A Germ Line Mutation in the Death Domain of DAPK-1 Inactivates ERK-induced Apoptosis
2007; Elsevier BV; Volume: 282; Issue: 18 Linguagem: Inglês
10.1074/jbc.m605649200
ISSN1083-351X
AutoresCraig Stevens, Yao Lin, M. Rodriguez Sanchez, Eliana Amin, Ellen Copson, Helen White, Vicky Durston, Diana Eccles, Ted R. Hupp,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
Resumop53 is activated genetically by a set of kinases that are components of the calcium calmodulin kinase superfamily, including CHK2, AMP kinase, and DAPK-1. In dissecting the mechanism of DAPK-1 control, a novel mutation (N1347S) was identified in the death domain of DAPK-1. The N1347S mutation prevented the death domain module binding stably to ERK in vitro and in vivo. Gel filtration demonstrated that the N1347S mutation disrupted the higher order oligomeric nature of the purified recombinant death domain miniprotein. Accordingly, the N1347S death domain module is defective in vivo in the formation of high molecular weight oligomeric intermediates after cross-linking with ethylene glycol bis(succinimidylsuccinate). Full-length DAPK-1 protein harboring a N1347S mutation in the death domain was also defective in binding to ERK in cells and was defective in formation of an ethylene glycol bis(succinimidylsuccinate)-cross-linked intermediate in vivo. Full-length DAPK-1 encoding the N1347S mutation was attenuated in tumor necrosis factor receptor-induced apoptosis. However, the N1347S mutation strikingly prevented ERK:DAPK-1-dependent apoptosis as defined by poly(ADP-ribose) polymerase cleavage, Annexin V staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling imaging. Significant penetrance of the N1347S allele was identified in normal genomic DNA indicating the mutation is germ line, not tumor derived. The frequency observed in genomic DNA was from 37 to 45% for homozygous wild-type, 41 to 47% for heterozygotes, and 12 to 15% for homozygous mutant. These data highlight a naturally occurring DAPK-1 mutation that alters the oligomeric structure of the death domain, de-stabilizes DAPK-1 binding to ERK, and prevents ERK:DAPK-1-dependent apoptosis. p53 is activated genetically by a set of kinases that are components of the calcium calmodulin kinase superfamily, including CHK2, AMP kinase, and DAPK-1. In dissecting the mechanism of DAPK-1 control, a novel mutation (N1347S) was identified in the death domain of DAPK-1. The N1347S mutation prevented the death domain module binding stably to ERK in vitro and in vivo. Gel filtration demonstrated that the N1347S mutation disrupted the higher order oligomeric nature of the purified recombinant death domain miniprotein. Accordingly, the N1347S death domain module is defective in vivo in the formation of high molecular weight oligomeric intermediates after cross-linking with ethylene glycol bis(succinimidylsuccinate). Full-length DAPK-1 protein harboring a N1347S mutation in the death domain was also defective in binding to ERK in cells and was defective in formation of an ethylene glycol bis(succinimidylsuccinate)-cross-linked intermediate in vivo. Full-length DAPK-1 encoding the N1347S mutation was attenuated in tumor necrosis factor receptor-induced apoptosis. However, the N1347S mutation strikingly prevented ERK:DAPK-1-dependent apoptosis as defined by poly(ADP-ribose) polymerase cleavage, Annexin V staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling imaging. Significant penetrance of the N1347S allele was identified in normal genomic DNA indicating the mutation is germ line, not tumor derived. The frequency observed in genomic DNA was from 37 to 45% for homozygous wild-type, 41 to 47% for heterozygotes, and 12 to 15% for homozygous mutant. These data highlight a naturally occurring DAPK-1 mutation that alters the oligomeric structure of the death domain, de-stabilizes DAPK-1 binding to ERK, and prevents ERK:DAPK-1-dependent apoptosis. The tumor suppressor protein p53 is a stress-activated DNA-binding protein and transcription factor that can induce a set of gene products implicated in growth arrest, apoptosis, redox balance, and cellular repair pathways (1Harris S.L. Gil G. Robins H. Hu W. Hirshfield K. Bond E. Bond G. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16297-16302Crossref PubMed Scopus (73) Google Scholar). Because p53 is mutated or inactivated frequently in human cancers, much effort is centered on determining the mechanisms whereby mutations inactivate the p53 protein, determining which gene products mediate the tumor suppressor activity of the protein, and identifying the enzymes that activate the protein as a tumor suppressor. It is important to determine whether the p53 "activating" or "inhibitory" enzymes are also themselves mutation targets that stimulate cancer development. One key paradigm developed for p53 is that its activity in unstressed cells is held in check by an ubiquitin-dependent degradation pathway that promotes the rapid turnover of the protein. A set of E3 2The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; ATM, ataxia telangiectasia mutated; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; AMPK, AMP kinase; TNF, tumor necrosis factor; TNFR, TNR receptor; HA, hemagglutinin; CMV, cytomegalovirus; PBS, phosphate-buffered saline; PARP, poly(ADP-ribose) polymerase; MAPK, mitogen-activate protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; CHK2, checkpoint kinase-2; DAPK-1, death-activated protein kinase-1; RSK, p90 ribosomal S6 kinase. ligases that can turnover p53 by promoting its ubiquitination include the ring-finger-containing proteins MDM2, COP-1, CHIP, and PirH2 (2Brooks C.L. Gu W. Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). 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Cell Biol. 2005; 17: 167-173Crossref PubMed Scopus (225) Google Scholar). Finally, the tumor suppressor protein DAPK-1 is a component of an oncogenic signaling pathway that mediates p53 activation (7Raveh T. Droguett G. Horwitz M.S. DePinho R.A. Kimchi A. Nat. Cell Biol. 2001; 3: 1-7Crossref PubMed Scopus (316) Google Scholar). The biochemical mechanism whereby DAPK-1 activates p53 is undefined, but biological studies have shown that DAPK-1 can induce myosin light chain phosphorylation (14Bialik S. Bresnick A.R. Kimchi A. Cell Death Differ. 2004; 11: 631-644Crossref PubMed Scopus (79) Google Scholar), autophagy (15Inbal B. Bialik S. Sabanay I. Shani G. Kimchi A. J. Cell Biol. 2002; 157: 455-468Crossref PubMed Scopus (422) Google Scholar, 16Gozuacik D. Kimchi A. Oncogene. 2004; 23: 2891-2906Crossref PubMed Scopus (1267) Google Scholar), and/or antagonize FAK (17Wang W.J. Kuo J.C. Yao C.C. Chen R.H. J. Cell Biol. 2002; 159: 169-179Crossref PubMed Scopus (141) Google Scholar). Thus, three distinct physiological stresses known to activate p53 are now linked to three signaling components of the calcium calmodulin kinase superfamily that have the common feature of being genetic components of tumor suppressor pathways ATM-CHK2, LKB-AMPK, and DAPK-1. In an attempt to begin to define the mechanism underlying how the tumor suppressor protein DAPK-1 functions at a molecular level, we have cloned the DAPK-1 gene and have identified a previously unidentified germ line mutation in the death domain. The death domain of DAPK-1 was identified originally in a genetic screen to be one of four functional domains required for DAPK-1 to exert its growth suppressive activity (18Raveh T. Berissi H. Eisenstein M. Spivak T. Kimchi A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1572-1577Crossref PubMed Scopus (68) Google Scholar). The death domain is thought to be a signaling module contained in pro-apoptotic proteins, including Fas, TNFR, FADD, and UNC5H2 (19Cohen O. Inbal B. Kissil J.L. Raveh T. Berissi H. Spivak-Kroizaman T. Feinstein E. Kimchi A. J. Cell Biol. 1999; 146: 141-148Crossref PubMed Scopus (0) Google Scholar, 20Llambi F. Laurenco F.C. Gozuacik D. Guix C. Pays L. Del Rio G. Kimchi A. Mehlen P. EMBO J. 2005; 24: 1192-1201Crossref PubMed Scopus (133) Google Scholar, 21Chen G. Goeddel D.V. Science. 2002; 296: 1634-1635Crossref PubMed Scopus (1505) Google Scholar). One recent report has indicated that the death domain of DAPK-1 forms a docking site required for its interaction with ERK (22Chen C.H. Wang W.J. Kuo J.C. Tsai H.C. Lin J.R. Chang Z.F. Chen R.H. EMBO J. 2005; 24: 294-304Crossref PubMed Scopus (187) Google Scholar), identifying the first binding protein for the death domain of DAPK-1. Our analysis on the effects of the novel germ line mutation on death domain function indicates the mutation alters death domain oligomerization and attenuates ERK docking and associated apoptotic signaling. These data highlight a post-translational mechanism whereby the DAPK-1 tumor suppressor activity can be quenched in response to signal transduction events. Further, the relatively high penetrance of the death domain mutation in germ line DNA from at least one population set identifies a signaling pathway mutation that might affect DAPK-1 activity in apoptotic diseases of the immune system, ischemic injury, and cancer. Potential study participants from the Southampton population with previously diagnosed truncating mutations of BRCA1 were identified from the data base of the Wessex Clinical Genetics Service, Southampton. Genomic DNA was obtained for 116 BRCA1 mutation carriers, 20 male and 96 female. Age at time of screening for BRCA1 mutations ranged between 29 and 57 years. The clinical histories of all subjects were reviewed to ascertain age at first and subsequent malignancies. 59 gene carriers had developed breast cancer, and a further 14 had ovarian cancer. 10 patients had undergone prophylactic bilateral oophorectomy (5 after developing breast cancer), and 3 patients had undergone bilateral risk reducing mastectomy. All malignancies occurred in female subjects; male subjects were therefore excluded from all analyses of cancer incidence. 102 anonymous genomic DNA samples (46 male, 56 female, age range 16-82), referred by the Wessex Clinical Genetics Service for genetic screening of non-neoplastic conditions, were obtained to provide an unmatched control group. Genotyping was performed using Pyrosequencing™ technology. Amplicons were generated in a 50-μl reaction volume with 15 pmol each of DAPK-Forward (5′-gtg cct tct cgc cat ga-3′) and DAPK-Reverse (biotin-5′-ata ggc ctc ctg gcc att-3′), as well as Reverse DAPK-SNP (codon 1347) (biotin-5′-ttg gga gcc ccg tta-3′), 0.2 mm dNTPs, 1.5 mm MgCl2, 1 × Buffer II (500 mm potassium chloride and 100 mm Tris-HCl, pH 8.3) (Applied Biosystems), 1 unit of AmpliTaq Gold (Applied Biosystems) using 10 ng of genomic DNA. PCR conditions were 94 °C for 7 min; 50 cycles with denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 30 s; 1 cycle at 72 °C for 7 min; and a final hold at 15 °C. Thermocycling was performed using a PTC-0225 DNA Engine Tetrad (MJ Research). Single-stranded bio-tinylated PCR products were prepared for Pyrosequencing using a Vacuum Prep Tool (Biotage AB). 