Bryostatin 1 induces ubiquitination and proteasome degradation of Bcl-2 in the human acute lymphoblastic leukemia cell line, Reh.
The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression.
Wall
N R
NR
Division of Hematology and Oncology, Department of Internal Medicine, Karmanos Cancer Institute at Wayne State University School of Medicine, Detroit, MI 48201, USA.
Mohammad
R M
RM
Reddy
K B
KB
Al-Katib
A M
AM
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D015153:Blotting, Western; D054713:Bryostatins; D002457:Cell Extracts; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006801:Humans; D007783:Lactones / Q000494:pharmacology*; D018942:Macrolides; D009097:Multienzyme Complexes / Q000378:metabolism*; D011233:Precipitin Tests; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
D000970:Antineoplastic Agents; D054713:Bryostatins; D002457:Cell Extracts; D007783:Lactones; D018942:Macrolides; D009097:Multienzyme Complexes; D019253:Proto-Oncogene Proteins c-bcl-2; D014452:Ubiquitins; C067713:lactacystin; C046785:bryostatin 1; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine