Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 331, Issue 4, Pages 1114-1119Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2005.04.027
Keywords
CARD12; lpaf; CARD domain; nucleotide-binding site; p-loop; leucine-rich repeats; NBS-LRR proteins; caspase-1 activation
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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 pro1L-beta 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. (c) 2005 Elsevier Inc. All rights reserved.
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