Role of Bcl-2 phosphorylation after microtubule disruption

为了阐明Bcl-2磷酸化的作用，我们建立了野生型和几种突变型表达Bcl-2的细胞系。我们发现跨膜区域了吗？-缺失的Bcl-2 (Bcl-2ΔTM)，不定位于线粒体，在细胞中被组成性磷酸化。我们在Bcl-2ΔTM蛋白内鉴定出Ser-87残基作为磷酸化位点。为了确定Bcl-2ΔTM磷酸化的激酶，我们使用了几种激酶抑制剂，发现MEK1抑制剂PD98059和U0126抑制了Bcl-2ΔTM的磷酸化。此外，体外激酶实验显示，MEK1的下游激酶ERK在Ser-87残基Bcl-2ΔTM处磷酸化。野生型Bcl-2和Bcl-2ΔTM均可与ERK结合。野生型Bcl-2可以与PP2A结合，因此，野生型Bcl-2没有组成性磷酸化。另一方面，Bcl-2ΔTM可以与ERK相互作用，但不能与PP2A相互作用。此外，Bcl-2蛋白在正常人血细胞中被组成性磷酸化，而Bcl-2在人肿瘤细胞系中不被磷酸化。

To clarify the role of Bcl-2 phosphorylation, we established the wild-type and several mutant-form of Bcl-2-expressing cell lines. We found that transmembrane region ?-deleted Bcl-2 (Bcl-2ΔTM), which was not localized at mitochondria, was constitutively phosphorylated in the cells. We identified Ser-87 residue within Bcl-2ΔTM protein as the phosphorylation site. To identify the responsible kinase for phosphorylation of Bcl-2ΔTM, we used several kinase inhibitors, and found that PD98059 and U0126, MEK1 inhibitors, suppressed phosphorylation of Bcl-2ΔTM. Moreover, in vitro kinase assay showed that ERK, a downstream kinase of MEK1, phosphorylated at Ser-87 residue of Bcl-2ΔTM. Both wild-type Bcl-2 and Bcl-2ΔTM could bind to ERK. Wild-type Bcl-2 could associate with PP2A, thus, wild-type Bcl-2 was not phosphorylated constitutively. On the other hand, Bcl-2ΔTM could interact with ERK, but not with PP2A. Moreover, Bcl-2 protein was constitutively phosphorylated in normal human blood cells, whereas Bcl-2 was not phosphorylated in human tumor cell lines.