). of DAPK2. Furthermore, overexpression of DAPK2 We have compared our interspeci®c map of WT and DCaM but not K52A mutant induced chromosome 9 with a composite mouse linkage map apoptosis. These results indicate that DAPK2 is that reports the map location of many uncloned mouse regulated by Ca2+/CaM and that the catalytic activity mutations (provided from Mouse Genome Database, a is crucial for mediating apoptosis. Taken together, computerized database maintained at The Jackson DAPK2 may be one of key molecules for linking the Laboratory, Bar Harbor, ME, USA). DAPK2 cytoplasmic Ca2+ to apoptotic execution. Future work mapped in a region of the composite map that lacks will be required to clarify the relationship between mouse mutations with a phenotype that might be Ca2+ -induced activation of DAPK2 and induction of expected for an alteration in this locus (data now apoptosis. Recent evidence has suggested the critical shown). The central region of mouse chromosome 9 link between the activation of the CaMK family and shares regions of homology with human chromosomes apoptosis. CaMKII is a multi-functional protein kinase 11q and 15q (summarized in Figure 8). In particular, that is expressed in various tissues and is activated in Csk has been mapped to 15q23-q25 and Rora to 15q22. response to several apoptotic stimuli including TNF-a The placement of DAPK2 between Csk and Rora in and UV-irradiation (Wright et al., 1997). Treatment of mouse suggests that the human homolog of DAPK2 cells with CaMKII inhibitors, protected cells from will map to 15q, as well. Recently, a region in 15q22 apoptosis induced by such stimuli. Activation of has been shown to be altered in human myeloid MLCK has been reported to be an essential leukemias (Roulston et al., 1998), suggeting a role for mechanism for the induction of apoptotic morpholo- DAPK2 in tumorigenesis. gical changes including membrane blebbing (Mills et In order to assess the biological functions of al., 1998). The MLCK inhibitors blocked the DAPK2, it is important to identify speci®c targets of membrane blebbing induced by serum removal in DAPK2 that are phosphorylated during cell death or PC12 cells. DAP kinase signi®cantly induced apopto- the upstream molecules that regulate the activity of sis upon overexpression and IFN-g-induced apoptosis DAPK2. To date, the speci®c targets for DAP-, ZIP- was inhibited by transfection of the kinase negative kinases and DRAKs or the upstream regulators have mutant of DAP kinase (Cohen et al., 1997). not yet been identi®ed. Study of cellular localization Furthermore, cells transfected with DAP kinase were may provide some insight to the identi®cation of such more sensitive to apoptosis induced by TNF-a and the molecules. Members of DAP kinase family are known cells costimulated with TNF-a and cycloheximide to dier in their intracellular localizations. DAPK2 was resulted in faster migration of the DAP kinase protein localized in the cytoplasm. DAP kinase was reported to on SDS ± PAGE, probably due to protein modi®cations be closely associated with cytoskeleton through the such as protein phosphorylation or cleavage by region located at the downstream to the ankyrin proteases (Inbal et al., 1997). These results suggest repeats (Cohen et al., 1997) (Figure 1f). On the other that DAP kinase may be a key signaling molecule in hand, ZIP kinase, DRAK1 and DRAK2 were reported not only IFN-g but also TNF-a-mediated apoptosis. to be localized to the nuclei (Kawai et al., 1998; Sanjo Although the catalytic domains among the DAP et al., 1998). These observations suggest a dierent kinase family including DAPK2, DAP kinase, ZIP substrate speci®city or dierent activation mechanisms kinase and DRAKs are closely related, the non- among the DAP kinase family. One possible in vivo catalytic C-terminal regions are dierent. DAP kinase target of DAPK2 is MLC. Recently, it has been contains several functional motifs characterized by the demonstrated that MLC phosphorylation was in- N-terminal kinase domain followed by the CaM- creased in cells undergoing apoptotic membrane binding domain, eight ankyrin repeats and the death blebbing and that MLC phosphorylation was blocked domain at the C-terminal end (Feinstein et al., 1996; by inhibitors of MLCK. Furthermore, the MLCK Cohen et al., 1997). ZIP kinase contains the leucine inhibitor ML-9 blocked membrane blebbing induced zipper domain at its C-terminus in addition to the N- by serum deprivation, and decreased MLC phosphor- terminal kinase domain (Kawai et al., 1998). These ylation (Mills et al., 1998). These results suggest that domains are known to mediate homodimerization or the DAP kinase family may play a key role in these interacting with other proteins containing these observations. domains. In fact, ZIP kinase was shown to interact The functional role of DAPK2 in apoptotic with the bZIP transcriptional factor ATF4, and to be signaling remains to be clari®ed. Determination of activated by the formation of homodimer through the the autophosphorylation site is one way to elucidate leucine zipper (Kawai et al., 1998). In the case of the activation mechanism and the biochemical func- DRAKs, they show homology in the non-catalytic C- tions of DAPK2. It is also important to understand terminal regions (24.1%) (Sanjo et al., 1998). Deletion whether DAPK2 is activated by apoptotic stimuli such studies revealed that the C-terminal domains were as TNF-a, Fas, IFNg, DNA-damage and stresses. required for regulation of the kinase activities of Furthermore, the use of caspase inhibitors may clarify DRAKs, suggesting that DRAKs may be associated the relationship between DAPK2-induced apoptosis with a regulatory molecule(s) through these domains. and the caspase activation. We examined the eect of However, the C-terminal domains of DRAKs show no the caspase inhibitor such as Z-VAD-fmk on DAPK2- homology to any other proteins or to the known induced apoptosis. We overexpressed DAPK2 WT or functional domains or motifs. It is also predicted that DCaM into 293 and NIH3T3 cells that had been the non-catalytic C-terminal domain of DAPK2 may pretreated with this inhibitor, and the ratio of the also function as an interaction domain. However, apoptotic cells were measured by X-Gal staining. In Apoptosis inducing kinase TKawaiet al 3479 these cells, however, treatment with this peptide did (Calbiochem) and 0.5 mM CaCl2 or 3 mM EGTA in the not aect the ability of DAPK2 to induce apoptosis presence of 3.0 mg of MLC (Sigma) for 30 min at 308C. The (our unpublished data), suggesting that DAPK2- reaction was terminated by adding Laemmli sample buer, induced apotosis is independent of the caspase and proteins bound to the beads were eluted by boiling. activation or DAPK2 functions downstream to the Proteins were separated on SDS ± PAGE and visualized by autoradiography. caspases. Recently, it has been reported that several For Western blot analysis, the cell lysates were dissolved types of apoptosis are not inhibited by the caspase by SDS ± PAGE, and transferred onto a nitrocellulose inhibitors (Xiang et al., 1996; Miller et al., 1997; membrane. The membrane was blocked, then incubated Lavoie et al., 1998; Quignon et al., 1998). DAPK2- with anti-Flag antibody. The membrane was then reacted induced cell death may contribute with these pathways. with peroxidase labeled anti-mouse Ig antibody. After To better understand these subjects, we are currently extensive washing, peroxidase activity was detected by using investigating the signaling pathway mediated by the enhanced chemiluminescence system (NEN). DAPK2. Calmodulin overlay assay Materials and methods COS-7 cells transiently transfected with the indicated plasmids were lysed in the buer containing 10 mM Tris-Cl Materials (pH 7.4), 0.5% Nonidet P-40, 150 mM NaCl and 1 mM EDTA. The whole cell lysates were dissolved by SDS ± Ca2+-Ionophore A23187 and Ionomycin were purchased PAGE and transferred onto a nitrocellulose membrane. The from Sigma. Thapsigargin was purchased from Calbiochem. ®lter was pre-incubated in CaM-binding buer (50 mM Tris-

