Lysine dimethylation in the BH4 domain of Bcl-2 and functional significance (P1306)

Abstract

Abstract Bcl-2 is an anti-apoptotic protein that can protect developing and mature T cells against a variety of apoptotic signals. The Bcl-2 family of proteins, compromised of anti-apoptotic as well as pro-apoptotic members, plays key roles in apoptosis; the balance of these members determines cell survival. Anti-apoptotic Bcl-2 contains four conserved Bcl-2 homology (BH) domains and the BH4 domain is indispensable for its ability to suppress cell death. Using mass spectroscopy to analyze a murine T cell line, we identified dimethylation of two lysine residues in the BH4 domain: K17 and K22. Mutation of K22 (but not K17) to alanine abrogated Bcl-2 binding to pro-apoptotic proteins Bax, Bak, Bad, Bim and Puma and greatly reduced Bcl-2 anti-apoptotic function in cytokine withdrawal. This assigns functional importance to K22 (which is conserved across species and other anti-apoptotic members) and suggests that dimethylation could affect its function. K22, based on structural models, appears to be involved in stabilizing peptide secondary structure, rather than directly interacting with pro-apoptotic proteins. We developed a monoclonal antibody that recognizes dimethylated K22 in the Bcl-2 sequence and detected expression in murine B cells (but not thymocytes). This suggests that Bcl-2 dimethylation may regulate B cell survival.