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.

Figure 1

Effects of vincristine and paclitaxel on MAPK activation. (A) Vincristine and paclitaxel induced JNK activation in a time- and dose-dependent manner. MCF-7 cells were treated with vincristine or paclitaxel for various times. The cell lysates were prepared and immunoprecipitated with 10 μg of polyclonal anti-JNK1 antibody followed by 20 μl of protein A Sepharose. The kinase reaction was performed by the procedures described in Materials and Methods. (Upper) The time course of kinase reaction. Represented is the autoradiograph of [γ-³²P]ATP incorporation into exogenous GST-c-Jun-(1–135). (Lower) The dose response of JNK activation. Anti-JNK1 immunocomplexes were obtained with lysates of cells treated with various doses of vincristine or paclitaxel for 30 min. (B) Paclitaxel-induced ERK activity. Cells were treated with paclitaxel (0.1 μM) for various time points, and Western blots were performed by using antibody specific for phosphorylated ERK (Upper) and anti-ERK2 antibody (Lower). (C) Paclitaxel had no effect on p38 activity. Cells were treated with paclitaxel (0.1 μM) for various time points, and Western blots were performed by using antibody specific for phosphorylated p38 (Upper) and anti-p38 antibody (Lower). For a positive control, cells were treated with short-wavelength UV light.

Figure 2

Block of JNK pathway inhibits paclitaxel-induced Bcl-2 phosphorylation and apoptosis. (A) JNK phosphorylates GST-Bcl-2 in vitro (Upper). MCF-7 cells were treated with paclitaxel (100 nM) for 4 h, and activated JNK1 was immunoprecipitated with either 5 μg or 10 μg anti-JNK1 antibody. GST-Bcl-2 (10 μg) was incubated with immunoprecipitated JNK1 in vitro (see Materials and Methods). The same blot was reprobed with anti-JNK1 antibody (Lower). (B) A dominant-negative mutant SEK1 (K→R) inhibits activation of the JNK pathway, Bcl-2 phosphorylation, and apoptosis induced by paclitaxel (100 nM). MCF-7 cells were transfected with a total of 2 μg/ml DNA of either empty vector (−) or mutated SEK1 (+) and incubated for 1 day. Cells were washed and incubated with fresh medium. After 8 h, the cells were incubated with 100 nM paclitaxel (+) or vehicle (−) for 30 min, 4 h, and 24 h for the measurement of JNK activation, Bcl-2 phosphorylation, and apoptosis, respectively. The JNK activity and immunoblottings were determined as described. (C) Effect of MKP-1 on JNK activation and Bcl-2 phosphorylation. MCF-7 cells were transfected with 30 μg anti-MKP-1 antisense (MKP-1-AS) or sense vector (MKP1-S) with Lipofectamine and incubated for 1 day. Cells were washed, and fresh medium was replaced. After 8 h, the cells were incubated with 100 nM paclitaxel (+) or vehicle (−) for 30 min, 4 h, and 24 h for the measurement of JNK activation, Bcl-2 phosphorylation, and apoptosis, respectively. (D) Effect of ERK and p38 inhibitors on JNK activation and Bcl-2 phosphorylation. MCF-7 cells were pretreated with inhibitors of ERK (PD098059), p38 (SB203580, SB202190), or negative control (SB202474) for 1 h. They were exposed to paclitaxel for 30 min, 4 h, and 24 h to determine JNK activation, Bcl-2 phosphorylation, and apoptosis, respectively.

Figure 3

Loop deletion mutant of Bcl-2 blocks paclitaxel-induced Bcl-2 phosphorylation and apoptosis. MDA-MB-231 cells were transiently transfected with a total of 2 μg/ml DNA of either empty vector (−) or Bcl-2 (WT, S70A, S51A, S87A, S105A, ΔTM, or Δloop) expression plasmid with Lipofectamine and incubated for 1 day. Cells were washed, and fresh medium was added. After 8 h, the cells were incubated with 100 nM paclitaxel (+) or vehicle (−) for 4 h and 24 h for the measurements of Bcl-2 phosphorylation (A) and apoptosis (B), respectively. Cells were stained with 4′,6-diamidino-2-phenylindole to examine apoptotic nuclei. (C) Phosphorylated Bcl-2 is dephosphorylated by PPase I, but not by PTPase in MDA-MB-231 cells. Cells were transfected with Bcl-2 (WT and mutants) as described above and treated with paclitaxel (100 nM) for 4 h. Cell lysates then were incubated with either PPase I (5 μM) or PTPase (5 μM).

Figure 4

Bcl-2 phosphorylation is linked to mitochondrial functions. MDA-MB-231 cells were transiently transfected with a total of 2 μg/ml of DNA of either empty vector (−) or Bcl-2 (WT, S70A, Δloop) expressing plasmid with Lipofectamine and incubated for 1 day. Cells were washed, and fresh medium was replaced. After 8 h, the cells were incubated with 100 nM paclitaxel (+) or vehicle (−) for 6, 12, and 24 h for the measurements of cytochrome c release (A), caspase-3 activation (B), and PARP cleavage (C), respectively. (A) The redistribution of cytochrome c in MDA/Neo, MDA/WT, MDA/S70A, and MDA/Δloop cells. The cells were mechanically lysed and separated into mitochondrial (M) and S100 (S) fractions. The amount of cytochrome c present in each fraction was determined by Western blot analysis. (B) Loop deletion mutant of Bcl-2 blocks paclitaxel-induced caspase-3 activation. Caspase-3 activity was measured as per manufacturer’s instructions. (C) Loop deletion mutant of Bcl-2 inhibits paclitaxel-induced PARP cleavage. Cell lysates were run on SDS/PAGE, and immunoblottings were performed by using anti-PARP antibody.