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WO1999066030A9 - Proteine apparentee a la dap-kinase - Google Patents

Proteine apparentee a la dap-kinase

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Publication number
WO1999066030A9
WO1999066030A9 PCT/US1999/013411 US9913411W WO9966030A9 WO 1999066030 A9 WO1999066030 A9 WO 1999066030A9 US 9913411 W US9913411 W US 9913411W WO 9966030 A9 WO9966030 A9 WO 9966030A9
Authority
WO
WIPO (PCT)
Prior art keywords
drp
seq
kinase
polypeptide
nucleotide sequence
Prior art date
Application number
PCT/US1999/013411
Other languages
English (en)
Other versions
WO1999066030A1 (fr
Inventor
Adi Kimchi
Original Assignee
Yeda Res & Dev
Mcinnis Patricia A
Adi Kimchi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yeda Res & Dev, Mcinnis Patricia A, Adi Kimchi filed Critical Yeda Res & Dev
Priority to AU44408/99A priority Critical patent/AU4440899A/en
Priority to GB0100660A priority patent/GB2354522B/en
Priority to US09/719,748 priority patent/US7026148B1/en
Priority to IL14003099A priority patent/IL140030A0/xx
Publication of WO1999066030A1 publication Critical patent/WO1999066030A1/fr
Publication of WO1999066030A9 publication Critical patent/WO1999066030A9/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins

Definitions

  • the present invention is directed to a DAP-kinase related protein.
  • tumor-suppressor genes One of the factors which determines the proliferation state of cells is the balance between the growth-promoting effects of proto-oncogenes and the growth-constraining effects of tumor-suppressor genes.
  • One mechanism by which tumor-suppressor genes exert their growth-constraining effect is by inducing a cell to undergo a physiological type of death. Such a controlled cell death is evident in a multitude of physiological conditions including metamorphosis, synaptogenesis of neurons, death of lymphocytes during receptor repertoire selection, and controlled homeostasis in the bone marrow and other proliferative tissues, etc. This cell death is regulated by the interaction of the cell with other cells or with cell products, for example through the activity of suitable cytokines.
  • cytokines have a double effect on the target cell. They can either inhibit the proliferation of the cell and/or give rise to cell death.
  • blockage or activation of expression of known tumor-suppressor genes was shown to counteract or enhance, respectively, cytokines inhibition of cells growth (Kimchi, 1992) but did not have any effect on the death- promoting action of cytokines.
  • the growth inhibitory response to cytokines, such as TGF- ⁇ was markedly reduced by the inactivation of the Rb gene, or the response to LL-6 was enhanced by introducing activated p53 genes (Pietenpol et aladmi 1990; Levy et al, 1993).
  • Apoptosis is a genetically controlled cell death process which is important in various developmental stages, as well as for cell maintenance and tissue homeostasis (Jacobson et al., 1997). During the last few years, many of the key players in this process have been identified, including receptors, adaptor proteins, proteases, and other positive and negative regulators (Green et al., 1998; White, 1996).
  • One of the positive mediators of apoptosis which has been cloned by the present inventors, is DAP-kinase (Deiss et al., 1995).
  • This protein was discovered by a functional approach to gene cloning, based on transfections of mammalian cells with anti-sense cDNA libraries and subsequent isolation of death-protective cDNA fragments (Deiss et al., 1995; Deiss et al., 1991; Kimchi, 1998; Kissil et al., 1998; Levy-Strumpf et al., 1998).
  • the anti-sense cDNA of DAP-kinase protected HeLa cells from interferon-gamma-induced cell death, and this property served as the basis for its selection.
  • DAP-kinase is a calcium/calmodulin-regulated 160 kDa serine/threonine protein kinase associated with actin microfilaments (Deiss et al., 1995; Cohen et al., 1997). Its structure contains at least two additional domains that might mediate interactions with other proteins: ankyrin repeats, and a typical death domain located at the C-terminal part of the protein (Deiss et al., 1995; Cohen et al., 1997).
  • DAP-kinase Overexpression of DAP- kinase in various cell lines results in cell death, and this death-promoting effect of DAP-kinase depends on at least three features: the catalytic activity, presence of the death domain, and the correct intracellular localization (Cohen et al., 1997; Cohen et al., 1999).
  • a tumor suppressive function was recently attributed to the DAP -Kinase, coupling the control of apoptosis to metastasis (Inbal et al., 1997).
  • MEKK-1 whose kinase activity is stimulated by caspase cleavage (Cardone et al., 1997). JNK may antagonize BCL-2 anti-apoptotic effects by phosphorylation (Park et al., 1997; Maundrell et al., 1997).
  • RIP serine/threonine kinase
  • DAP -Kinase serine/threonine kinase
  • RIP was shown to positively mediate apoptosis in cell cultures (Stanger et al., 1995). However, in vivo studies in RIP-deficient mice demonstrated its ability to exert anti-apoptotic effects by mediating the TNF- ⁇ - induced TNF- ⁇ activation (Kelliher et al., 1998).
  • Other RLP members, RTP2 and RLP 3 were also recently identified and shown to possess pro-apoptotic effects (McCarthy et al., 1998; Sun et al., 1998; Yu et al., 1999).
  • AKT protein kinase
  • ZIP-kinase One protein, named ZIP-kinase, was found to be 80% identical to DAP-kinase within the kinase domain, yet it lacks the CaM-regulatory domain and the other domains and motifs characteristic of DAP-kinase.
  • Zip-kinase contains a leucine zipper domain at the C-terminus and is localized to the nucleus (Kawai et al., 1998; Kogel et al. 1998). The activation of ZIP kinase occurs by a different mechanism involving homo- dimerization, mediated by its leucine zipper domain.
  • ZIP-kinase is a nuclear protein, which instead of being regulated by a calmodulin-binding domain, is activated by homo-dimerization of its leucine-zipper motifs (Kogel et al., 1998).
  • DRAKl and DRAK2 are closely related to each other, and which share 50% identity with the kinase domain of DAP-kinase, were also recently characterized.