3 μl of Streptavidin Sepharose™ HP (Amersham Biosciences) was added to 37 μl of binding buffer (10 mm Tris-HCl, pH 7.6, 2 m NaCl, 1 mm EDTA, 0.1% Tween 20) and mixed with 20 μl of PCR product and 20 μl of high purity water for 10 min at room temperature using a Variomag Monoshaker (Camlab). The beads containing the immobilized templates were captured onto the filter probes after applying the vacuum, and then washed with 70% ethanol for 5 s, denaturation solution (0.2 m NaOH) for 5 s, and washing buffer (10 mm Tris-acetate, pH 7.6) for 5 s. The vacuum was switched off, and the beads were released into a PSQ 96 well plate containing 45 μl of annealing buffer (20 mm Tris-acetate, 2 mm MgAc2, pH 7.6), 0.3 μm DAPK sequencing primer (5′-GTGCCTTCTCGCCATGA-3′). The samples were heated to 80 °C for 2 min and then allowed to cool to room temperature. Pyrosequencing reactions were performed according to the manufacturer's instructions using the PSQ 96 SNP Reagent Kit (Biotage AB), which contained the enzyme and substrate mixture and nucleotides. Assays were performed using the nucleotide dispensation order AGTACGCGC. The sample genotype was determined using SNP Software (Biotage AB). The C terminus of DAPK-1 (amino acids 1313-1431) was cloned into the Gateway system vector pDONR221 (Invitrogen) using the following primers: Fwd 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGAAACTGAGTCGCCTGCTGGACCCG-3′ and Rev 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGTCACCGGGATACAACAGAGCTAAT-3′. This vector was used as a template to construct DAPK-1 polymorphic variants using the in vitro mutagenesis system QuikChange (Stratagene) as recommended by the manufacturer. HA-tagged ERK and MEK(EE) expression vectors were a gift of Steve Keyse (Dundee University, UK) HA-tagged DAPK-1 was a gift of Adi Kimchi (Weizmann Institute, Israel), and this allele (Asn-1347:Leu-1392) was mutated to derive the "founding" allele (Asn-1347:Phe-1392) and the "SNP" allele (Ser-1347:Phe-1392). The sequences of the primers used for mutagenesis are (bases which introduce amino acid change are underlined): amino acid 1347 S>N (Fwd 5′-CAAAGTACAACACCAATAACGGGGCTCCCAAG-3′ and Rev 5′-CTTGGGAGCCCCGTTATTGGTGTTGTACTTTG-3′) and amino acid 1392 F>L (Fwd 5′-GGATGCCGCAGACCTTTTGCTGAAGGCATCC-3′ and Rev 5′-GGA TGC CTT CAG CAA AAG GTC TGC GGC ATCC-3′). For expression of glutathione S-transferase or His-tagged fusion protein in Escherichia coli, pDONR221-DAPK-1 vectors were recombined with the pDEST15/pDEST17 vector (Invitrogen) as recommended by the manufacturer. HA-tagged ERK was described previously, and p55-TNFR expression construct was obtained from David Dornan (Genentech). For expression in mammalian cells, the pDONR-DAPK-1 vectors were used as a template for PCR cloning into the p3XFLAG-myc-CMV-26 expression vector (Sigma), which has an N-terminal 3×FLAG tag and a C-terminal myc tag. The primers were designed to include EcoR1 and BglII restriction sites, and PCR products were cloned into the FLAG-myc vector at the same sites. The primers used were: Fwd 5′-TTGAATTCAAAACTGAGTCGCCTGCTGGACCCG-3′ and Rev 5′-TTAGATCTATCCGGGATACAACAGAGCTAATGGA-3′. HCT116 wt cells were grown in McCoy's medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) at 37 °C in a 5% CO2/H2O-saturated atmosphere. HEK293 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen). Cells for transient transfection were plated out 24 h before transfection at ∼1.5 × 106 cells per 100-mm dish or 5 × 105 cells per 60-mm dish. For Lipofectamine 2000 transfection (Invitrogen), 2 μl of Lipofectamine was used for every 1 μg of DNA transfected. Cells were harvested after a further incubation of 24-36 h. Cells were lysed in ice-cold extraction buffer (50 mm Tris (pH 7.4), 150 mm NaCl2, 5 mm EDTA, 0.5% Nonidet P-40, 5 mm NaF, 1 mm sodium vanadate, 1× protease inhibitor mixture) for 30 min and centrifuged at 13,000 rpm for 15 min to remove insoluble material. The protein content of cell extracts was measured using Bio-Rad reagent (Bio-Rad). Typically, 