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• W2007318620 abstract "In this study, two alternatively spliced forms of the mouse death-associated protein kinase (DAPK) have been identified and their roles in apoptosis examined. The mouse DAPK-α sequence is 95% identical to the previously described human DAPK, and it has a kinase domain and calmodulin-binding region closely related to the 130–150 kDa myosin light chain kinases. A 12-residue extension of the carboxyl terminus of DAPK-β distinguishes it from the human and mouse DAPK-α. DAPK phosphorylates at least one substrate in vitro and in vivo, the myosin II regulatory light chain. This phosphorylation occurs preferentially at Ser-19 and is stimulated by calcium and calmodulin. The mRNA encoding DAPK is widely distributed and detected in mouse embryos and most adult tissues, although the expression of the encoded 160-kDa DAPK protein is more restricted. Overexpression of DAPK-α, the mouse homolog of human DAPK has a negligible effect on tumor necrosis factor (TNF)-induced apoptosis. Overexpression of DAPK-β has a strong cytoprotective effect on TNF-treated cells. Biochemical analysis of TNF-treated cell lines expressing mouse DAPK-β suggests that the cytoprotective effect of DAPK is mediated through both intrinsic and extrinsic apoptotic signaling pathways and results in the inhibition of cytochrome c release from the mitochondria as well as inhibition of caspase-3 and caspase-9 activity. These results suggest that the mouse DAPK-β is a negative regulator of TNF-induced apoptosis. In this study, two alternatively spliced forms of the mouse death-associated protein kinase (DAPK) have been identified and their roles in apoptosis examined. The mouse DAPK-α sequence is 95% identical to the previously described human DAPK, and it has a kinase domain and calmodulin-binding region closely related to the 130–150 kDa myosin light chain kinases. A 12-residue extension of the carboxyl terminus of DAPK-β distinguishes it from the human and mouse DAPK-α. DAPK phosphorylates at least one substrate in vitro and in vivo, the myosin II regulatory light chain. This phosphorylation occurs preferentially at Ser-19 and is stimulated by calcium and calmodulin. The mRNA encoding DAPK is widely distributed and detected in mouse embryos and most adult tissues, although the expression of the encoded 160-kDa DAPK protein is more restricted. Overexpression of DAPK-α, the mouse homolog of human DAPK has a negligible effect on tumor necrosis factor (TNF)-induced apoptosis. Overexpression of DAPK-β has a strong cytoprotective effect on TNF-treated cells. Biochemical analysis of TNF-treated cell lines expressing mouse DAPK-β suggests that the cytoprotective effect of DAPK is mediated through both intrinsic and extrinsic apoptotic signaling pathways and results in the inhibition of cytochrome c release from the mitochondria as well as inhibition of caspase-3 and caspase-9 activity. These results suggest that the mouse DAPK-β is a negative regulator of TNF-induced apoptosis. myosin light chain kinase calmodulin regulatory light chain death-associated protein kinase tumor necrosis factor kilobase(s) poly(A)DP-ribose polymerase doxycycline polyacrylamide gel electrophoresis base pair(s) 4-morpholinepropanesulfonic acid p-nitroaniline nuclear factor κB Apoptosis is a carefully regulated cellular event with important roles in a number of processes that occur during development and contribute to tissue homeostasis. Dysregulation of apoptosis can result in cancer, autoimmune diseases, and neurodegenerative disorders. A large number of signaling molecules involved in regulating the commitment and progression of apoptosis have been identified, and their complex interactions are being investigated. There is now also considerable evidence that many protein kinases have roles in apoptosis and may help regulate the signaling pathways that ultimately determine the critical balance in the choice between life and death (1Anderson P. Microbiol. Mol. Biol. Rev. 1997; 61: 33-46Crossref PubMed Scopus (165) Google Scholar, 2Utz P.J. Anderson P. Cell Death Differ. 2000; 7: 589-602Crossref PubMed Scopus (143) Google Scholar). Myosin II motor activities have been implicated in the general regulation of morphological changes that occur during the execution phase of apoptosis (3Mills J.C. Stone N.L. Erhardt J. Pittman R.N. J. Cell Biol. 1998; 140: 627-636Crossref PubMed Scopus (410) Google Scholar). In smooth and nonmuscle cells, the phosphorylation of myosin II by myosin light chain kinase (MLCK)1 is a key event leading to the activation of myosin II motor activities and the production of forces for contraction, migration, adhesion, and cytokinesis (4Kamm K.E. Stull J.T. J. Biol. Chem. 2001; 276: 4527-4530Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). It is now known that other protein kinases in addition to the conventional calcium/calmodulin (Ca2+/CaM)-dependent MLCKs can phosphorylate myosin II regulatory light chain (RLC). These kinases include p21-activated kinase (PAK), rho-activated kinase (RHOK), and death-associated protein kinase (DAPK) (5Chew T.L. Masaracchia R.A. Goeckeler Z.M. Wysolmerski R.B. J. Muscle Res. Cell. Motil. 1998; 19: 839-854Crossref PubMed Scopus (168) Google Scholar, 6Goeckeler Z.M. Wysolmerski R.B. J. Cell Biol. 1995; 130: 613-627Crossref PubMed Scopus (377) Google Scholar, 7Van Eyk J.E. Arrell D.K. Foster D.B. Strauss J.D. Heinonen T.Y. Furmaniak-Kazmierczak E. Cote G.P. Mak A.S. J. Biol. Chem. 1998; 273: 23433-23439Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Thus, multiple signaling pathways converge at the myosin regulatory light chain, and it is likely that each of these pathways modulates myosin motor activities to generate forces necessary for the formation or disassembly of signaling complexes and their intracellular trafficking. A recent study has shown that myosin II motor activities activated by the conventional Ca2+/CaM-dependent MLCK has an important role in regulating the translocation of at least one death receptor, TNFR-1, to the plasma membrane (8Jin Y. Atkinson S.J. Marrs J.A. Gallagher P.J. J. Biol. Chem. 2001; 276: 30342-30349Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), suggesting an additional role in regulation of the apoptotic response in cells. DAPK is a Ca2+/CaM-dependent Ser/Thr protein kinase that was identified as a positive mediator of interferon-γ-induced apoptosis in HeLa cells (9Deiss L.P. Feinstein E. Berissi H. Cohen O. Kimchi A. Genes Dev. 1995; 9: 15-30Crossref PubMed Scopus (530) Google Scholar, 10Cohen O. Feinstein E. Kimchi A. EMBO J. 1997; 16: 998-1008Crossref PubMed Scopus (376) Google Scholar). Several other kinases related to DAPK have also been identified, and all have strong sequence homology that is restricted to the kinase domain of DAPK (11Kogel D. Prehn J.H. Scheidtmann K.H. Bioessays. 2001; 23: 352-358Crossref PubMed Scopus (85) Google Scholar). This kinase family includes ZIP/DLK, DAPK-related apoptosis-inducing kinases 1 and 2, DAPK2, and dystrophin-related protein-1 (12Kawai T. Matsumoto M. Takeda K. Sanjo H. Akira S. Mol. Cell. Biol. 1998; 18: 1642-1651Crossref PubMed Scopus (197) Google Scholar, 13Kogel D. Plottner O. Landsberg G. Christian S. Scheidtmann K.H. Oncogene. 1998; 17: 2645-2654Crossref PubMed Scopus (100) Google Scholar, 14Sanjo H. Kawai T. Akira S. J. Biol. Chem. 1998; 273: 29066-29071Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Kawai T. Nomura F. Hoshino K. Copeland N.G. Gilbert D.J. Jenkins N.A. Akira S. Oncogene. 