title: Nucleotide binding to CARD12 and its role in CARD12-mediated caspase-1 activation.
abstract: CARD12 (Ipaf/Clan) is an important regulator of caspase-1 activation. It belongs to the family of the nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins. The NBS domain of the NBS-LRR proteins contains putative ATP/GTPase-specific P-loop and Mg2+-binding site motifs. However, the nucleotide-binding properties and the function of the NBS domain are unknown. We developed a nucleotide-binding assay and investigated nucleotide binding to CARD12. We find that the NBS domain of CARD12 contains a nucleotide-binding pocket with specificity for ATP/dATP. A point mutation in the P-loop (K175R) of the NBS domain abolishes ATP/dATP binding. We further demonstrate that the nucleotide-binding site is required for CARD12-mediated caspase-1 activation. CARD12 self-association and association with procaspase-1 in transfected cells were markedly decreased by the P-loop mutation K175R. Furthermore, the P-loop mutation greatly reduced caspase-1 activation-dependent proIL-1beta processing. Thus, CARD12 function is dependent on the nucleotide-binding site. Our data provide insights into the molecular mechanisms of CARD12-mediated caspase-1 activation.
authors.affiliation: Millennium Pharmaceuticals, Inc., 35 Landsdowne Street, Cambridge, MA 02139, USA. chafenlu@berkeley.edu
authors.forename: Chafen
authors.initials: C
authors.lastname: Lu
authors.forename: Anlai
authors.initials: A
authors.lastname: Wang
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Marion
authors.initials: M
authors.lastname: Dorsch
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Ocain
authors.forename: Yajun
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020170:Caspase 1 / Q000378:metabolism*; D004789:Enzyme Activation; D017354:Point Mutation; D011485:Protein Binding
chemical_list: D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D002135:Calcium-Binding Proteins; C435692:NLRC4 protein, human; D000255:Adenosine Triphosphate; D020170:Caspase 1