Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis

At high concentrations, the tubule poison paclitaxel is able to kill cancer cells that express Bcl-2; it inhibits the antiapoptotic activity of Bcl-2 by inducing its phosphorylation. To localize the site on Bcl-2 regulated by phosphorylation, mutant forms of Bcl-2 were constructed. Mutant forms of B cl-2 with an alteration in serine at amino acid 70 (S70A) or,vith deletion of a 60-aa loop region between the alpha 1 and alpha 2 helices (Delta loop Bcl-2, which also deletes amino acid 70) were unable to be phosphorylated b y paclitaxel treatment of MDA-MB-231 cells into which the genes for the mut ant proteins were transfected, The Delta loop mutant completely inhibited p aclitaxel-induced apoptosis, In cells expressing the S70A mutant, paclitaxe l induced about one-third the level of apoptosis seen with wild-type Bcl-2, To evaluate the role of mitogen-activated protein kinases (MAPKs) in Bcl-2 phosphorylation, the activation of c-jun N-terminal kinase (JNK), extracel lular signal-regulated kinase (ERK, and p38 was examined. Paclitaxel induce d apoptosis was associated with phosphorylation of Bcl-2 and activation of ERK and JNK MAPKs. If JNK activation was blocked by transfections with eith er a stress-activated protein kinase kinase dominant-negative (K-->R) gene (which prevents the activation of a kinase upstream of JNK) or MAPK phospha tase-l gene (which dephosphorylates and inactivates JNK), Bcl-2 phosphoryla tion did not occur, and the cells were not killed by paclitaxel. By contras t, neither an ERK inhibitor (PD098059) nor p38 inhibitors (SB203580 and SB2 02190) had an effect on Bcl-2 phosphorylation, Thus, our data show that the antiapoptotic effects of Bcl-2 can be overcome by phosphorylation of Ser-7 0; forms of Bcl-2 lacking the loop region are much more effective at preven ting apoptosis than wild-type Bcl-2 because they cannot be phosphorylated, JNK, but not ERK or p38 MAPK, appear to be involved in the phosphorylation of Bcl-2 induced by paclitaxel.