Hu Z B, Minden M D, McCulloch E A
The Ontario Cancer Institute and Princess Margaret Hospital, Toronto, Canada.
Blood. 1998 Sep 1;92(5):1768-75.
Serine phosphorylation of bcl-2 has been reported after treatment of cells with protein kinase C, okadaic acid, taxol, and other chemotherapeutic agents that attack microtubules. We report here that bcl-2 is phosphorylated on serine in acute myeloblastic leukemia (AML) blasts exposed to all trans retinoic acid (ATRA). Two-dimension gels (isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) disclosed a novel acidic isoform of bcl-2 in ATRA-treated blast cells from a continuous line and from two AML patients; when the cell lysates were digested with lambda-phosphatase, bcl-2 reverted to the control position, indicating that it was phosphorylated. Metabolic labeling experiments using 32Pi showed that, while control bcl-2 was labeled, incorporation was greatly increased when cells were treated with ATRA. A comparison of bcl-2 from blasts treated with ATRA or taxol showed that bcl-2 was phosphorylated on serine in cells treated with either agent; however, both qualitative and quantitative differences were seen. Qualitatively, the phosphorylated isoform from taxol-treated cells was slightly larger than the native isoform and could be distinguished on 10% to 20% SDS-polyacrylamide gradient gels, while the phosphorylated bcl-2 after ATRA ran as a single band on gradient gels at the same position as control bcl-2. Quantitatively, all bcl-2 from ATRA-treated cells was in the phosphorylated isoform, while after taxol, both phosphorylated and native bcl-2 was present; incorporation of 32Pi into bcl-2 was stimulated to greater extent in ATRA-treated compared with taxol-treated cells. We used immunoprecipitation experiments to ask if bcl-2 phosphorylated after ATRA or taxol had altered capacity to dimerize with bax. No change in dimerization was demonstrated. We conclude that: bcl-2 is phosphorylated on serine after treatment of AML blasts with ATRA; bcl-2 phosphorylation after ATRA is different from that seen after taxol; bcl-2 phosphorylated after either agent retains capacity to dimerize with bax. The ATRA or taxol-induced phosphorylation of bcl-2 can also be seen in blast cells obtained from AML patients.
在用蛋白激酶C、冈田酸、紫杉醇以及其他攻击微管的化疗药物处理细胞后，已报道bcl-2会发生丝氨酸磷酸化。我们在此报告，在暴露于全反式维甲酸（ATRA）的急性髓细胞白血病（AML）原始细胞中，bcl-2在丝氨酸上发生了磷酸化。二维凝胶（等电聚焦随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳[SDS-PAGE]）显示，在来自一个连续细胞系和两名AML患者的经ATRA处理的原始细胞中，bcl-2出现了一种新的酸性异构体；当细胞裂解物用λ-磷酸酶消化时，bcl-2恢复到对照位置，表明它被磷酸化了。使用32Pi进行的代谢标记实验表明，虽然对照bcl-2被标记，但在用ATRA处理细胞时，标记掺入量大大增加。对经ATRA或紫杉醇处理的原始细胞中的bcl-2进行比较显示，在用任何一种药物处理的细胞中，bcl-2都在丝氨酸上发生了磷酸化；然而，在定性和定量方面都存在差异。定性地说，紫杉醇处理细胞中的磷酸化异构体比天然异构体略大，在10%至20%的SDS-聚丙烯酰胺梯度凝胶上可以区分，而ATRA处理后的磷酸化bcl-2在梯度凝胶上以与对照bcl-2相同的位置呈单一条带。定量地说，来自经ATRA处理细胞的所有bcl-2都处于磷酸化异构体形式，而在紫杉醇处理后，磷酸化和天然的bcl-2都存在；与紫杉醇处理的细胞相比，32Pi掺入bcl-2在经ATRA处理的细胞中受到的刺激程度更大。我们使用免疫沉淀实验来询问经ATRA或紫杉醇处理后磷酸化的bcl-2与bax二聚化的能力是否发生了改变。未证明二聚化有变化。我们得出结论：用ATRA处理AML原始细胞后，bcl-2在丝氨酸上发生磷酸化；ATRA处理后bcl-2的磷酸化与紫杉醇处理后不同；经任何一种药物处理后磷酸化的bcl-2仍保留与bax二聚化的能力。在从AML患者获得的原始细胞中也可以看到ATRA或紫杉醇诱导的bcl-2磷酸化。
