Bryostatin 1 induces ubiquitination and proteasome degradation of Bcl-2 inthe human acute lymphoblastic leukemia cell line, Reh

The ubiquitin-mediated proteolytic system has been implicated in the turnov er of a number of intracellular proteins. In the present study, we investig ated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquit in-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and i mmunoblotting analyses revealed that Bcl-2 is ubiquitinated following expos ure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryosta tin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the contr ol after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 prot ein again rapidly decreases to 6% Of its prebryostatin 1 level at which tim e a plateau is reached and maintained for another 72 h. Furthermore, ubiqui tin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 trea ted 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, w hich inhibits the proteinase activities of the proteasome, inhibited the br yostatin 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 treat ment. Proteolytic activity reached its highest point by 3 h, and subsequent ly returned to control levels by 12 h, post-bryostatin 1 treatment. In addi tion, bryostatin 1 treatment of the Reh cell line decreased expression of b cl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. W e speculate that this decrease in mRNA together with increased 26S proteoly tic activity accounts for the initial rapid decrease recorded in Bcl-2 prot ein. These findings indicate that bryostatin 1 treatment of Reh ALL cells d ecreases 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.