Cl pH 7.5, 150 mM NaCl, 1 mM CaCl2 and 3.0% skim-milk) cDNA cloning for 1 h. The membrane was then incubated with biotinylated- CaM (Calbiochem) in the CaM-binding buer for 12 h at The Expression Sequence Tags (EST) clones were identi®ed room temperature, and washed three times in CaM-binding by the BLAST program with the nucleotide sequence of the buer. The membrane was further reacted with Avidin- catalytic domain of DAP kinase as the query. Two EST peroxidase (Bio-Rad) for 1 h. After washing ®ve times, clones (GenBank accession numbers; AA858002, W82116) peroxidase activity was detected by using the enhanced showed signi®cant homology to DAP kinase. Full length chemiluminescence system. The same lysates were subjected cDNA was obtained using the RACE protocol from human to the Western blot analysis with anti-Flag antibody. skeletal muscle and mouse lung cDNA libraries according to the manufacturer's instructions (Marathon cDNA amplifica- tion kit; Clontech). The primer sequence was made available X-Gal staining of cells upon request. NIH3T3 or 293 cells (36105) were transiently cotransfected with 3.0 mg of the DAPK2 construct along with 0.3 mgof Northern blot analysis LacZ expression plasmid (pEF-BOS LacZ) as an indicator of Full length cDNA of human DAPK2 was used to probe transfection. After 36 h, transfected cells were visualized by human Multiple Tissue Northern blots containing 2 mgof X-Gal staining as described previously (Kawai et al., 1998). poly (A)+ RNA isolated from various tissues according to the manufacture's instructions (Clontech). Cellular localization of Flag-DAPK2 protein Cells and cell culture COS-7 cells (56104) grown on the cover slip were transiently transfected with 1.0 mg of the plasmid. After 24 h, cells were Monkey COS-7, human 293 and murine NIH3T3 cells were ®xed and doubly stained with anti-Flag antibody and DAPI cultured in Dulbecco's Modi®ed Eagle Medium (DMEM) (Wako) as described previously (Sanjo et al., 1998). (Gibco BRL) supplemented with 10% fetal bovine serum

(Gibco BRL) in the presence of 5% CO2 at 378C. Interspeci®c mouse backcross mapping Plasmids Interspeci®c backcross progeny were generated by mating N-terminal Flag-tagged human DAPK2 was obtained by (C57BL/6J x M. spretus) F1 females and C57BL/6J males as

PCR and ligated into a mammalian expression vector pEF- described (Copeland and Jenkins, 1991). A total of 205 N2 BOS (Kawai et al., 1998). To construct DAPK2 K52A and mice were used to map the DAPK2 locus (see text for details). DAPK2 DCaM mutant, site-directed mutagenesis was DNA isolation, restriction enzyme digestion, agarose gel performed using a site-directed mutagenesis kit according to electrophoresis, Southern blot transfer and hybridization the manufacturer's instructions (Clontech). Sequence of the were performed essentially as described (Jenkins et al., primer was available upon request. 1982). All blots were prepared with Hybond-N+ nylon membrane (Amersham). The probe, an *300 bp BamHI/ XbaI fragment from the 3'UTR of the mouse cDNA, was In vitro kinase assay and Western blot analysis labeled with a32P-dCTP using a random primed labeling kit One million 293 cells were transiently transfected with the (Stratagene); washing was done to a ®nal stringency of plasmids by Lipofection according to the manufacturer's 1.06SSCP, 0.1% SDS, 658C. A fragment of 6.5 kb was instructions (Mirus Co.). Twenty-four hours after transfec- detected in HindIII digested C57BL/6J DNA and a fragment tion, cells were lysed in the lysis buer containing 10 mM of 9.1 kb was detected in HindIII digested M spretus DNA. Tris-Cl (pH 7.4), 1.0% Triton X-100, 150 mM NaCl and 1 m The presence or absence of the 9.1 kb HindIII M. spretus- EDTA. The cell lysates were immunoprecipitated with anti- speci®c fragment was followed in backcross mice. Flag M2 antibody (Sigma) as described previously (Kawai et A description of the probes and RFLPs for the loci linked al., 1998). In vitro kinase reaction was carried out in 20 mlof to DAPK2 including Atm, Csk and Rora has been reported the kinase buer containing 10 mCi [g-32P]ATP, 50 mM previously (Giguere et al., 1995; Pecker et al., 1996).

