N-TERMINUS CLEAVAGE OF BCL-2 BY A NOVEL CELLULAR NON-ICE CYSTEINE PROTEINASE

The bcl-2 protein plays an essential role in preventing cell death. it s activity is regulated through association with bcl-2 homologous and nonhomologous proteins and also by serine phosphorylation. We now repo rt that bcl-2 can be proteolytically cleaved towards its N-terminus by a cysteine proteinase present in RL-7 lymphoma cell lysates, yielding a major product of apparent MW 20 kDa, different from the products of bcl-2 cleavage by HIV protease. Moreover, bcl-2 proteins mutated for Asp residues at positions 31 and 34 were efficiently cleaved by RL-7 c eIl lysates, indicating that this proteolytic activity is distinct fro m the caspase-3 that cleaves bcl-2 at Asp 34. This bcl-2 cleaving acti vity is inhibited by E-64 and is therefore distinct from the proteinas es of the ICE/Ced-3 family (caspases), whereas reciprocally, ICE (casp ase-1) is unable to cleave bcl-2. It is optimally active at pH 5, a fe ature distinguishing it from calpain, another non-ICE cysteine protein ase which has been associated with apoptosis. This novel bcl-2 cleavin g protease, although constitutively present in RL-7 cells and resting peripheral blood lymphocytes (PBL) was upregulated following induction of apoptosis in RL-7 cells or mitogen activation in PBL. The N-termin us of bcl-2 which contains the BH4 domain that binds the kinase Raf-1 and the phosphatase calcineurin is essential for anti-apoptotic activi ty. its cleavage might provide a novel post-translational mechanism fo r regulating bcl-2 function and could amplify ongoing programmed cell death.