Nucleotide binding to CARD12 and its role
in CARD12-mediated caspase-1 activation
Chafen Lu
*
, Anlai Wang, Lin Wang, Marion Dorsch, Timothy D. Ocain,
Yajun Xu
Millennium Pharmaceuticals, Inc., 35 Landsdowne Street, Cambridge, MA 02139, USA
Received 30 March 2005
Available online 15 April 2005
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 Mg
2+
-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-med-
iated 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-1b
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.
Ó 2005 Elsevier Inc. All rights reserved.
Keywords: CARD12; Ipaf; CARD domain; Nucleotide-binding site; P-loop; Leucine-rich repeats; NBS-LRR proteins; Caspase-1 activation
CARD12, also known as Ipaf and Clan, belongs to the
new family of nucleotide-binding site and leucine-rich re-
peat (NBS-LRR) proteins. Members of the NBS-LRR
protein family, including NOD1 and NOD2, play impor-
tant roles in apoptosis, cytokine processing, and the acti-
vation of the transcription factor NF-jB [1–5]. CARD12
is an activator of caspase-1, which mediates the matura-
tion of inflammatory cytokines IL-1b and IL-18. It has
previously been shown that CARD12 directly interacts
with procaspase-1 and induces proteolytic activation of
procaspase-1 in transfected cells [6,7]. A recent finding
that macrophages from CARD12-deficient mice fail to
activate caspase-1 in response to Salmonella typhimurium
infection establishes a role of CARD12 in caspase-1
activation in vivo [8]. CARD12 has also been reported
to interact with the pro-apoptotic adaptor protein
ASC [6,9]. Co-expression of CARD12 with ASC induces
NF-jB activation and apoptosis [9,10].
The NBS-LRR proteins contain three distinct
domains: an N-terminal caspase-activating and recruit-
ment domain (CARD) or pyrin-like domain (PYD), a
central NBS domain, and a C-terminal LRR domain
[4,5] (also see Fig. 1). The CARD and PYD domains
are protein–protein interaction domains that mediate
association with other CARD or PYD-containing pro-
teins. The C-terminal LRR domain appears to be in-
volved in the recognition of microbes and their
products. It has recently been shown that NOD1 and
NOD2 use their C-terminal LRR domain to recognize
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2005.04.027
*
Corresponding author. Present address: University of California,
Berkeley, Molecular and Cell Biology, 16 Barker Hall MC 3202,
Berkeley, CA 94720, USA. Fax: +1 510 643 2352.
E-mail address: chafenlu@berkeley.edu (C. Lu).
www.elsevier.com/locate/ybbrc
Biochemical and Biophysical Research Communications 331 (2005) 1114–1119
BBRC