HEPES pH 7.5, 10 mM MgCl2,3mM MnCl2,1mM CaM Recombination distances were calculated using Map Man- Apoptosis inducing kinase TKawaiet al 3480 ager, version 2.6.5. Gene order was determined by minimizing Acknowledgments the number of recombination events required to explain the We thank T Aoki and M Hyuga for excellent secretarial allele distribution patterns. assistance, and Deborah B Householder for excellent technical assistance. This work was supported by grants from the Ministry of Education of Japan and in part by the Accession numbers National Cancer Institute, DHHS, under contract with The nucleotide sequence data reported in this paper will ABL. appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with accession numbers AB018001 (human DAPK2) and AB018002 (mouse DAPK2).

References

Anderson P. Microbiol. Mol. Biol. Rev., 61, 33 ± 46. Lavoie JN, Nguyen M, Marcellus RC, Branton PE and Askew D, Ashmun R, Simmons B and Cleveland J. (1991). Shore GC. (1998). J. Cell. Biol., 140, 637 ± 645. Oncogene, 6, 1915 ± 1922. Lowe SW, Schmitt EM, Smith SW, Osborne BA and Jacks T. Bossy-Wetzel E, Bakiri L and Yaniv M. (1997). EMBO J., (1993). Nature, 362, 847 ± 849. 16, 1695 ± 1709. McCarthyJV,NiJandDixitVM.(1998).J. Biol. Chem., Cardone M, Salvesen GS, Widmann C, Johnson GL and 273, 16968 ± 16975. Frisch SM. (1997). Cell, 90, 315 ± 323. Miller TM, Moulder KL, Knudson CM, Creedon DJ, Cohen O, Feinstein E and Kimchi A. (1997). EMBO J., 16, Deshmukh M, Korsmeyer SJ and Johnson Jr EM. 998 ± 1008. (1997). J. Cell. Biol., 139, 205 ± 217. Copeland NG and Jenkins NA. (1991). Trends Genet., 7, Mills JC, Stone NL, Erhardt J and Pittman RN. (1998). J. 113 ± 118. Cell. Biol., 140, 627 ± 636. Deiss LP, Feinstein E, Berissi H, Cohen O and Kimchi A. Nagata S. (1997). Cell, 88, 355 ± 356. (1995). Genes Dev., 9, 15 ± 30. Pecker I, Avraham KB, Gilbert DJ, Savitsky K, Rotman G, Derijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T, Karin Harnik R, Fukao T, Schrok E, Hirotsune S, Tagle DA, M and Davis RJ. (1994). Cell, 76, 1025 ± 1037. Collins FS, Wynshaw-Boris A, Ried T, Copeland NG, Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Jenkins NA, Shiloh Y and Ziv Y. (1996). Genomics, 35, Brooks M, Waters CM, Penn LZ and Hancock DC. 39 ± 45. (1992). Cell, 69, 119 ± 128. Quignon F, De Bels F, Koken K, Feunteun J, Ameisen JC Feinstein E, Wallach D, Boldin M, Varfolomeev E and and The H. (1998). Nature Genet., 20, 259 ± 265. Kimchi A. (1996). Trends Biochem. Sci., 20, 342 ± 344. Roulston D, Espinosa R, Nucifora G, Larson RA, Le-Beau Giguere V, Beatty B, Squire J, Copeland NG and Jenkins