73These studies indicate that the role of caspase-2 in developmental
cell death is redundant or can be compensated by other caspases
and/or that caspase-2 has context and tissue specifi c roles [ 24 ].
Studies with Casp2 −/− mice indicate that caspase-2 has important
regulatory functions in non-apoptotic contexts [ 2 , 28 , 29 ]. For
example, Casp2 −/− mice display premature aging-related traits [ 30 –
32 ] and accumulation of oxidative stress [ 32 ]. More recently, our
studies have shown that caspase-2 is important in DNA damage
signaling, genomic stability, and tumor suppression [ 21 , 29 ].
A unique feature of caspase-2 is its nuclear localization [ 5 , 6 ,
33 ]. While caspase-2 has been shown to translocate from nucleus to
cytosol during apoptosis [ 34 , 35 ], it also constitutively localizes to
the nucleus during cell death signaling [ 5 , 6 ]. Therefore, the trans-
location of caspase-2 during cell death is not necessarily an indica-
tion of its activation and function. The nuclear function of caspase-2
remains poorly understood, although it has been suggested that
nuclear localization is important for caspase-2 functions in the DNA
damage signaling response and genomic stability [ 21 ].
The activation of caspases is required for the initiation and
execution of apoptosis. As for other caspases, the most common
method to assess caspase-2 activation is by measuring the cleavage
of specifi c peptide substrates which are conjugated to a fl uorogenic
tag. Cleavage of the peptide releases the tag, and fl uorescence
emission is directly correlated to the amount of substrate cleaved
and therefore an indication of the extent of caspase activity. The
optimal substrate specifi city for caspase-2 has been determined
using peptide combinatorial libraries [ 15 , 36 ], and the synthetic
peptide VDVAD is commonly used to assess caspase-2 activity.
However, it is important to note that other caspases, including
caspase-3, the most abundant effector apoptotic caspase, are also
able to cleave this peptide substrate [ 15 , 36 ]. Therefore, when
assaying caspase-2 activity from an apoptotic cell lysate, it is not
exclusively indicative of caspase-2 activation alone. Unfortunately,
there are no biochemical assays that uniquely determine caspase-2
enzyme activity in cells or tissue lysates.
Purifi ed active recombinant caspase-2 protein is a useful bio-
chemical tool to assess caspase-2-mediated substrate cleavage in vitro,
using cell extracts or in vitro-synthesized proteins. There are cur-
rently only a few published substrates which are cleaved by caspase- 2
in apoptotic cells [ 2 ], but there are no known specifi c substrates
which can be used as a readout for caspase-2 specifi c activity and
function in vivo. The use of recombinant caspase-2 can help to vali-
date specifi c substrates in vitro. In addition to using peptide sub-
strates,^35 S-labeled caspase-2 substrates can be synthesized in vitro
and their cleavage by recombinant caspase-2 determined by fl uorog-
raphy. Similarly, auto-cleavage of caspase-2 can be assessed by incuba-
tion of recombinant protein with^35 S-labeled pro-caspase- 2 (Fig. 2 ).
Assays for Studying Caspase-2