Department of Pharmacology and Cardiovascular Research Institute, Chonbuk National University, and Research Center for Pulmonary Disorder, Chonbuk National University Hospital, Jeonju, South Korea.
Mol Cell Biol. 2010 Apr;30(7):1800-13. doi: 10.1128/MCB.01357-09. Epub 2010 Feb 1.
Bax inhibitor 1 (BI-1), a transmembrane protein with Ca2+ channel-like activity, has antiapoptotic and anticancer activities. Cells overexpressing BI-1 demonstrated increased cell adhesion. Using a proteomics tool, we found that BI-1 interacted with gamma-actin via leucines 221 and 225 and could control actin polymerization and cell adhesion. Among BI-1-/- cells and cells transfected with BI-1 small interfering RNA (siRNA), levels of actin polymerization and cell adhesion were lower than those among BI-1+/+ cells and cells transfected with nonspecific siRNA. BI-1 acts as a leaky Ca2+ channel, but mutations of the actin binding sites (L221A, L225A, and L221A/L225A) did not change intra-endoplasmic reticulum Ca2+, although deleting the C-terminal motif (EKDKKKEKK) did. However, store-operated Ca2+ entry (SOCE) is activated in cells expressing BI-1 but not in cells expressing actin binding site mutants, even those with the intact C-terminal motif. Consistently, actin polymerization and cell adhesion were inhibited among all the mutant cells. Compared to BI-1+/+ cells, BI-1-/- cells inhibited SOCE, actin polymerization, and cell adhesion. Endogenous BI-1 knockdown cells showed a similar pattern. The C-terminal peptide of BI-1 (LMMLILAMNRKDKKKEKK) polymerized actin even after the deletion of four or six charged C-terminal residues. This indicates that the actin binding site containing L221 to D231 of BI-1 is responsible for actin interaction and that the C-terminal motif has only a supporting role. The intact C-terminal peptide also bundled actin and increased cell adhesion. The results of experiments with whole recombinant BI-1 reconstituted in membranes also coincide well with the results obtained with peptides. In summary, BI-1 increased actin polymerization and cell adhesion through Ca2+ regulation and actin interaction.
Bax 抑制剂 1（BI-1）是一种具有 Ca2+通道样活性的跨膜蛋白，具有抗凋亡和抗癌活性。过度表达 BI-1 的细胞表现出增强的细胞黏附。使用蛋白质组学工具，我们发现 BI-1 通过亮氨酸 221 和 225 与γ-肌动蛋白相互作用，并可以控制肌动蛋白聚合和细胞黏附。在 BI-1-/-细胞和转染 BI-1 小干扰 RNA（siRNA）的细胞中，肌动蛋白聚合和细胞黏附水平低于 BI-1+/+细胞和转染非特异性 siRNA 的细胞。BI-1 作为一个渗漏的 Ca2+通道，但肌动蛋白结合位点的突变（L221A、L225A 和 L221A/L225A）并没有改变内质网内的 Ca2+，尽管删除 C 端基序（EKDKKKEKK）也没有改变。然而，在表达 BI-1 的细胞中激活了储存操作的 Ca2+内流（SOCE），但在表达肌动蛋白结合位点突变体的细胞中没有激活，即使那些具有完整的 C 端基序的细胞也没有激活。同样，在所有突变细胞中，肌动蛋白聚合和细胞黏附均受到抑制。与 BI-1+/+细胞相比，BI-1-/-细胞抑制了 SOCE、肌动蛋白聚合和细胞黏附。内源性 BI-1 敲低细胞也表现出类似的模式。即使删除了四个或六个带电荷的 C 端残基，BI-1 的 C 端肽（LMMLILAMNRKDKKKEKK）也能聚合肌动蛋白。这表明 BI-1 中的肌动蛋白结合位点 L221 到 D231 负责肌动蛋白相互作用，而 C 端基序仅起支持作用。完整的 C 端肽也能使肌动蛋白成束，并增加细胞黏附。在膜中重建的全长重组 BI-1 进行的实验结果与肽的实验结果也非常吻合。总之，BI-1 通过 Ca2+调节和肌动蛋白相互作用增加了肌动蛋白聚合和细胞黏附。