  • the DRAKl and DRAK2 proteins also lack the CaM-regulatory domain.
  • DAP-Kinase-related 1 protein which is a novel homologue of DAP-kinase, has been isolated.
  • This novel calmodulin-dependent kinase is a 42kDa serine/threonine kinase which shows a high degree of homology to DAP-kinase both in its catalytic domain and its calmodulin-regulatory region.
  • the catalytic domain of DRP-1 is also homologous to recently identified ZLP -kinase and, to a lesser extent, to the catalytic domains of DRAK1/2.
  • DRP-1 is localized to the cytoplasm as shown by immunostaining and cellular fractionation assays. In vitro kinase assays indicate that wild type DRP-1, but not a kinase inactive mutant, undergoes autophosphorylation and phosphorylates an external substrate in a Ca2+/CaM-dependent manner. Ectopically expressed DRP-1 is able to induce apoptosis in various types of cells; with this killing being dependent on its kinase activity.
  • DAPk DD The dominant negative form of DAP -Kinase (DAPk DD) is a potent blocker of apoptosis induced DRP-1.
  • DRP-1 may be a death-promoting protein functioning in the biochemical pathway which involves DAP (death-associated protein)-kinase (e.g., forming a cascade of sequential kinases, one directly activating the other).
  • DAP death-associated protein
  • the two kinases may operate to promote cell death in parallel pathways.
  • the present invention provides for a DRP-1 protein and functional homologues thereof having at least 85% sequence identity to the DRP-1 sequence of SEQ ID NO:2. Also provided is a fragment of DRP-1, which either is capable of inducing cell death or lacks such capability but instead is capable of inhibiting the activity of DRP-1 or a functional homologue thereof to induce cell death, and a homologous fragment which has at least 85% sequence identity thereto and which has the same properties.
  • the present invention further provides an isolated DNA molecule encoding for such DRP-1 protein, functional homologues thereof, or fragments thereof. Also included within the scope of the present invention are isolated DNA molecules which hybridize to the nucleotide sequence encoding DRP-1 protein under moderately or highly stringent conditions and encode a calmodulin-dependent serine/threonine kinase having the property of being capable of inducing cell death.
  • compositions comprising the DRP-1 protein, functional homologues and fragments thereof, and an antibody which specifically recognizes DRP-1 but does not cross-react with DAP kinase or ZIP kinase.
  • Yet another aspect of the present invention is directed to a single stranded RNA molecule complementary to at least a portion of the mRNA encoding the DRP-1 protein of SEQ ID NO:2.
  • This single stranded antisense RNA molecule can be used in a method of neutralizing DRP-1 mRNA by hybridizing to the DRP-1 mRNA to prevent its translation into DRP-1 protein.
  • the present invention also provides a method for screening individuals for predisposition to cancer.
  • Figure 1 shows the nucleotide (SEQ LD NO: 1) and amino acid (SEQ LD NO: 2) sequence of the DAP-kinase homologue, DRP-1.
  • the initiation (ATG) and stop (TAA) codons are boxed.
  • the polyadenylation signal (ATTAAA) is underlined.
  • the kinase domain and the calmodulin regulatory regions are in bold or underlined by a dash, respectively.
  • Figures 2A-2B show the multiple sequence alignment of the serine/threonine kinase domains (Fig.
  • DAP-kinase-related proteins DAP-kinase (SEQ ID NO:3), ZLP-kinase (SEQ ID NO:4), DRP-1 (corresponding to residues 13-275 of SEQ ID NO:2), DRAKl (SEQ ID NO:5) and DRAK 2 (SEQ LD NO:6), conducted according to Hanks and Quinn (1991) with identical amino acids boxed and homologous amino acids shown with gray shading, and the multiple sequence alignment of the calmodulin regulatory regions (Fig. 2B) of DAP-kinase-related proteins, DAP-kinase (SEQ ID NO:3), ZLP-kinase (SEQ ID NO:4), DRP-1 (corresponding to residues 13-275 of SEQ ID NO:2), DRAKl (SEQ ID NO:5) and DRAK 2 (SEQ LD NO:6), conducted according to Hanks and Quinn (1991) with identical amino acids boxed and homologous amino acids shown with gray shading, and the multiple sequence alignment
  • DAP-kinase (SEQ LD NO: 7), DRP-1 (corresponding to residues 292 to 320 of SEQ ID NO:2), smMLCK (SEQ LD NO:8), CaMKIIa (SEQ LD NO:9), CaMKI (SEQ LD NO:10), CaMKIV (SEQ LD NO: 11), and ZJR-Kinase (SEQ LD NO: 12) conducted manually, keeping the conserved (boxed) regions aligned to each other.
  • the corresponding region of ZLP-Kinase which does not contain homology to DAP-Kinase and ZIP-Kinase CAM-regulatory regions is given at the bottom of Fig. 2B.
  • Figure 3 A shows Northern blot analysis of polyA+RNA extracted from various cell lines for mRNA expression of DRP-1
  • Figure 3B show Western blot analysis of in vitro transcription and translation of DRP-1
  • Figure 3C shows protein expression of DRP-1 in HeLA cells on an immunoblot.
  • Figures 4A and 4B show control COS-7 cells and cellular localization of DRP-1 in COS-7 cells, respectively, and Figure 4C shows a Western blot of fractions from a detergent extraction of COS-7 cells transfected with a pCDNA3 vector expressing either FLAG-tagged DRP-1 or DAP-Kinase.
  • Figure 5 A shows in vitro kinase activity of DRP-1 and Figure 5B shows a Western blot of DRP-1 proteins.
  • Figures 6A-6B show fluorescent microscope images of 293 cells transfected by pCDNA3-luciferase as a negative control (Fig. 6A), by pCDNA3- ⁇ CaM DAP-Kinase as positive control (Fig. 6B), by pCDNA -DRP-1 (Fig. 6C), and by pCDNA3-K42ADRP-l (Fig. 6D). Apoptotic cells are indicated by arrows.
  • Figure 7 shows the scores of apoptotic cells in a graph of the percentage of apoptotic cells resulting from the transfections of Figs. 6A-6D.
  • FIGs 8 A and 8B show DRP-1 protein expression in 293 transfected cells in immunoblots to anti-FLAG antibodies (Fig. 8A) and anti-vinculin antibodies (Fig. 8B).
  • Figure 9A shows that DAP kinase death domain protects from DRP-1 induced apoptosis
  • Figure 9B shows an immunoblot of DRP-1 protein expression in 293 transfected cells.
  • Figure 10A shows a schematic representation of a series of generated deletion mutant
  • Figure 10B shows an immunoblot containing extracts of 293 cells transiently transfected with GFP and the series of deletion mutants, (DRP-1 fragments, cloned in pCDNA3, and tagged with HA epitope at the C-terminus), as in Figs. 8A and 8B are probed with anti-HA antibodies for DRP-1 detection and anti-vinculin antibodies to quantitate the loaded protein amounts.
  • pCDNA3 2 -luciferase is the negative control.
  • Figure 11 A shows fluorescent microscope images of the transiently transfected cells of Fig.
  • Figure 1 IB shows a graph of the score in percent apoptotic cells in Fig. 11 A resulting from co-transfections of 293 cells with l-2 ⁇ g HA-tagged wild type DRP-1 or various deletion mutants of DRP-1 after 24 hours (average S.D. calculated from triplicates of 100 cells each).
  • Figures 12A and 12B show by Western analysis that the C-terminal part of DRP-1 is required for its homo-dimerization.
  • wild type DRP-1 is shown to undergo specific homo-dimerization.
  • the lanes correspond to the following co-transfections (5 ⁇ g of DRP-1 constructs and 20 g of RFXl- ⁇ Smal constructs/9mm plate): (1) DRP-1- FLAG+RFXl- ⁇ Smal-HA (control to rule-out nonspecific attachment of DRP-1 to HA beads or to an irrelevant gene). (2) RFX- ⁇ SmaI-FLAG+DRP-l-HA(control to rule out nonspecific attachment of DRP-1 to FLAG beads or to an irrelevant gene).
  • DRP-1 -FLAG+DRP-1- HA Both LP directions and their Western blottings are shown.
  • Figure 12B truncation of C-terminal 40 amino acids of DRP-1 is shown to abolish its homo-dimerization.
  • the lanes correspond to the following co-transfections (5 ⁇ g of each construct/90mm plate): (1) DRP-1 - FLAG+DRP-1-HA (2) DRP-1 -FLAG+DRP-1- ⁇ 40-HA (3) DRP-1-FLAG+DRP-1- ⁇ 73-HA (4) DRP-1-FLAG+DRP-1- ⁇ 85-HA.
  • the lower panel quantitate the immunoprecipitation efficiency of DRP-1 -FLAG by the anti-FLAG antibodies.
  • the present invention is based on the discovery by the present inventor of a novel serine/threonine kinase with remarkable homology to the catalytic and CaM-regulatory domains of DAP-kinase.
  • This kinase named DAP-kinase-related protein 1 (DRP-1)
  • DAP-kinase-related protein 1 DAP-kinase-related protein 1
  • DAP-1 DAP-kinase-related protein 1
  • SEQ ID NO: 1 amino acid sequences of this
  • DRP-1 protein are shown in Fig 1. It is composed of 1742 nucleotides. The predicted initiation and stop codons are boxed, and the polyadenylation signal is underlined. The protein kinase domain is shown in bold letters and corresponds to amino acid residues 13 to 275 of SEQ ID NO:2. This protein displays 80% identity with the catalytic domain of DAP- kinase. The calmodulin-regulatory region is underlined with a dashed line; this region displays high homology to the corresponding region in DAP-kinase.
  • DAP-kinase-related I protein does not carry all of the other motifs and protein modules characteristic of DAP- kinase.
  • the mRNA expression levels transcribed from this gene are low.
  • ZLP-kinase Another protein, ZLP-kinase, which by virtue of its sequence homology to the kinase domain of DAP-Kinase, is also a member of the DAP-Kinase-related proteins subfamily, was recently identified (Kawai et al., 1998; Kogel et al., 1998). Unlike DAP- Kinase and DRP-1, ZIP-kinase is a nuclear protein, which instead of being regulated by a calmodulin-binding domain, is activated only by homo-dimerization via its leucine-zipper motifs (Kawai et al., 1998).
  • DRP-1 K42A a kinase inactive mutant of DRP-1 (DRP-1 K42A)
  • DRP-1 K42A did not induce apoptosis, although it was expressed at a similar level in the transfected cells.
  • DRP-1 K42A is indeed unable to phosphorylate MLC or under autophosphorylation.
  • DRP-1 which lacks the CaM-regulatory region
  • DAP-Kinase a truncated form of DRP-1 which lacks the CaM-regulatory region
  • ⁇ CaM the CaM-regulatory region of DAP-Kinase
  • Such dependence on the catalytic activity for the apoptotic function is apparent also in the other members of DAP-kinase-related proteins (Kawai et al., 1998; Sanjo et al., 1998).
  • the present invention thus provides for the polypeptide of DRP-1 and for a calmodulin-dependent serine/threonine kinase homologue having the properties of DRP-1, such as the ability to phosphorylate protein in a calcium/calmodulin dependent manner and the ability to induce programmed cell death or apoptosis, and having at least 85% sequence identity to the amino acid sequence SEQ LD NO: 2 of DRP-1.
  • the calmodulin- dependent serine/threonine kinase homologue has at least 90% sequence identity, and more preferably, at least 95% sequence identity to SEQ ID NO:2.
  • the Clustal-X program referred to in the previous paragraph is the Windows interface for the ClustalW multiple sequence alignment program (Thompson et al., 1994).
  • sequences are aligned using Version 9 of the Genetic Computing Group's GDAP (global alignment program), using the default (BLOSUM62) matrix (values -4 to +11) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (per each additional consecutive null in the gap).
