title: DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity.
abstract: DAP-kinase was initially identified as a gene whose anti-sense-mediated reduced expression protected HeLa cells from interferon-gamma-induced programmed cell death. It was cloned in our laboratory by a functional gene selection approach. According to its amino acid sequence, this 160 kDa protein was predicted to be a novel type of calmodulin-regulated serine/threonine kinase which carries ankyrin repeats and the death domain. In this work we have shown that the kinase was autophosphorylated and capable of phosphorylating an exogenous substrate in a Ca2+/calmodulin-dependent manner. We proved that calmodulin binds directly to the recombinant kinase, and generated a constitutively active kinase mutant by the deletion of the calmodulin-regulatory domain. By immunostaining and biochemical fractionations we demonstrated that the kinase is localized to the cytoskeleton, in association with the microfilament system, and mapped a region within the protein which is responsible for binding to the cytoskeleton. Several assays attributed a cell death function to the gene. Ectopic expression of wild-type DAP-kinase induced the death of target cells, and the killing property depended strictly on the status of the intrinsic kinase activity. Conversely, a catalytically inactive mutant that carried a lysine to alanine substitution within the kinase domain, displayed dominant-negative features and protected cells from interferon-gamma-induced cell death. DAP-kinase is therefore a novel cytoskeletal-associated cell death serine/threonine kinase whose activation by Ca2+/calmodulin may be linked to the biochemical mechanism underlying the cytoskeletal alterations that occur during cell death.
authors.affiliation: Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: O
authors.initials: O
authors.lastname: Cohen
authors.forename: E
authors.initials: E
authors.lastname: Feinstein
authors.forename: A
authors.initials: A
authors.lastname: Kimchi
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002384:Catalysis; D003599:Cytoskeleton / Q000201:enzymology*; D064550:Death-Associated Protein Kinases; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D002147:Calmodulin; D011994:Recombinant Proteins; D007371:Interferon-gamma; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium