phleomycin-d1 and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

The serine/threonine kinase, B-RAF, is frequently mutated in melanoma and is required for cell proliferation. Proteasomal turnover of cyclins and cyclin-dependent kinase inhibitors via E3 ubiquitin ligases regulates cell cycle progression. We previously showed that B-RAF regulates Cks1, a co-factor for the F-box protein Skp2. Recently, a second F-box protein cofactor was identified, alphaB-crystallin, that binds Fbx4 and promotes cyclin D1 degradation. Here, we demonstrate that alphaB-crystallin is down-regulated in mutant B-RAF melanoma cells compared to melanocytes in a B-RAF and MEK-dependent manner. In a subset of lines, MEK inhibition was sufficient to up-regulate alphaB-crystallin protein levels; whereas in other lines combined MEK and proteasome inhibition was required. alphaB-crystallin knockdown partially stabilized cyclin D1 in melanocytes. Expression of alphaB-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. Together, these data show that alphaB-crystallin is highly expressed in melanocytes contributing, in part, to cyclin D1 turnover. Furthermore, alphaB-crystallin is down-regulated in a B-RAF-dependent manner in melanoma cells and its re-expression regulates cyclin D1 turnover after DNA damage.

Topics: alpha-Crystallin B Chain; Bleomycin; Butadienes; Cells, Cultured; Cyclin D1; Cycloheximide; DNA Damage; Etoposide; Humans; Leupeptins; Melanocytes; Melanoma; Mutant Proteins; Mutation; Nitriles; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Structure-Activity Relationship; Tetracycline