  • BLOSUM62 default matrix
  • percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the claimed sequence.
  • the present invention also provides for a fragment of the DRP-1 protein of SEQ LD NO: 2 which either maintains the ability to induce cell death or lacks this ability but instead is capable of inhibiting the cell killing ability of DRP-1 protein or its functional homologue described above.
  • the 40 amino acid C-terminal tail (residues 321 to 360 of SEQ LD NO:2) is critical to induction of cell death. As the action of DRP-1 is dependent on dimerization, the 40 amino acid tail, by itself, can inhibit the ability of DRP-1 to induce cell death by interfering with and preventing DRP-1 from dimerizing.
  • the catalytic domain by itself (without the calmodulin regulatory domain and the 40 amino acid C-terminal tail, e.g., amino acid residues 13 to 275 of SEQ ID NO:2), is super-killing.
  • One of ordinary skill in the art can readily obtain fragments of the full length sequence of the present invention using N-terminal amino peptidases or C-terminal carboxypeptidases. Each fragment can then be readily tested to see if it possesses one of the two functions described herein for such fragments, without undue experimentation.
  • fragments of DRP-1 having the above-mentioned properties fragments having an amino acid sequence with at least 85% sequence identity to the above fragments of DRP-1, preferably with at least 90% sequence identity, and more preferably with at least 95% sequence identity, and maintaining the cell death induction or inhibition properties of the original fragment, are also comprehended by the present invention.
  • chemical derivative contains additional chemical moieties not normally part of the DRP-1 amino acid sequence. Covalent modifications of the amino acid sequence are included within the scope of this invention. Such modifications may be introduced into DRP-1 or fragments thereof by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxylmethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo- beta-(5-imidazoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo- 2-oxa- 1 , 3 -diazole.
  • Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Parabromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for derivatizing alpha-amino acid-containing residues include imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methyliosurea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methyliosurea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3- butanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine, as well as the arginine epsilon-amino group.
  • Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'N-C-N-R 1 ) such asl-cyclohexyl-3-[2-morpholinyl-(4-ethyl)] carbodiimide or 1- ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • the present invention also comprehends an isolated DNA molecule which includes a nucleotide sequence encoding the DRP-1 protein of SEQ LD NO:2, a functional homologue thereof as described above, or a fragment of DRP-1 which either maintains the ability of DRP-1 to induce cell death or lacks this ability but is instead capable of inhibiting the cell killing ability of DRP-1 protein, as defined above.
  • the isolated DNA molecule according to the present invention is also intended to comprehend a DNA molecule which hybridizes under moderately stringent, preferably highly stringent, conditions to the nucleotide sequence encoding DRP-1 (corresponding to nucleotides 62 to 1141 of SEQ LD NO: 1) and which encodes a polypeptide which maintains the cell death induction properties of DRP-1.
  • the present invention further comprehends isolated DNA molecules which hybridize under moderately stringent, preferably highly stringent, conditions to a nucleotide sequence which encodes for a fragment of DRP-1 which either maintains the ability of DRP-1 to induce cell death (i.e., nucleotides 98 to 886 of SEQ ID NO: 1 encoding the catalytic kinase domain of DRP-1) or lacks the ability but is instead capable of inhibiting the cell killing ability of DRP-1 protein (i.e., nucleotides 1022 to 1141 of SEQ LD NO:l encoding the 40 amino acid C- terminal tail of DRP-1).
  • polypeptides encoded by any nucleic acid such as DNA or RNA, which hybridizes to the nucleotide sequence of nucleotides 62 to 141 of SEQ ID NO: 1 under moderately stringent or highly stringent conditions are considered to be within the scope of the present invention as long as the encoded polypeptide maintains the ability of DRP-1 to induce cell death.
  • stringency conditions are a function of the temperature used in the hybridization experiment, the molarity of the monovalent cations and the percentage of formamide in the hybridization solution.
  • Tm melting temperature
  • Tm 81.5°C + 16.6 (LogM) + 0.41 (%GC) - 0.61 (% form) - 500/L
  • M is the molarity of monovalent cations
  • %GC is the percentage of G and C nucleotides in the DNA
  • % form is the percentage of formamide in the hybridization solution
  • L is the length of the hybrid in base pairs.
  • highly stringent conditions are those which provide a Tm which is not more than 10°C below the Tm that would exist for a perfect duplex with the target sequence, either as calculated by the above formula or as actually measured.
  • Modely stringent conditions are those which provide a Tm which is not more than 20 °C below the Tm that would exist for a perfect duplex with the target sequence, either as calculated by the above formula or as actually measured.
  • examples of highly stringent (5-10°C below the calculated or measured Tm of the hybrid) and moderately stringent (15-20°C below the calculated or measured Tm of the hybrid) conditions use a wash solution of 2 X SSC (standard saline citrate) and 0.5% SDS (sodium dodecyl sulfate) at the appropriate temperature below the calculated Tm of the hybrid.
  • the ultimate stringency of the conditions is primarily due to the washing conditions, particularly if the hybridization conditions used are those which allow less stable hybrids to form along with stable hybrids. The wash conditions at higher stringency then remove the less stable hybrids.
  • a common hybridization condition that can be used with the highly stringent to moderately stringent wash conditions described above is hybridization in a solution of 6 X SSC (or 6 X SSPE (standard seline-phosphate-EDTA)), 5 X Denhardt's reagent, 0.5% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA at a temperature approximately 20° to 25° C below the Tm. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel, 1987, 19989. Additional aspects of the present invention are vectors which carry the isolated
  • the present invention further provides for antisense RNA complementary to at least a portion of a messenger RNA (mRNA or "sense" RNA) molecule which is the transcription product of the DNA sequence encoding the DRP-1 protein of SEQ LD NO:2.
  • mRNA or "sense" RNA messenger RNA
  • the antisense DRP-1 sequence can be chemically synthesized or it can be expressed in host cells. However, when expressed in host cells, the expressed antisense RNA must be stable (i.e., does not undergo rapid degradation).
