Srivastava R K, Mi Q S, Hardwick J M, Longo D L
Laboratory of Immunology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3775-80. doi: 10.1073/pnas.96.7.3775.
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.
在高浓度下，微管毒素紫杉醇能够杀死表达Bcl-2的癌细胞；它通过诱导Bcl-2磷酸化来抑制其抗凋亡活性。为了定位Bcl-2上受磷酸化调节的位点，构建了Bcl-2的突变体形式。在氨基酸70位丝氨酸发生改变（S70A）的Bcl-2突变体形式，或在α1和α2螺旋之间缺失一个60个氨基酸的环区域（缺失环Bcl-2，其也缺失氨基酸70）的Bcl-2突变体形式，在用紫杉醇处理转染了突变蛋白基因的MDA-MB-231细胞后，无法被磷酸化。缺失环突变体完全抑制了紫杉醇诱导的细胞凋亡。在表达S70A突变体的细胞中，紫杉醇诱导的细胞凋亡水平约为野生型Bcl-2的三分之一。为了评估丝裂原活化蛋白激酶（MAPKs）在Bcl-2磷酸化中的作用，检测了c-Jun氨基末端激酶（JNK）、细胞外信号调节激酶（ERK）和p38的激活情况。紫杉醇诱导的细胞凋亡与Bcl-2磷酸化以及ERK和JNK MAPKs的激活相关。如果通过转染应激激活蛋白激酶激酶显性阴性（K→R）基因（其阻止JNK上游激酶的激活）或MAPK磷酸酶-1基因（其使JNK去磷酸化并使其失活）来阻断JNK激活，Bcl-2磷酸化不会发生，细胞也不会被紫杉醇杀死。相比之下，ERK抑制剂（PD098059）和p38抑制剂（SB203580和SB202190）对Bcl-2磷酸化均无影响。因此，我们的数据表明，Bcl-2的抗凋亡作用可通过Ser-70的磷酸化来克服；缺乏环区域的Bcl-2形式在预防细胞凋亡方面比野生型Bcl-2更有效，因为它们不能被磷酸化。JNK，而非ERK或p38 MAPK，似乎参与了紫杉醇诱导的Bcl-2磷酸化。
