The VTLISFG motif in the BH1 domain plays a significant role in regulating the degradation of Mcl-1.
Mcl-1 is a member of the Bcl-2 family protein; its degradation is required for the initiation of apoptosis. The mechanism, however, is not yet clearly known. Previously, it was reported that Mcl-1 is degraded through the ubiquitination-mediated pathway and the PEST domain is the motif responsible for promoting this degradation. We found evidence that this may not be true. We generated several Mcl-1 deletion mutants and examined their effects on protein stability. Deletion of the PEST domain did not prevent the degradation of Mcl-1 during apoptosis. The BH1 domain, but not the PEST, BH3 or BH2 domain, exhibited a short half-life. A peptide named "F3" (VTLISFG) in the C-terminus of the BH1 domain appears to be critical for the rapid turnover of Mcl-1. Deletion of F3 from GFP-Mcl-1-ΔPEST retarded the degradation of this mutant. F3 appeared to be the minimum functional sequence of the degradation motif, since deletion of a single residue was sufficient to abrogate its short half-life. Fusion of F3 with p32 resulted in the degradation of p32 during UV-induced apoptosis, while wild type p32 was not affected. Taken together, these findings suggest that F3 (VTLISFG), instead of PEST, is the major motif responsible for the degradation of Mcl-1 during apoptosis.
Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong ; Shenzhen Middle School, 18 Shenzhong Street, North Renmin Road, Luohu District, Shenzhen, Guangdong Province, P.R. China, 518025.
Kang
K
Xiao
Shenzhen Middle School, 18 Shenzhong Street, North Renmin Road, Luohu District, Shenzhen, Guangdong Province, P.R. China, 518025.
Pengxuan
P
Chen
Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Donald Choy
DC
Chang
Apoptosis; BH domain, Bcl-2 homology domain; BSA, bovine serum albumin; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2; Bim, Bcl-2-interacting mediator; CCD, charge-coupled device; Caspase, cysteine aspartase; Degradation motif; EGFP, enhanced green fluorescent protein; EIF2, eukaryotic translation initiation factor 2; EYFP, enhanced yellow fluorescent protein; GCN2, general control nonrepressed 2; GSK-3β, glycogen synthase kinase-3β; HECT, homologous to E6-AP carboxylterminus; HRP, horseradish peroxidase; MEM, minimum essential medium; Mcl-1; Mcl-1, myeloid cell leukaemia sequence 1; Mule, Mcl-1 ubiquitin ligase E3; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PERK, PKR-like ER kinase; PEST domain; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; TM domain, transmembrane domain; UV, ultraviolet light; h, hour; kD, kilodaltons; pDNA, plasmid DNA; β-TrCP, β-transducin repeat-containing protein