  • the antisense DRP-1 RNA will essentially specifically only hybridize to the sense DRP-1 mRNA and form a stable double- stranded RNA molecule that is essentially non-translatable.
  • the antisense DRP-1 RNA prevents the expressed sense DRP-1 mRNA from being translated into active DRP-1 protein.
  • a vector-borne antisense DRP-1 sequence may carry either the entire DRP-1 gene sequence or merely a portion thereof as long as the antisense DRP-1 sequence is capable of hybridizing to "sense" DRP-1 mRNA to prevent its translation into DRP-1 protein.
  • an "antisense" sequence of the present invention can be defined as a sequence which is capable of specifically hybridizing to "sense" DRP-1 mRNA to form a non-translatable double-stranded RNA molecule.
  • the antisense DRP-1 sequence need not hybridize to the entire length of the DRP-1 mRNA. Instead, it may hybridize to selected regions, such as the 5 -untranslated sequence, the coding sequence, or the 3 '-untranslated sequence of the "sense" mRNA.
  • the antisense DRP-1 sequence is preferably at least 17, more preferably at least 30, base pairs in length.
  • shorter sequences may still be useful, i.e., they either fortuitously do not hybridize to other mammalian sequences, or such "cross-hybridization" does not interfere with the metabolism of the cell in a manner and to a degree which prevents the accomplishment of an object of this invention.
  • Standard methods such as described in Sambrooke et al., (1989) can be used to systematically remove an increasingly larger portion of the antisense DRP-1 sequence from a plasmid vector. Besides the full length antisense DRP-1 sequence, a series of staggered deletions may be generated, preferably at the 5 '-end of the antisense DRP-1 sequence.
  • the antisense RNA according to the present invention can be used in a method to neutralize a mRNA molecule, which is the transcription product of the DNA sequence encoding the DRP-1 protein of SEQ ID NO:2, by allowing the antisense RNA to hybridize to the DRP-1 mRNA to prevent its translation into DRP-1 protein.
  • a further aspect of the present invention is directed to a composition, such as a pharmaceutical composition, which contains DRP-1, functional homologues or fragments thereof and a pharmaceutically-acceptable excipient, carrier, diluent, or auxiliary agent.
  • an antibody which specifically recognizes DRP-1 or functional homologues thereof is part of the present invention as long as the antibody does not cross-react with DAP- Kinase or ZLP-kinase.
  • an antibody that specifically recognizes the unique 40 amino acid C-terminal tail of DRP-1, which is not present in DAP-Kinase or ZLP-kinase is a preferred embodiment of the antibody according to the present invention.
  • Such an antibody can be used for diagnostic imaging, purification of DRP-1 etc.
  • antibody or “antibodies” as used herein are intended to include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof such as the Fab or F(ab') 2 fragments.
  • mAbs monoclonal antibodies
  • the DNA encoding the variable region of the antibody can be inserted into other antibodies to produce chimeric antibodies (see, for example, U.S. Patent 4,816,567) or into T-cell receptors to produce T-cells with the same broad specificity (Eshhar et al., 1990; Gross, et al., 1989). Single chain antibodies can also be produced and used.
  • Single chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising a pair of amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain (linked V H -V L or single chain F v ). Both V H and V L may copy natural monoclonal antibody sequences or one or both of the chains may comprise a CDR-FR construct of the type described in U.S. Patent 5,091,513 (the entire contents of which are hereby incorporated herein by reference). The separate polypeptides analogous to the variable regions of the light and heavy chains are held together by a polypeptide linker.
  • antibody or “antibodies” are also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab') 2 , which are capable of binding antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., 1983).
  • Fab and F(ab') 2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of DRP-1 or functional homologues thereof according to the methods used for intact antibody molecules.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • the present invention comprehends not only the intact antibodies or fragments, but also any molecule which includes an antigen binding portion of an antibody such that the molecule is capable of binding to the antigen. It is well within the skill of the art for the artisan to make e.g., fusion proteins which include antigen binding portions of an antibody fused to any other material which is desired to be carried to the antigen binding site, such as marker molecules, toxins, etc.
  • the antibodies, or fragments of antibodies, of the present invention may be used to quantitatively or qualitatively detect the presence of DRP-1 or functional homologues according to the present invention in a sample.
  • the antibody according to the present invention may also be used for the isolation and purification of DRP-1 or homologues and fragments thereof, such as in an affinity column where the antibodies are immobilized on a solid phase support or carrier.
  • solid phase support or carrier any support capable of binding antigen or antibodies.
  • supports, or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • One of the ways in which the DRP-1 -specific antibody can be detectably labeled is by linking the same to an enzyme and used in an enzyme immunoassay (EIA).
  • EIA enzyme immunoassay
  • This enzyme when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay (RIA) (Chard, T., "An Introduction to Radioimmune Assay and Related Techniques” (In: Work, T.S., et al, Laboratory Techniques in Biochemistry in Molecular Biology. North Holland Publishing Company, New York (1978), incorporated by reference herein).
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a liquid scintillation counter or by autoradiography.
  • Radioactively labeled antibodies or antibody fragments can also be used for their capacity to kill cells bound by such antibodies, or cells in the immediate vicinity which are exposed to the radiation from such antibodies. It is also possible to label the antibody with a fluorescent compound, a chemiluminescent or bioluminescent compound.
  • the antibody molecules of the present invention may also be adapted for utilization in an immunometric assay (also known as a "two-site” or “sandwich” assay) which is well know in the art.
  • an immunometric assay also known as a "two-site” or “sandwich” assay
  • programmed cell death is used to denote a physiological type of cell death which results from activation of some cellular mechanisms, i.e., death which is controlled by the cell's machinery.
  • Programmed cell death may, for example, be the result of activation of the cell machinery by an external trigger, e.g., a cytokine, which leads to cell death.
  • an external trigger e.g., a cytokine
  • apoptosis is also used interchangeably with programmed cell death.
  • tumor in the present specification denotes an uncontrolled growing mass of abnormal cells. This term includes both primary tumors, which may be benign or malignant, as well as secondary tumors, or metastases, which have spread to other sites in the body.
  • DRP-1 can be used to inhibit growth and metastasis of tumors.
  • Tumor cells are exposed to a variety of death-inducing signals which, in combination with DAP-kinase-related I, can lead to death of the tumor cells.
  • DAP-kinase-related I can lead to death of the tumor cells.
  • invading tumor cells must resist programmed cell death that is induced by interactions with cytotoxic T lymphocytes, natural killer cells, and macrophages, and with the cytokines which these hematopoietic cells secrete (e.g., LFNs, TNF, LL-1 ⁇ ).
  • Tumor cells must also resist the apoptotic cell death induced by nitric oxide anions produced by the endothelial cells, and withstand mechanical shearing forces caused by hemodynamic turbulence. Moreover, during the intravasation or extravasation processes, and during growth in a foreign hostile microenvironment, locally produced inhibitory cytokines (e.g., TGF- ⁇ or loss of cell-matrix interactions (e.g., detachment from the basement membranes) also trigger apoptotic cell death. DRP-1 is useful in promoting death of tumor cells.
  • the protein may be administered to patients, in particular, to cancer patients, which administration may cause death of the tumor cells.
  • the protein may be administered per se, or may be administered by an expression vector comprising a DNA molecule of the present invention.
  • DRP-1 displays 80% identity with the catalytic domain of DAP-kinase and has a region which displays a high homology to the calmodulin-regulatory region of DAP-kinase, it is expected that DRP-1 has enzymatic kinase activity, which is calmodulin-dependent.
  • DRP-1 has use as an enzyme and may be used, for example, as the enzyme in any in vitro enzymatic reaction which requires the presence of a kinase enzyme. Accordingly, DRP-1 can be used in vitro to catalyze phosphorylation reactions as a kinase.
  • DRP-1 is capable of inducing apoptotic cell death when overexpressed in various cell lines. This ectopic cell death is blocked specifically by the death domain of DAP-kinase, suggesting possible crosstalk between these two kinases. Thus, DRP-1 may also be used for promoting the death of normal or tumor cells and for suppressing the metastatic activity of tumor cells. A particular application of the death-promoting aspect is in therapy of diseases or disorders associated with uncontrolled, pathological cell growth, e.g., cancer (primary tumors and metastasis), psoriasis, autoimmune disease and others.
  • cancer primary tumors and metastasis
  • psoriasis psoriasis
  • DAP-kinase-related protein I of the present invention may be used in the same manner as disclosed in detail in U.S. applications 08/810,712 and 08/631,097, as well as WO 95/10630.
  • DRP-1 DNA molecules are used in order to screen individuals for predisposition to cancer.
  • the screening is carried out by comparing the sequence of each of the DAP-kinase-related I DNA molecules to each of the respective DAP genes in the individual, or by following RNA and/or protein expression.
  • the absence of a DAP-kinase-related I gene, a partial deletion or any other difference in the sequence that indicates a mutation in an essential region, or the lack of a DRP-1 RNA and/or protein which may result in a loss of function may lead to a predisposition for cancer.
  • a battery of related DAP and DRP-1 genes maybe used, as well as different antibodies.
  • DAP-kinase related product I molecules may also be used for prognostic purposes. For example, if a tumor cell lacks DRP-1 activity, this may reflect high chances of developing metastasis.
  • DRP-1 positive cells may be more susceptible to control by chemotherapeutic drugs that work by inducing apoptosis, so that the choice of treatment modalities may be made based upon the DRP-1 state of the cells.
  • the DAP-kinase-related product can be used to screen individuals for predisposition to cancer.
  • a method for detecting the absence of a DRP-1 gene, a partial deletion or a mutation (i.e., point mutation, deletion or any other mutation) in the DRP-1 genes of an individual, or the absence of a DRP-1 RNA or protein comprising probing genomic DNA, cDNA, or RNA from the individual with a DNA probe or a multitude of DNA probes having a complete or partial sequence of the DRP-1 genes, or probing protein extracts with specific antibodies.
  • a particular application of the screening aspect of this invention is in the screening for individuals having a predisposition to cancer, an absence of the gene, or a detected mutation or deletion indicating that the individual has such a predisposition.
  • One example of a method in accordance with the screening aspect typically comprises the following steps:
  • (c) providing conditions for hybridization to determine whether the DRP-1 gene is present or absent, i.e., whether there is a match between the sequence of the DNA probe or probes and a sequence in the DNA of said sample or a mismatch, a mismatch indicating a deletion or a mutation in the endogenous DNA and a predisposition to cancer in the tested individual.
  • screening aspect of the invention include, but are not limited to, Northern blots, RNase protection assays, and various PCR procedures.
  • the mutation in the DRP-1 gene indicating a possible predisposition to cancer, can also be detected by the aid of appropriate antibodies which are able to distinguish between a mutated, a non-functional and a normal functional DRP-1 gene product.
  • mutations that abolish protein translation or transcription due to promoter inactivation can be detected with the aid of antibodies that are used to react with protein cell extracts. Screening is also possible with respect to metastases.
  • DRP-1 DAP-kinase-related protein
  • DRP-1 is a 42kDa Ca 2 7CaM-regulated serine/threonine kinase which shows high degree of homology to DAP (Death Associated Protein)-kinase.
  • the homology spans over the catalytic domain and the calmodulin-regulatory region, whereas the rest C-terminal part of the protein differs completely from DAP-kinase and displays no homology to any known protein.
  • the catalytic domain is also homologous to the recently identified ZLP-kinase and to a lesser extent to the catalytic domains of DRAKl II, thus forming together a novel subfamily of serine/threonine kinases.
  • DRP-1 is localized to the cytoplasm as shown by immunostaining and cellular fractionation assays. In vitro kinase assays indicate that wild type DRP-1, but not a kinase inactive mutant, undergoes autophosphorylation and phosphorylates an external substrate in a Ca 2 7CaM-dependent manner. Ectopically expressed DRP-1 is able to induce apoptosis in various types of cells.
  • DRP-1 Cell killing by DRP-1 is dependent on two features: the intact kinase activity and the presence of C-terminal 40 amino acids shown to be involved in self-dimerization of the kinase. Interestingly, further deletion of the CaM-regulatory region overrided the indispensable role of the C-terminal tail and generated a "super-killer" mutant. Finally, a dominant negative fragment of DAP-kinase encompassing the death domain is a potent blocker of apoptosis induced by DRP-1. This implies a possible functional connection between DAP-kinase and DRP-1. The experiments conducted in this study and the results obtained are presented below.
  • a PCR fragment of 364 bp was obtained from a ⁇ gtll human spleen cDNA library (Clontech) using primers from the deduced DRP-1 sequence, 1047-GGCCGGATGAGGACCTGAGG-1066 (SEQ LD NO:13) and
  • RNA prepared by a standard procedure from various cell lines.
  • RNA was translated in reticulocyte lysate (TNT® T7 Quick Coupled Transcription Translation System; Promega) by conventional procedures, with [ 3S S] methionine (Amersham) as a labeled precursor.
  • TNT® T7 Quick Coupled Transcription Translation System Promega
  • [ 3S S] methionine Amersham
  • the reaction product was then run on 12% SDS-PAGE gel, followed by sodium salicylate incubation for signal amplification. The gel was dried and exposed to X-ray film.
  • In vitro Kinase Assay 293 cells were transfected by a FLAG-tagged wild type DRP-1, DRP-1 K42A mutant, or mock-transfected. Cell lysates of 293 transfected cells were prepared as previously described (Deiss et al., 1995). Immunoprecipitation of DRP-1 or DRP-1 K42A mutant from 150 ⁇ g total extract was done with 20 ⁇ l anti-FLAG M2 gel (LBI, Kodak) in 500 ⁇ l of PLB supplemented with protease and phosphatase inhibitors for 2h at 4°C.
  • reaction buffer 50 mM HEPES pH 7.5, 20 mM MgCl 2 , and 0.1 mg/ml BSA.
  • the proteins bound to the beads were incubated for 15 min at 30°C in 50 ⁇ l of reaction buffer containing 15 ⁇ Ci [ ⁇ 32p] ATP (3 pmole), 50 ⁇ M ATP, 5 ⁇ g MLC (Sigma), and where indicated, also l ⁇ M bovine calmodulin (Sigma), 0.5 mM CaCl 2 , or 3mM EGTA in the absence of calmodulin/CaCl 2 .
  • Protein sample buffer was added to terminate the reaction, and after boiling, the proteins were analyzed on 11% SDS-PAGE. The gel was blotted onto a nitrocellulose membrane and 32 P- labeled proteins were visualized by autoradiography.
  • DRP-1 transfected or mock-transfected COS-7 cells were plated on glass cover-slips (13 mm diam.). After 48 hours, the cells were fixed/permeabilized in 3% formaldehyde for 5 min, methanol 5 min, acetone 2 min. The cells were blocked in 10% NGS for 30 min and incubated with anti-FLAG antibodies (dilution 1:100; IBI, Kodak) in 10% NGS for 60 min. Rhodamine-conjugated goat anti-mouse secondary antibodies (dilution 1:200; Jackson Immuno Research Lab.) and the nucleic acid dye, Oligreen (dilution 1:5000; Molecular Probes), for nuclear staining were then applied. The coverslips were mounted in Mowiol and observed under fluorescence microscope.
  • cell death protection assays we used a mixture containing 1.2 ⁇ g of cell death inducing plasmid (either DRP-1 or ⁇ CaM DAPk mutant), 0.5 ⁇ g of a plasmid to be tested for cell death protection (expressing DAPk-DD, DN FADD or luciferase as negative control), and 0.5 ⁇ g of pEGFP-NI plasmid.
  • Cells were counted and photographed 24 hours after transfection. In each transfection, three fields, each consisting of at least 100 GFP-positive cells, were scored for apoptotic cells according to their morphology. When indicated, cell lysates were prepared from the transient transfection at 24 hours, for protein analysis.
  • the transfections of Rat embryo fibroblasts (REF) and FACS analysis of transfected fibroblasts for DNA content distribution were done as previously described (Kissil et al., 1998).
  • 293 cells grown in 90mm plates were co-transfected with 5 ⁇ g FLAG-tagged or HA-tagged DRP-1 and 20 ⁇ g of HA-tagged or FLAG-tagged RFX1- ⁇ Smal, respectively, or with DRP-l-HA and DRP-1-FLAG, 5 ⁇ g each.
  • Immunoprecipitation of DRP-1 or RFXl- ⁇ Smal from lmg total extract was done using anti-FLAG M2 gel or anti- HA as described above. Detection of bound proteins was done using anti-HA antibodies
  • the nucleotide sequence of human DRP-1 has been submitted to the GenBankTM/EBI Data Bank (accession no. AF052941).
  • the murine DRP-1 is also deposited at the GenBankTM EBI Data Bank (accession no. AF052942).
  • EST databases were searched using the BLASTTM program. Two ESTs of human and murine origin showed remarkable amino acid homology to the catalytic domains of DAP-kinase and the recently identified protein ZLP-kinase (79.5% and 80.2% identity, respectively). A second EST search was performed using the 5' and the 3' ends of the human EST, which identified a few more overlapping ESTs. A putative novel cDNA sequence was generated and used to design primers for cloning the full length cDNA. PCR performed on human spleen cDNA library amplified a 364 bp fragment that was further used to screen the same library. The full length cDNA was then isolated, subcloned into BlueScript vector, and sequenced.
  • the isolated cDNA was found to be 1742 bp long and to contain a serine/threonine kinase domain with all of the 12 characterized subdomains present (Park et al., 1997, Fig. 1 A). Sequence alignment indicated that the catalytic domain of DRP-1 has 80%sequence identity to that of DAP-kinase and ZLP-kinase, yet less 50% sequence identity to the newly identified DRAK proteins (Fig. 2A). Like DAP-kinase but unlike ZLP-kinase, DRP-1 carries a typical CaM-regulatory region adjacent to its catalytic domain, as shown in Figures 1 and 2B.