1999; 18: 3471-3480Crossref PubMed Scopus (109) Google Scholar, 16Inbal B. Shani G. Cohen O. Kissil J.L. Kimchi A. Mol. Cell. Biol. 2000; 20: 1044-1054Crossref PubMed Scopus (134) Google Scholar). These kinases have been shown in vitro to phosphorylate RLC isolated from skeletal muscle (10Cohen O. Feinstein E. Kimchi A. EMBO J. 1997; 16: 998-1008Crossref PubMed Scopus (376) Google Scholar, 13Kogel D. Plottner O. Landsberg G. Christian S. Scheidtmann K.H. Oncogene. 1998; 17: 2645-2654Crossref PubMed Scopus (100) Google Scholar, 15Kawai T. Nomura F. Hoshino K. Copeland N.G. Gilbert D.J. Jenkins N.A. Akira S. Oncogene. 1999; 18: 3471-3480Crossref PubMed Scopus (109) Google Scholar, 16Inbal B. Shani G. Cohen O. Kissil J.L. Kimchi A. Mol. Cell. Biol. 2000; 20: 1044-1054Crossref PubMed Scopus (134) Google Scholar) or smooth muscle myosin (17Murata-Hori M. Suizu F. Iwasaki T. Kikuchi A. Hosoya H. FEBS Lett. 1999; 451: 81-84Crossref PubMed Scopus (114) Google Scholar), but to date no in vivo substrates have been identified. The significance of RLC phosphorylation by DAPKs is unknown, although DAPK, dystrophin-related protein-1, and ZIP/DLK are or can become associated with the actomyosin cytoskeleton (10Cohen O. Feinstein E. Kimchi A. EMBO J. 1997; 16: 998-1008Crossref PubMed Scopus (376) Google Scholar, 16Inbal B. Shani G. Cohen O. Kissil J.L. Kimchi A. Mol. Cell. Biol. 2000; 20: 1044-1054Crossref PubMed Scopus (134) Google Scholar,18Page G. Kogel D. Rangnekar V. Scheidtmann K.H. Oncogene. 1999; 18: 7265-7273Crossref PubMed Scopus (93) Google Scholar). Similar to other apoptotic regulators, ectopic overexpression of the DAPK family members induces morphological and biochemical changes associated with apoptosis, and this family of protein kinases is considered to be positive regulators of apoptosis. Although the signaling pathway through which members of the DAPK family promote apoptotic cell death is not understood, it has been shown that DAPK acts upstream of p53 to regulate p53 activity in a p19ARF-dependent manner (19Raveh T. Droguett G. Horwitz M.S. DePinho R.A. Kimchi A. Nat. Cell. Biol. 2001; 3: 1-7Crossref PubMed Scopus (313) Google Scholar). This study describes the cloning and characterization of two alternatively spliced mouse DAPKs. Mouse DAPK-α and DAPK-β are highly related to the previously described proapoptotic human DAPK. However, ectopic overexpression of the murine DAPK-α or DAPK-β does not promote apoptosis as previously shown for the human DAPK (9Deiss L.P. Feinstein E. Berissi H. Cohen O. Kimchi A. Genes Dev. 1995; 9: 15-30Crossref PubMed Scopus (530) Google Scholar, 10Cohen O. Feinstein E. Kimchi A. EMBO J. 1997; 16: 998-1008Crossref PubMed Scopus (376) Google Scholar). In addition, in TNF-treated cells, overexpression of DAPK-β is cytoprotective and suppresses caspase-3 and -9 activity and mitochondrial cytochrome c release. Together these studies show that DAPK can protect cells from apoptosis. A cDNA probe encoding the kinase domain of the mouse 130-kDa MLCK (20Herring B.P. Dixon S. Gallagher P.J. Am. J. Physiol. 2000; 279: C1656-C1664Crossref PubMed Google Scholar) was used in a low stringency screen of a mouse AT2 cardiac myocyte gt11 cDNA library, generously provided by the Indiana University School of Medicine. Positive isolates were subcloned and sequenced. One cDNA identified from this library screen had significant homology to the kinase domain of MLCK and was extended by subsequent screens to yield a 4.9-kb cDNA encoding the full-length DAPK-α. Sequencing of several other positive cDNAs obtained from subsequent library screens identified a cDNA encoding a DAPK-β that was distinguished at the 3′ end by a putative alternative splice that would result in extending the carboxyl-terminal coding region of DAPK by 12 residues. Two monoclonal anti-human DAPK (BD/Transduction Laboratories, clone 17, and Sigma, clone 55) were used at dilutions of 1:250 and 1:10,000, respectively, and gave similar results. Omniprobe polyclonal and monoclonal antibodies (Santa Cruz Biotechnology) and Xpress tag antibody (Invitrogen), all of which recognize the Xpress epitope tag (Invitrogen), were used at a dilution of 1:1,000 and 1:2,500, respectively. A polyclonal antibody to purified myosin II regulatory light chains was generated and characterized in this laboratory. The cytochrome c antibody is from PharMingen (San Diego, CA), and the poly(A)DP-ribose polymerase (PARP) antibody (Santa Cruz Biotechnology) recognizes both full-length and caspase-cleaved PARP. MDCK or HeLa cell lines expressing either DAPK-α or DAPK-β under the control of a tetracycline-inducible transactivator were constructed by the transfection of a pCDNA4/TO plasmid containing DAPKs into MDCK or HeLa cells already expressing tetracycline-VP16 transactivator (21Barth A.I. Pollack A.L. Altschuler Y. Mostov K.E. Nelson W.J. J. Cell Biol. 1997; 136: 693-706Crossref PubMed Scopus (206) Google Scholar, 22Gaush C.R. Hard W.L. Smith T.F. Proc. Soc. Exp. Biol. Med. 1966; 122: 931-935Crossref PubMed Scopus (259) Google Scholar). Stable zeocin resistant cell lines were selected and characterized for tetracycline-regulated expression of DAPKs. A similar strategy was used to generate MDCK cell lines expressing mutant DAPK-α (K42A) and DAPK-β (K42A). The exogenously expressed mouse wild-type and mutant DAPKs have an amino-terminal Xpress epitope tag that is recognized by both the Omniprobe and Xpress tag antibodies. Parental cells and cell lines expressing DAPKs were maintained routinely in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. To regulate expression of DAPK in the MDCK and HeLa cell lines, Doxycycline (Dox) is increased from 0 (repression) to 2 (maximal induction) µg/ml. Under maximal induction conditions, stable and approximately equal expression levels of the DAPKs were achieved in each of the cell lines. For each experiment, controls included MDCK and HeLa parental cells expressing only the tetracycline transactivator and cells under maximal repression (0 µg/ml Dox for MDCK and HeLa cells). In all cases the repressed cells gave similar results as the parental cell line. Overexpression of DAPKs did not have a deleterious effect on growth, doubling time, or morphology. Western blotting and immunofluorescence were used routinely to monitor the expression of the exogenous DAPKs to ensure that the cell lines were expressing each DAPK at equal levels. Cells were maintained at subconfluent levels (∼30–50% density) during analysis. MDCK cells are sensitive to TNF and do not require inhibition of protein synthesis with cyclohexamide (8Jin Y. Atkinson S.J. Marrs J.A. Gallagher P.J. J. Biol. Chem. 2001; 276: 30342-30349Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). A biochemical apoptotic response is apparent in MDCK cells within 8 h of exposure to TNF; however, morphological changes including apoptotic blebbing are not readily apparent until between 24 and 48 h of treatment. HeLa cells were treated with 10 ng/ml TNF in the presence of 10 µg/ml cyclohexamide to induce apoptosis, which becomes morphologically apparent within 2–3 h. Western blotting was performed as described previously (23Gallagher P.J. Jin Y. Killough G. Blue E.K. Lindner V. Am. J. Physiol. 