50 μg of cell extract was immunoblotted. Samples were resolved by denaturing gel electrophoresis, typically 4-12% precast gels (Novex) and electrotransferred to Hybond C-extra nitrocellulose membrane (Amersham Biosciences), blocked in PBS-10% nonfat milk for 30 min, then incubated with primary antibody overnight at 4 °C in PBS-5% nonfat milk-0.1% Tween-20. After washing (3 × 10 min) in PBS-Tween 20, the blot was incubated with secondary antibody, either horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (Dako, 1:5000), for 1 h at room temperature in PBS-5% nonfat milk-0.1% Tween 20. After washing (3 × 10 min) in PBS-Tween 20, proteins were visualized by incubation with ECL reagent (Sigma). Equal protein loading was confirmed with Ponceau S staining. FLAG antibody (M2) and FLAG M2-conjugated agarose were purchased from Sigma. Myc antibody (9E10) was purchased from Cancer Research UK. HA and PARP antibodies were purchased from Cell Signaling. The anti-HA antibody is from Upstate (07-221), the anti-ERK anti-body (9102), anti-phospho-ERK (9101), and anti-PARP antibody (9542) are from Cell Signaling. 16 h post-transfection, HCT116wt cells expressing FLAG-myc-tagged DAPK-1 proteins were cross-linked with 2 mm EGS (Pierce) for 1 h at 37 °C in a 5% CO2/H2O-saturated atmosphere. Cross-linking was terminated by washing cells in PBS and harvesting. For immunoprecipitation of cross-linked proteins, 30 μl of FLAG M2 beads (Sigma) were incubated overnight at 4 °C with rotation, together with 500 μl of cell extract (∼1 mg) prepared in extraction buffer (50 mm Tris (pH 7.4), 150 mm NaCl2, 5 mm EDTA, 0.5% Nonidet P-40, 5 mm NaF, 1 mm sodium vanadate, 1× protease inhibitor mixture). The bead pellets were then washed five times in lysis buffer before resuspension in 100 μl of FLAG peptide elution buffer (0.25 μg/μl FLAG peptide (Sigma) resuspended in TBS (50 mm Tris, pH 7.5, 150 mm NaCl) and mixed with constant rotation for 2 h. Eluted proteins were then resuspended in 3× SDS-loading buffer and analyzed by denaturing gel electrophoresis and immunoblotting with antibodies specific to the myc tag. For immunoprecipitation of exogenous HA-ERK from HCT116 wt cells, HA antibody (Cell Signaling) was incubated with 30 μl of washed Protein G beads (Sigma) overnight at 4 °C with constant rotation, together with cell extract (∼1 μg) diluted to a volume of 500 μl in extraction buffer (50 mm Tris (pH 7.4), 150 mm NaCl2, 5 mm EDTA, 0.5% Nonidet P-40, 5 mm NaF, 1 mm sodium vanadate, 1× protease inhibitor mixture). The bead pellets were then washed five times in extraction buffer before being resuspended in 3× SDS-loading buffer and analyzed by denaturing gel electrophoresis and immunoblotting. To monitor PARP cleavage, HEK293 cells or HCT116 wt cells were seeded and transfected 24 h later with vectors encoding the respective death domain modules (5 μg, Asn-1347:Phe-1392 or Ser-1347: Phe-1392), empty vector control, together with p55-TNFR (0.5 μg), or DAPK-1, ERK, and MEK(EE) vectors (1 μg of each). 16 h post-transfection cells were harvested and immunoblotted for PARP cleavage of FLAG-tagged death domain expression. TUNEL Assay—HCT116wt or HEK293 cells were seeded directly onto cover slips in 6-well plates. 18 h later cells were transfected with the indicated amounts of expression vectors for a further 24 h. Following transfection cells were fixed in 1% paraformaldehyde for 10 min at room temperature. Apoptotic cells were labeled using Apoptag Plus Fluorescein In Situ Apoptosis Detection Kit S7111 (Chemicon) according to the manufacturer's instruction and viewed by fluorescence microscopy. Annexin V staining—HCT116wt cells were seeded into 6-well plates. 18 h later cells were transfected with the indicated amounts of expression vectors for a further 24 h. To include any floating cells, media was collected into fluorescence-activated cell sorting tubes and centrifuged at 1700 rpm for 4 min to leave a cell pellet. Media was then discarded from tubes. Petri dishes were washed with 2 ml of PBS, PBS was discarded, and then 1.5 ml of trypsin was added to each plate and the mixture was incubated until cells became detached. Following cell detachment 1.5 ml of McCoy's 5A medium supplemented with 10% fetal calf serum was added to each plate to stop the trypsin. The cell suspensions were then added to each of the relevant cell pellets from the floating cells. Samples were centrifuged at 1700 rpm for 4 min, pellets were resuspended in 1 ml of McCoy's 5A medium supplemented with 10% fetal calf serum, and the mixture was incubated for 5 min. Samples were centrifuged for a further 4 min at 1700 rpm prior to resuspension in 1 ml of ice-cold PBS. Samples were centrifuged for a further 4 min at 1700 rpm. Apoptotic cells were detected using TACS Annexin V-FITC Apoptosis Detection Kit (R&D Systems) according to the manufacturer's instruction and analyzed by flow cytometry. PARP Cleavage—HEK293 cells were seeded into 6-cm plates. 18 h later cells were transfected with the indicated amounts of expression vectors (DAPK-1, ERK, and MEK (EE)) for a further 24 h. Cells were lysed in ice-cold TNN buffer (150 mm NaCl, 50 mm Tris, pH 8, 0.1% Nonidet P-40, phosphatase inhibitor mixture, 5 mm NaF, and 1 mm sodium orthovanadate) for 30 min on ice prior to centrifugation at 13,000 rpm for 15 min at 4 °C. Cleared lysates were then resolved by SDS-gel electrophoresis, and proteins were detected using Rabbit polyclonal antibody to PARP (Cell Signaling #9542). To examine effects of the death domain mutation on its oli-gomerization in vitro, a Superdex 200 10/300 GL (Tricon) high performance gel filtration column was equilibrated with buffer (PBS, 5% glycerol, 1 mm benzamidine pre-filtrated with a Millipore filter 0.22 μm). Sample containing the purified recombinant death domain protein (800 μg) was injected and eluted into fractions of 1 ml each. The amount of protein eluted into each fraction was then quantified using Bradford assay. The equipment employed in the gel filtration was AKTA fast protein liquid chromatography (Amersham Biosciences) and fraction collector FRAC-950 (Amersham Biosciences). The software used was UNICORN version 4.10 (Amersham Biosciences). Molecular weight markers were obtained from Sigma. A 96-well microtiter plate (Corning Inc.) was coated with purified DAPK death domain protein diluted in 0.1 m Na2HCO3, pH 8.0, and incubated overnight at 4 °C. Each well was washed 6× with PBS containing 0.1% Tween 20 (PBS-T) followed by incubation for 1 h at room temperature with gentle agitation in PBS-T supplemented with 3% bovine serum albumin. The wells were washed 6× with PBS-T prior to incubation with appropriate amounts of purified ERK protein (Upstate) diluted in PBS-T 3% bovine serum albumin for 1 h at room temperature. After 1 h incubation the plate was washed again 6× with PBS-T and incubated with antibody specific to ERK (p44/42 MAPK anti-body #9102, Cell Signaling) for 1 h at room temperature. Following a further 6× washes with PBS-T wells were incubated with secondary rabbit horseradish peroxidase antibodies followed by further washing and ECL. The results were quantified using Fluoroskan Ascent FL equipment (Labsystems) and analyzed with Ascent Software version 2.4.1 (Labsystems). Asn → Ser Mutations at Codon 1347 in the Death Domain Attenuate ERK Binding—Upon cloning and sequencing of the DAPK-1 gene cloned from total RNA of a tumor cell line, we identified two mutations in the death domain that differed from the original wild-type sequence (Fig. 1). These two mutations include an Asn → Ser mutation at codon 1347 and an Leu → Phe mutation at codon 1392. To determine whether the Asn → Ser mutation at codon 1347 or the Leu → Phe mutation at codon 1392 alters the function of the death domain, biochemical characterization was initiated. The only direct biochemical function defined for the death domain of DAPK-1 is that it functions as an ERK docking site leading to phosphorylation at amino acid 735 (22Chen C.H. Wang W.J. Kuo J.C. Tsai H.C. Lin J.R. Chang Z.F. Chen R.H. EMBO J. 2005; 24: 294-304Crossref PubMed Scopus (187) Google Scholar). Mutation of the ERK docking site on DAPK-1 from 1392-LXL-1394 to 1392-AXA-1394 attenuates ERK binding (22Chen C.H. Wang W.J. Kuo J.C. Tsai H.C. Lin J.R. Chang Z.F. Chen R.H. EMBO J. 2005; 24: 294-304Crossref PubMed Scopus (187) Google Scholar). This latter characterization utilized the Leu-1392 allele of DAPK-1 that differs from the gene we have cloned harboring the Phe-1392 codon. It is possible that the L to F mutation at codon 1392 alters ERK binding either positively or negatively, and we first determined whether the L to F mutation altered ERK binding. The transfection of the minimal death domain alleles, Asn-1347:Leu-1392 or Asn-1347:Phe-1392, did not alter the ability of the death domain to co-immunoprecipitate with transfected HA-tagged ERK (Fig. 2A, lanes 2 and 3 versus 1). These data suggest that the original identification of ERK as a death domain binding protein at the LXL motif is not altered by the Phe-1392 substitution.FIGURE 2The N1347S mutation in the death domain of DAPK-1 attenuates ERK binding. A, ERK binding to codon 1392 death domain variants. HA-tagged ERK and FLAG-tagged death domain expression vectors were transfected into cells as indicated under "Experimental Procedures." ERK was immunoprecipitated with an anti-HA antibody and immunoblotted for (i) total ERK protein (HA-immunoblot) and (ii) immunoprecipitated death domain using an anti-FLAG IgG immunoblot. The input death domain was quantified using an anti-FLAG IgG, as indicated (top panel). B, steady-state levels of the Asn-1347 and Ser-1347 death domain modules. Vectors encoding the indicated Asn-1347 and Ser-1347 alleles of the death domain were transfected into cells and immunoblotted to examine for general changes in steady state levels. C, examination of ERK binding to codon 1347 death domain mutants. HA-tagged ERK and FLAG-tagged Asn-1347 and Ser-1347 alleles of the death domain expression vectors were transfected into cells as indicated under "Experimental Procedures." Total ERK and death domain were immunoblotted, and the amount of proteins present after immunoprecipitation with an anti-HA antibody was determined by immunoblotting with an anti-HA IgG for ERK and an anti-FLAG IgG for the death domain. The top panel represents total protein in lysate by direct blotting, and the bottom panel represents protein purified in complex by co-precipitation. D, purification of recombinant death domain miniproteins. Death domain miniproteins were expressed and purified using nickel-chelate chromatography as indicated under "Experimental Procedures." Purified proteins were resolved using SDS-PAGE and stained with Coomassie Blue (lane 2, Asn-1347 death domain; lane 4, mutant Ser-1347 death domain). E, quantitation of complex formation between ERK and death domain miniproteins. Purified death domain miniproteins were adsorbed on the solid phase and increasing amounts of ERK proteins were titrated into reactions and added to microtiter wells to allow complex formation. The amount of ERK bound is represented as relative light units as a function of increasing ERK protein titrated.View Large Imag
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