Abstract
The apoptosis-linked gene ALG-2 encodes a Ca(2+)-binding protein of the penta EF-hand family. To investigate the Ca(2+) binding properties of the recombinant ALG-2 protein, we have cloned ALG-2 cDNA from mouse liver mRNA. Sequence analysis showed that two types of clones were present. One (named ALG-2,5) corresponds to the published ALG-2 sequence (Vito, P., Lacana, E., and D'Adamio, L. (1996) Science 271, 521-525); the second (named ALG-2,1) is 6 nucleotides shorter, and the corresponding protein lacks the amino acid residues Gly(121) and Phe(122). Both transcripts are present in mouse tissues in the same 2:1 molar ratio. The ALG-2,5 and ALG-2,1 recombinant proteins are fully soluble in the metal-free form but can be precipitated from bacterial lysates by Ca(2+). In the presence of Tween the Ca(2+) binding profiles display two high affinity sites with [Ca(2+)](0.5) values of 1.2 and 3.1 microM for ALG-2,5 and ALG-2,1, respectively, plus one low affinity site. Using the yeast two-hybrid system we demonstrate that both proteins have a strong tendency to form homo- and heterodimers. In contrast to ALG-2, 5, the ALG-2,1 isoform does not interact with the target protein AIP-1, earlier described to play a role in apoptosis (Vito, P., Pellegrini, L., Guiet, C., and D'Adamio, L. (1999) J. Biol. Chem. 274, 1533-1540). We propose that the minor sequence difference between ALG-2,5 and ALG-2,1 affects the Ca(2+) binding properties and function of the proteins.
Highlights
In many experimental systems it has been shown that an increased intracellular Ca2ϩ concentration, generated for instance by ionophores, induces apoptotic cell death [1]
Because in the ALG-2 depleted clones protected from apoptosis caspases were normally activated following T-cell receptor engagement, it was concluded that the ALG-2 protein acts either downstream of caspases or in a caspase-independent way [14]
ALG-2 expression is not enhanced in apoptotic cells, indicating that its activity is regulated by protein modification or by ligand interaction
Summary
In many experimental systems it has been shown that an increased intracellular Ca2ϩ concentration, generated for instance by ionophores, induces apoptotic cell death [1]. To investigate the Ca2؉ binding properties of the recombinant ALG-2 protein, we have cloned ALG-2 cDNA from mouse liver mRNA. Because truncated AIP-1, which is still able to bind ALG-2, can block apoptosis, it was postulated that AIP-1 cooperates with the ALG-2 protein to mediate the Ca2ϩ-dependent signaling leading to cell death.
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