  • DRP-1 As compared with other kinases such as CaKIIa and MLCK, DRP-1 has the highest homology to DAP- kinase in this region, as shown in Figure 2B. The remaining short stretch of amino acids at the C-terminal part of DRP-1 (40 amino acid tail) displays no homology to any known protein.
  • RNA expression of DRP-1 and Tissue Distribution was prepared from various cell lines and hybridized to a probe designed from the less conserved region of DRP-1. A single weak band of 1.9 kb appeared in some cell lines, in a Northern blot analysis of poly A+RNA (3 micrograms) extracted from various cell lines (Fig. 3 A), suggesting that the mRNA is expressed at low amounts in HeLa, 293 and MCF-7 cells.
  • the mRNA was hybridized with a radiolabeled human DRP-1 probe. The position of the transcript is indicated by an arrow.
  • a protein of 42 kDa was evident upon immunoblot analysis of the cell lysates with anti-FLAG antibodies, shown in Figure 3C.
  • the cells were harvested and lysed.
  • the extracted proteins were separated by SDS-PAGE and then immunoblotted with anti-FLAG antibodies.
  • DRP-1 is a cytoplasmic protein with minor association with insoluble matrix components.
  • DRP-1 functions as a kinase as predicted from the amino acid sequence
  • an in vitro kinase assay was performed using myosin light chain (MLC) as an exogenous substrate. This substrate was chosen because it is phosphorylated by DAP-kinase (Cohen et al., 1997).
  • MLC myosin light chain
  • DRP-1 was transfected into human kidney 293 cells, immunoprecipitated, and incubated with MLC in the presence and absence of Ca2+ and calmodulin. Both MLC phosphorylation and DRP-1 autophosphorylation were evident, as can be seen from Figure 5 A.
  • FIG. 5 A shows the autophosphorylation of DRP-1 and MLC phosphorylation, respectively, as seen after exposure of X-ray film.
  • Figure 5B shows the DRP-1 proteins by incubation of the same blot with anti-FLAG antibodies and ECL detection.
  • DRP-1 Induces Apoptosis in a Variety of Cell Lines
  • Apoptotic cells were scored after 24 hours. Overexpression of the DRP-1 resulted in massive apoptotic cell death (50-60%), as compared to the basal level of apoptotic cells caused by transfection of the non-relevant gene luciferase, shown in Figures 6A-6B and 7.
  • the fluorescent microscopic images correspond to 293 cells transfected by pCDNA3-luciferase as negative control (Fig. 6A), pCDNA#-deltaCaM DAP-kinase as positive control (Fig. 6B), pCDNA3 -DRP-1 (Fig. 6C), pCDNA3-K42A DRP-1 (Fig. 6D).
  • graphs show the percentage of apoptotic cells resulting from the above-mentioned transfections (average ⁇ S.D. calculated from triplicates of 100 cells each). The scores were taken from the same experiment shown in Figures 6A-6D.
  • DAP Kinase Death Domain Protects From DRP-1 Induced Apoptosis
  • DAPk DD dominant-negative DAP- kinase death domain
  • DRP-1 ⁇ 40 lacks the most C-terminal part of DRP-1 which displays no homology to any known protein.
  • DRP-1 ⁇ 73 lacks, in addition to that, the CaM-regulatory region of DRP- 1, and DRP-1 ⁇ 85 contains only the catalytic domain.
  • the wild type DRP-1 and the various truncation mutants of DRP-1 were transfected into 293 cells. Induction of apoptotic cell death was assayed as mentioned above in DRP-1 induced apoptosis. Overexpression of the wild type DRP-1 resulted in apoptosis (25%) while the DRP-1 ⁇ 40 had no effect in these assays. On the other hand, further truncations of the CaM-regulatory region, yielded mutants ( ⁇ 73, ⁇ 85) which acted as "super-killers" (-90% apoptosis) (Figs. 11 A and 1 IB). This experiment was repeated three times with reproducible results.
  • FIG. 12 A see LP anti- FLAG panel, lanes 2 or 1+2, respectively). Also non-specific binding of DRP-1-FLAG to HA bead or to RFX- ⁇ Smal protein could not be detected (Fig. 12 A, see UP anti-HA panel, lanes 1 or 1+2, respectively). Western analyses confirmed the expression of all proteins in these cell extracts (Fig. 12A, see Western panels).
  • DRP-1 -FLAG was co-expressed in conjugation with the various deletion mutants of DRP-1 tagged by HA. A strong binding of DRP-1 -FLAG to the wild type DRP-l-HA was detected, whereas the binding to DRP-1 ⁇ 40 was mostly abolished (Fig. 12B, upper LP panel, compare lane 1 to 2-4).
  • RLP2 is a novel NF-kappaB-activating and cell death-inducing kinase, J. Biol. Chem.. 273, 16968-75, 1998. Meinkoth et al., Anal. Biochem.. 138, 267-284, 1984.

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Abstract

On a isolé une nouvelle protéine laquelle est un nouvel homologue de DAP-kinase. Cette nouvelle kinase dépendante de la calmoduline est une protéine stimulant la mort cellulaire fonctionnant dans la voie biochimique impliquant la DAP (protéine associée à la mort)-kinase (par exemple, formant une cascade de kinases séquentielles, une activant directement l'autre). Dans un autre mode de réalisation, les deux kinases peuvent favoriser la mort cellulaire dans des voies parallèles.
PCT/US1999/013411 1998-06-15 1999-06-15 Proteine apparentee a la dap-kinase WO1999066030A1 (fr)

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US09/719,748 US7026148B1 (en) 1998-06-15 1999-06-15 DAP-kinase related protein
IL14003099A IL140030A0 (en) 1998-06-15 1999-06-15 Polypeptides dna molecules encoding them

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