Bmf contributes to the onset of anoikis by translocating from cytoskeleton to mitochondria when cells lose attachment to the extracellular matrix. However, the structural details of Bmf cytoskeleton tethering and the control of Bmf release upon loss of anchorage remained unknown. Here we showed that cell detachment induced rapid and sustained activation of p38 MAPK in mammary epithelial cell lines. Inhibition of p38 signaling or Bmf knockdown rescued anoikis. Activated p38 MAPK could directly phosphorylate Bmf at multiple sites including a non-proline-directed site threonine 72 (T72). Crystallographic studies revealed that Bmf T72 directly participated in DLC2 binding and its phosphorylation would block Bmf/DLC2 interaction through steric hindrance. Finally, we showed that phosphomimetic mutation of T72 enhanced Bmf apoptotic activity in vitro and in a knock-in mouse model. This work unraveled a novel regulatory mechanism of Bmf activity during anoikis and provided structural basis for Bmf cytoskeleton tethering and dissociation.

当细胞失去与细胞外基质的附着时，Bmf 通过从细胞骨架转移到线粒体来促进失巢凋亡的发生。然而，Bmf 细胞骨架束缚的结构细节和 Bmf 在失去锚定时释放的控制仍然未知。在这里，我们发现细胞脱离诱导乳腺上皮细胞系中 p38 MAPK 的快速和持续激活。抑制 p38 信号或 Bmf 敲低挽救了失巢凋亡。激活的 p38 MAPK 可以在多个位点直接磷酸化 Bmf，包括非脯氨酸定向位点苏氨酸 72 (T72)。晶体学研究表明，Bmf T72 直接参与 DLC2 结合，其磷酸化会通过空间位阻阻断 Bmf/DLC2 相互作用。最后，我们发现 T72 的拟磷突变增强了体外和敲入小鼠模型中的 Bmf 凋亡活性。这项工作揭示了失巢凋亡期间 Bmf 活性的一种新调节机制，并为 Bmf 细胞骨架的束缚和解离提供了结构基础。