2000; 279: C1078-C1087Crossref PubMed Google Scholar). Equivalent amounts of total cellular protein or immunoprecipitates were fractionated by electrophoresis through an SDS-polyacrylamide gel and transferred to nitrocellulose. Immunoreactive proteins on Western blots were visualized using the Supersignal West Dura or West Pico detection systems (Pierce) according to manufacturer directions. Cell extracts were prepared from cells or tissues by homogenization in a lysis buffer containing 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 m NaCl, 10 mm sodium phosphate, pH 7.2, 2 mm EDTA, 50 mm sodium fluoride, 0.2 mm sodium vanadate, 20 µg/ml leupeptin, 40 µg/ml aprotinin, 60 µg/mlN-tosyl-l-phenylalanyl chloromethyl ketone, 60 µg/mlNα-p-tosyl-l-lysine-chloromethyl ketone, and 100 µg/ml phenylmethylsulfonyl fluoride. After SDS-PAGE and transfer to nitrocellulose, Western blotting was performed using the appropriate anti-DAPK or Xpress antibodies. RNA was prepared from mouse tissues and cell lines using the Totally RNA kit (Ambion). For Northern blotting, 20 µg of total RNA/lane was fractionated on a 1.2% agarose gel. The RNA was transferred to Brightstar Plus positively charged nylon membrane (Ambion) using a vacuum blotter (Bio-Rad), UV cross-linked using a Stratalinker (Stratagene), and prehybridized for 60 min at 65 °C. A 550-bp 32P-labeled antisense riboprobe (cRNA) corresponding to bp 3370–3920 was transcribed from DAPK cDNA using the Maxiscript kit (Ambion). The probe was added to the blot in prehybridization buffer at a concentration of 1 × 106 cpm/ml buffer and hybridized overnight at 65 °C. Final wash conditions were 15 mmsodium citrate, pH 7.0, 0.15 m NaCl (0.1× SSC), and 0.1% SDS at 65 °C for 10 min. The blot was rinsed in 2× SSC and exposed to X-OMAT AR film with an intensifying screen for 1 week. RNase protection was performed using the reagents and protocols from the RPA III kit (Ambion). A 507-bp 32P-labeled probe was prepared using the Maxiscript kit (Ambion) from aStuI-linearized template from DAPK-β. The probe generated contains an additional 43 nucleotides from the pGEM polylinker. A β-actin probe was used as a positive control. The hybridization of the cRNA probe and total RNA was performed at 42 °C overnight followed by a 30-min RNase digestion (2.5 units/ml RNase A and 100 units/ml RNase T1) and ethanol precipitation. Protected fragments were separated on a 6% acrylamide/7 m urea gel and exposed to film with an intensifying screen at −70 °C for 3 days. Transiently transfected COS-1 cells expressing Xpress-tagged DAPK were washed with PBS, and lysates were prepared in lysis buffer (20 mm MOPS, pH 7, 1% Nonidet P-40, 10% glycerol, 0.5 mm EGTA, 50 mm MgCl2, 0.3 m NaCl, 6 µg/mlNα-p-tosyl-l-lysine-chloromethyl ketone and N-tosyl-l-phenylalanyl chloromethyl ketone, 10 µg/ml (p-amidinophenyl)methanesulfonyl fluoride, 20 µg/ml leupeptin, 40 µg/ml aprotinin, and 1 mm Pefabloc SC). The lysate was clarified by centrifugation, and the supernatant was precleared using protein A-Sepharose. DAPK was immunoprecipitated by the addition of protein A beads precomplexed with rabbit anti-mouse IgG and monoclonal omniprobe antibody. Immune complexes bound to protein A-Sepharose 4B were washed twice with lysis buffer and then twice with kinase assay buffer (50 mm MOPS, pH 7, 10 mm magnesium acetate, and 1 mm dithiothreitol). Protein A-Sepharose beads containing the immunopurified DAPK were resuspended in kinase assay buffer, and equivalent volumes were used for in vitro RLC phosphorylation reactions. Assays to determine the specific activity of the recombinant murine 160-kDa DAPKs were performed as described previously (24Gallagher P.J. Herring B.P. Trafny A. Sowadski J. Stull J.T. J. Biol. Chem. 1993; 268: 26578-26582Abstract Full Text PDF PubMed Google Scholar). The amount of DAPK immunoprecipitated per assay was estimated by ligand blotting with biotin-conjugated calmodulin using purified 150-kDa bovine tracheal MLCK as a standard. RLCs were phosphorylated in 50-µl kinase assay buffer (50 mm MOPS, pH 7, 10 mm MgAc, 0.6 mm CaCl2, 1 mm dithiothreitol, 1.2 µm CaM, and 22.5 µm chicken gizzard RLC) containing [γ-32P]ATP (200 cpm/pmol) diluted in 1 mm ATP for 15 min. To determine whether both forms of DAPK undergo autophosphorylation, the immunoprecipitated DAPK was analyzed following SDS-PAGE gel and autoradiography after completion of the kinase assay. To determine whether DAPK phosphorylates myosin regulatory light chains at the activating sites (Ser-19 and Thr-18) and RLC associated with myosin II, kinase assays were performed using either 1 µg of purified mutant recombinant regulatory light chain or 10 µg of myosin II, and the results were analyzed following SDS-PAGE and autoradiography. The myosin II was purified from partially fractionated human platelets, and Western blotting using isoform-specific myosin antibodies confirmed that both nonmuscle myosin IIA and IIB were present. The phosphorylation of RLCs was determined as described (25Gallagher P.J. Herring B.P. Griffin S.A. Stull J.T. J. Biol. Chem. 1991; 266: 23936-23944Abstract Full Text PDF PubMed Google Scholar, 26Gallagher P.J. Herring B.P. Stull J.T. J. Muscle Res. Cell. Motil. 1997; 18: 1-16Crossref PubMed Scopus (174) Google Scholar). Briefly, the cellular proteins were precipitated with 10% trichloroacetic acid; the pellets were washed with acetone and dissolved in 8 m urea, 20 mm Tris, 23 mm glycine, and 10 mm dithiothreitol. Western blotting with an anti-myosin II RLC antibody was used to identify unphosphorylated, monophosphorylated, and diphosphorylated forms of RLC after fractionation through a 10% glycerol-polyacrylamide gel and transfer to nitrocellulose. The relative abundance of each RLC band was determined by scanning densitometry. The scan data were used to calculate the myosin II RLC phosphorylation index as described previously (8Jin Y. Atkinson S.J. Marrs J.A. Gallagher P.J. J. Biol. Chem. 2001; 276: 30342-30349Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). For transient expression analysis, Cos or HeLa cells were seeded at 1 × 105 cells/30-mm dish, transfected with vectors as indicated for expression of DAPKs, and an empty vector (pCDNA4TO, mock) together with a vector encoding β-galactosidase. Transfections were carried out using Fugene 6 (Roche, Indianapolis, IN) according to manufacturer protocol. At 24 h after transfection, HeLa cells were treated with TNF (10 ng/ml) and cyclohexamide (10 µg/ml) for 3 h and then fixed and stained for β-galactosidase expression. Control transfections (−TNF), were incubated for 48 h before analysis. The percentage of apoptotic cells were determined by scoring the number of transfected (LacZ+) cells having apoptotic morphology with condensed cytoplasm and several apparent plasma membrane blebs. At least 100 LacZ-positive blue cells were counted in each well, and each experiment was independently repeated eight times. To examine the expression levels of DAPK and LacZ proteins, cells treated in parallel were lysed in SDS lysis buffer (1% SDS and 50 mm Tris, pH 7.4) and examined by Western blotting to detect DAPK or β-galactosidase expression. Quantification of apoptotic cell death in conditionally regulatable HeLa or MDCK cell lines expressing DAPKs was performed by seeding the cells at 5 × 104 cells/well in 6-well tissue culture dishes and culturing the cells in the presence (+) or absence (−) of Dox for 24 h to induce stable levels of the exogenous DAPKs. At 24 h post-seeding, TNF (10 ng/ml) or vehicle (Me2SO, final concentration <0.01%) was added (t = 0). At the indicated times, viable attached cells were identified using trypan blue exclusion and counted. Cell viability is expressed as the percentage of the surviving TNF-treated cells compared with the surviving control cells not treated with TNF. Caspase-8, caspase-3, and caspase-9 activity were determined after extraction of the cells with CHAPS lysis buffer (0.1% CHAPS, 100 mm NaCl, 100 µm EDTA, 10 mmdithiothreitol, and 50 mm HEPES, pH 7.4). After centrifugation, equal amounts of total cellular proteins were incubated at 37 °C in assay buffer (CHAPS lysis buffer plus 10% glycerol), and the assay was initiated by the addition of either 200 µm Ac-IETD-pNA (caspase-8), 200 µmAc-DEVD-pNA (caspase-3), or 200 µm Ac-LEHD-pNA (caspase-9) (Calbiochem, La Jolla, CA). The change in absorbance at 405 nm with time was monitored by spectrophotometry and converted to caspase activity (pmol/min/mg of total protein). Purep-nitroaniline (pNA, Calbiochem) was used for calibration of the standard A405 curve. For every cell sample, the background was determined by adding the caspase-specific inhibitors, Ac-IETD-CHO (caspase-8), Ac-DEVD-CHO (caspase-3), or Ac-LEHD-CHO (caspase-9) (Calbiochem) as negative control. DNA fragmentation was analyzed by enzymatic detachment of the adherent cells, which were pooled with floating cells and then fixed in 5% acetic acid/95% ethanol at −20 °C and stained with 50 µg/ml propidium iodide (Sigma). Cells were analyzed with a Becton-Dickinson (Mountain View, CA) FACStar plus, and the data were computed with CellQuest. At least 10,000 cells were counted for each condition. Hela or MDCK cells were scraped into PBS supplemented with a protease inhibitor mixture (20 µg/ml leupeptin, 40 µg/ml aprotinin, 60 µg/mlN-tosyl-l-phenylalanyl chloromethyl ketone, 60 µg/mlNα-p-tosyl-l-lysine-chloromethyl ketone, 100 µg/ml phenylmethylsulfonyl fluoride, and 100 µg/ml (p-amidinophenyl)methanesulfonyl fluoride) and lysed by passage through a 23-gauge needle 10 times. Cytosol (devoid of mitochondria) and pellet (including mitochondria and nuclei) fractions were separated by centrifugation at 14,000 rpm for 30 min. Western blotting of both fractions was used to determine the relative amounts of cytochrome c and NF-κB. Each analysis was repeated at least three times, and the relative levels of expression were quantified by densitometry. A low stringency screen of a mouse AT2 cardiomyocyte cDNA library using the kinase domain of the mouse 130-kDa MLCK identified two cDNA clones that represent the murine homologs of human DAPK. The two mouse DAPK clones represent alternative splice forms, differing only at their carboxyl termini. A sequence alignment of the mouse and human DAPKs (Fig. 1A) shows that the murine DAPKs are ∼95% identical to the previously described human DAPK (9Deiss L.P. Feinstein E. Berissi H. Cohen O. Kimchi A. Genes Dev. 1995; 9: 15-30Crossref PubMed Scopus (530) Google Scholar). The murine DAPK-β appears to be an alternatively spliced DAPK that has a unique carboxyl terminus that extends DAPK-α by 12 residues. All the structural features between the mouse and human DAPKs are highly conserved including the kinase, calmodulin binding, ankyrin repeats, P-loops, and death domain (Fig. 1 B). Within the kinase domain (residues 13–263) there are two nonconserved residues, and throughout the remainder of the molecule there are ∼25 nonconserved changes in the sequence (Fig. 1 A). The significance of these findings, if any, is not known. The kinase domain of DAPK is ∼40–50% identical to the 130–150-kDa conventional MLCK, and importantly, residues involved in binding and phosphorylation of myosin II RLC and activation by Ca2+/CaM are highly conserved (24Gallagher P.J. Herring B.P. Trafny A. Sowadski J. Stull J.T. J. Biol. Chem. 1993; 268: 26578-26582Abstract Full Text PDF PubMed Google Scholar). The full-length cDNAs of DAPK-α and DAPK-β or two mutants with an alanine substitution at the ATP-reactive lysine within the kinase domain, K42A (αK42A, βK42A), which is predicted to inactivate kinase activity, were cloned into pcDNA3His such that the predicted translation start site at the amino terminus was fused in frame to the Xpress/hexahistidine tag, and the plasmids were transfected into Cos cells for expression. Fig.2A shows the results of a Western blot to detect the wild-type recombinant DAPKs using either a DAPK-specific antibody or the Omniprobe monoclonal antibody (Santa Cruz Biotechnology) to detect the Xpress-tagged DAPK. Blots reacted with the anti-DAPK antibody revealed that the molecular mass of the mouse DAPKs is indistinguishable from the 160-kDa endogenous DAPK present in Cos cells. A smaller, proteolytic breakdown product of ∼100 kDa is also detected with this antibody. There is also no apparent size difference between DAPK-α and DAPK-β isoforms (∼160 and 161 kDa, respectively) when they are separated on a 5% SDS-PAGE gel. Identical results were obtained for the mutant cDNAs (data not shown). Ribonuclease protection assays were used to confirm the presence of mRNAs for both isoforms of DAPK. DAPK-β has a deletion of 485 bp that occurs immediately prior to the UGA stop codon. The splicing results in a deletion of 485 nucleotides of mRNA present in the 3′-untranslated region of DAPK-α and the extension of the open reading frame for an additional 12 residues to generate DAPK-β. The DAPK-β cDNA was linearized at a StuI site located at bp 4506; the resulting template allowed us to generate a cRNA probe that distinguishes the DAPK-α and DAPK-β isoforms. The results of the ribonuclease protection analysis are shown in Fig. 2 B. Two protected fragments of 507 and 445 bp were detected, corresponding to the predicted sizes of the32P-labeled cRNA probe protected by the mRNAs encoding DAPK-β and DAPK-α, respectively. Both of these protected fragments appear in adult liver, whole embryos at days 10, 15, and 19, and embryonic heart (at days 12 and 15), showing that DAPK-β is expressed during early development as well as in adult liver tissue. Northern blotting with a probe corresponding to bp 3370–3920 (residues 1033–1215) of the DAPK cDNA identified a predominant mRNA of ∼6.0 kb in mouse embryos obtained at days 10, 15, and 19 and many adult tissues tested as well as one cell line (Fig.3A). The relative levels of the 6.0-kb mRNA were highest in the embryo tissues and in adult bladder, uterus, vas deferens, liver, kidney, and 3T3 mouse fibroblasts. Western blotting with a monoclonal anti-DAPK antibody (Transduction Laboratories) revealed the presence of a 160-kDa band that is detectable in several adult tissues including bladder, uterus, vas deferens, lung, liver, and kidney (Fig. 3 B). The 100-kDa protein is a proteolytic breakdown product of DAPK. Comparison of the Northern and Western blotting revealed that for several tissues a 6.0-kb mRNA is present, but no DAPK protein is detectable. These tissues generally had lower relative levels of the 6.0-kb mRNA, suggesting that either DAPK is expressed in skeletal muscle, testes, stomach, colon, and ileum but at levels below the detectable limit of the antibody or expression is post-transcriptionally regulated. The kinase activity of the human DAPK has been determined in vitro using commercially available myosin II RLC purified from rabbit skeletal muscle as a substrate (10Cohen O. Feinstein E. Kimchi A. EMBO J. 1997;" @default.
• W2007318620 created "2016-06-24" @default.
• W2007318620 creator A5015465631 @default.
• W2007318620 creator A5020784022 @default.
• W2007318620 creator A5027609235 @default.
• W2007318620 creator A5031894095 @default.
• W2007318620 creator A5040261229 @default.
• W2007318620 creator A5090804157 @default.
• W2007318620 date "2001-10-01" @default.
• W2007318620 modified "2023-09-29" @default.
• W2007318620 title "Identification of a New Form of Death-associated Protein Kinase That Promotes Cell Survival" @default.
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