title: Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis.
abstract: 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 Bcl-2 with an alteration in serine at amino acid 70 (S70A) or with deletion of a 60-aa loop region between the alpha1 and alpha2 helices (Deltaloop Bcl-2, which also deletes amino acid 70) were unable to be phosphorylated by paclitaxel treatment of MDA-MB-231 cells into which the genes for the mutant proteins were transfected. The Deltaloop mutant completely inhibited paclitaxel-induced apoptosis. In cells expressing the S70A mutant, paclitaxel 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), extracellular signal-regulated kinase (ERK), and p38 was examined. Paclitaxel-induced apoptosis was associated with phosphorylation of Bcl-2 and activation of ERK and JNK MAPKs. If JNK activation was blocked by transfections with either a stress-activated protein kinase kinase dominant-negative (K-->R) gene (which prevents the activation of a kinase upstream of JNK) or MAPK phosphatase-1 gene (which dephosphorylates and inactivates JNK), Bcl-2 phosphorylation did not occur, and the cells were not killed by paclitaxel. By contrast, neither an ERK inhibitor (PD098059) nor p38 inhibitors (SB203580 and SB202190) 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-70; forms of Bcl-2 lacking the loop region are much more effective at preventing 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.
authors: Laboratory of Immunology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.
authors: R K
authors: RK
authors: Srivastava
authors: Q S
authors: QS
authors: Mi
authors: J M
authors: JM
authors: Hardwick
authors: D L
authors: DL
authors: Longo
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001943:Breast Neoplasms; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D008928:Mitochondria / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases; D016297:Mutagenesis, Site-Directed; D017239:Paclitaxel / Q000633:toxicity*; D010766:Phosphorylation; D017354:Point Mutation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012694:Serine; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000633:toxicity
chemical_list: D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D012694:Serine; D014750:Vincristine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D000409:Alanine; D017239:Paclitaxel