title: A germ line mutation in the death domain of DAPK-1 inactivates ERK-induced apoptosis.
abstract: 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.
authors.affiliation: Cancer Research UK p53 Signal Transduction Group, University of Edinburgh, South Crewe Road, Edinburgh EH4 2XR.
authors.forename: Craig
authors.initials: C
authors.lastname: Stevens
authors.forename: Yao
authors.initials: Y
authors.lastname: Lin
authors.forename: Maria
authors.initials: M
authors.lastname: Sanchez
authors.forename: Eliana
authors.initials: E
authors.lastname: Amin
authors.forename: Ellen
authors.initials: E
authors.lastname: Copson
authors.forename: Helen
authors.initials: H
authors.lastname: White
authors.forename: Vicky
authors.initials: V
authors.lastname: Durston
authors.forename: Diana M
authors.initials: DM
authors.lastname: Eccles
authors.forename: Ted
authors.initials: T
authors.lastname: Hupp
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D053938:DNA Fragmentation; D064550:Death-Associated Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases / Q000172:deficiency / Q000235:genetics; D005260:Female; D015894:Genome, Human; D018095:Germ-Line Mutation; D006801:Humans; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism; D019683:Penetrance; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D017304:Annexin A5; D051017:Apoptosis Regulatory Proteins; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases