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

R. K. Srivastava, Q. S. Mi, J Marie Hardwick, D. L. Longo

Research output: Contribution to journalArticle

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 α1 and α2 helices (Δloop 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 Δloop 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.

Original languageEnglish (US)
Pages (from-to)3775-3780
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number7
DOIs
StatePublished - Mar 30 1999

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Paclitaxel
Phosphorylation
Phosphotransferases
Apoptosis
Extracellular Signal-Regulated MAP Kinases
Mitogen-Activated Protein Kinases
Mitogen-Activated Protein Kinase Phosphatases
Dual Specificity Phosphatase 1
Amino Acids
JNK Mitogen-Activated Protein Kinases
Poisons
Mitogen-Activated Protein Kinase 1
p38 Mitogen-Activated Protein Kinases
Mutant Proteins
Heat-Shock Proteins
Protein Kinases
Serine
Genes
Transfection
Neoplasms

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Deletion of the loop region of Bcl-2 completely blocks paclitaxel- induced apoptosis. / Srivastava, R. K.; Mi, Q. S.; Hardwick, J Marie; Longo, D. L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, No. 7, 30.03.1999, p. 3775-3780.

Research output: Contribution to journalArticle

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