MM and Rowley JD. (1998). Blood, 92, 2879 ± 2885. NA. (1995). Genomics, 28, 596 ± 598. Sanjo H, Kawai T and Akira S. (1998). J. Biol. Chem., 273, Hakem R, Hakem A, Duncan GS, Henderson JT, Woo M, 29066 ± 29071. Soengas MS, Elia A, de la Pompa JL, Kagi D, Khoo W, Shi L, Nishioka CWK, Th'ng J, Bradbury EM, Litch®eld Potter J, Yoshida R, Kaufman SA, Lowe SW, Penninger DW and Greenberg AH. (1994). Science, 263, 1143 ± 1145. JM and Mak TW. (1998). Cell, 94, 339 ± 352. Steller H. (1995). Science, 267, 1445 ± 1449. Hibi M, Lin A, Smeal T, Minden A and Karin M. (1993). Thompson JD, Higgins DG and Gibson TJ. (1994). Nuc. Genes Dev., 7, 2135 ± 2148. Acid. Res., 22, 2673 ± 4680. Ichijo H, Nishida E, Irie K, Dijke PT, Saitoh M, Moriguchi Thornberry NA and Lazebnik Y. (1998). Science, 281, T, Takagi M, Matsumoto K, Miyazono K and Gotoh Y. 1312 ± 1316. (1997). Science, 275, 90 ± 94. Tian Q, Taupin JL, Elledge S, Robertson M and Anderson P. Inbal B, Cohen O, Polak-Charcon S, Kopolovic J, Vadai E, (1995). J. Exp. Med., 182, 865 ± 874. Eisenbach L and Kimchi A. (1997). Nature, 390, 180 ± 184. VerheijM,BoseR,LinXH,YaoB,JarvisWD,GrantS, Inohara N, Peso L, Koseki T, Chen S and Nunez G. (1998). Birrer MJ, Szabo E, Zon LI, Kyriakis JM, Haimovitz- J. Biol. Chem., 273, 12296 ± 12300. Freidman A, Fuks Z and Kolesnick RN. (1996). Nature, Jenkins NA, Copeland NG, Taylor BA and Lee BK. (1982). 380, 75 ± 79. J. Virol., 43, 26 ± 36. White E. (1996). Genes Dev., 10, 1±15. Kawai T, Matsumoto M, Takeda K, Sanjo H and Akira S. Widman C, Gerwins P, Johnson NL, Japre MB and Johnson (1998). Mol. Cell. Biol., 18, 1642 ± 1651. GL. (1998). Mol. Cell. Biol., 18, 2416 ± 2429. Kissil JL, Feinstein E, Cohen O, Jones PA, Tsai YC, Wright SC, Schellenberger U, Ji L, Wang H and Larrick JW. Knowles MA, Edymann ME and Kimchi A. (1997). (1997). FASEB J., 11, 843 ± 849. Oncogene, 15, 405 ± 407. Xia Z, Dickens M, Raingeaud J, Davis RJ and Greenberg KuidaK,ZhengTS,NaS,KuanC,YangD,KarasuyamaH, ME. (1995). Science, 270, 1326 ± 1331. Rakic P and Flavell RA. (1996). Nature, 384, 368 ± 372. Xiang J, Chao DT and Korsmeyer SJ. (1996). Proc. Natl. Kuida K, Haydar TF, Kuan CY, Gu Y, Taya C, Acad.Sci.USA,93, 14559 ± 14563. Karasuyama H, Su MS, Rakic P and Flavell RA. (1998). Yuan J, Shaham L, Ledoux S, Ellis HM and Horvitz HR. Cell, 94, 325 ± 337. (1993). Cell, 75, 641 ± 652. Lahti JM, Xiang J, Heath LS, Campana D and Kidd VJ. (1995). Mol. Cell. Biol., 15, 1±11. Lassignal Johnson N, Gardner AM, Diener KM, Lange- Cartner CA, Gleavy J, Jarpe MB, Minden A, Karin M, Zon LI and Johnson GL. (1996). J. Biol. Chem., 271, 3229 ± 3237.