title: Polyubiquitination and proteasomal turnover controls the anti-apoptotic activity of Bcl-B.
abstract: Anti-apoptotic Bcl-2 family members can contribute to tumorigenesis and may convey resistance to anti-cancer regimens. Therefore, they are important targets for novel therapeutics, particularly Bcl-2 homology (BH)3 mimetics. Bcl-B (BCL-2-like protein-10) is a relatively understudied member of the Bcl-2 protein family. Its physiological function is unknown, but it has been proven to have an anti-apoptotic activity and to act as a tumor promoter in mice. In human, high Bcl-B protein expression levels correlate with poor prognosis in various carcinomas and predict treatment resistance in acute myeloid leukemia. We here report that protein expression level and anti-apoptotic activity of Bcl-B are dictated by its ubiquitination. We demonstrate that Bcl-B is polyubiquitinated at steady state, in a unique loop between the BH1 and BH2 domains. Mutagenesis identified lysine (K)128 as an acceptor site for polyubiquitin chains, and K119 and K120, but not K181, as potential ubiquitination sites. Mass spectrometry confirmed K128 as a ubiquitination site and defined the polyubiquitin chains as K48-linked, which was confirmed by linkage-specific antibodies. Accordingly, Bcl-B proved to be an instable protein that is subject to ubiquitin-dependent proteasomal degradation at steady state. At equal mRNA expression, protein expression of a lysineless, nonubiquitinated Bcl-B mutant was fivefold higher than that of wild-type Bcl-B, demonstrating that ubiquitination is a key determinant for Bcl-B protein expression levels. Ubiquitination controlled the anti-apoptotic capacity of Bcl-B, in response to a variety of conventional and novel anti-cancer drugs. Certain anti-cancer drugs, known to reduce Mcl-1 protein levels, likewise downregulated Bcl-B. Together, these data demonstrate that polyubiquitination and proteasomal turnover dictate the expression level and anti-apoptotic capacity of Bcl-B.
authors.affiliation: Division of Immunology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands.
authors.forename: B
authors.initials: B
authors.lastname: van de Kooij
authors.forename: R W
authors.initials: RW
authors.lastname: Rooswinkel
authors.forename: F
authors.initials: F
authors.lastname: Kok
authors.forename: M
authors.initials: M
authors.lastname: Herrebout
authors.forename: E
authors.initials: E
authors.lastname: de Vries
authors.forename: M
authors.initials: M
authors.lastname: Paauwe
authors.forename: G M C
authors.initials: GM
authors.lastname: Janssen
authors.forename: P A
authors.initials: PA
authors.lastname: van Veelen
authors.forename: J
authors.initials: J
authors.lastname: Borst
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D006207:Half-Life; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D058990:Molecular Targeted Therapy; D025821:Polyubiquitin / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011487:Protein Conformation; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; C117144:BCL2-like 10 protein; D019253:Proto-Oncogene Proteins c-bcl-2; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D008239:Lysine
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