Yes-associated protein (YAP) is a transcriptional coactivator that is essential for the malignancy of various cancers. We have previously shown that YAP activity is positively regulated by phosphatidylserine (PS) in recycling endosomes (REs). However, the mechanism by which YAP is activated by PS in REs remains unknown. In the present study, we examined a group of protein phosphatases (11 phosphatases) that we had identified previously as PS-proximity protein candidates. Knockdown experiments of these phosphatases suggested that PPP1R12A, a regulatory subunit of the myosin phosphatase complex, was essential for YAP-dependent proliferation of triple-negative breast cancer MDA-MB-231 cells. Knockdown of PPP1R12A increased the level of phosphorylated YAP, reduced that of YAP in the nucleus, and suppressed the transcription of CTGF (a YAP-regulated gene), reinforcing the role of PPP1R12A in YAP activation. ATP8A1 is a PS-flippase that concentrates PS in the cytosolic leaflet of the RE membrane and positively regulates YAP signalling. In subcellular fractionation experiments using cell lysates, PPP1R12A in control cells was recovered exclusively in the microsomal fraction. In contrast, a fraction of PPP1R12A in ATP8A1-depleted cells was recovered in the cytosolic fraction. Cohort data available from the Cancer Genome Atlas showed that high expression of PPP1R12A, PP1B encoding the catalytic subunit of the myosin phosphatase complex, or ATP8A1 correlated with poor prognosis in breast cancer patients. These results suggest that the “ATP8A1-PS-YAP phosphatase” axis in REs facilitates YAP activation and thus cell proliferation.

Yes相关蛋白（YAP）是一种转录共激活因子，对于各种癌症的恶性肿瘤至关重要。我们之前已经表明，YAP 活性在回收内体 (RE) 中受到磷脂酰丝氨酸 (PS) 的正向调节。然而，RE 中 PS 激活 YAP 的机制仍不清楚。在本研究中，我们检查了一组蛋白磷酸酶（11 种磷酸酶），我们之前已将其鉴定为 PS 邻近蛋白候选物。这些磷酸酶的敲低实验表明，PPP1R12A（肌球蛋白磷酸酶复合物的调节亚基）对于三阴性乳腺癌 MDA-MB-231 细胞的 YAP 依赖性增殖至关重要。敲低 PPP1R12A 会增加磷酸化 YAP 的水平，降低细胞核中 YAP 的水平，并抑制 CTGF（YAP 调节基因）的转录，从而增强了 PPP1R12A 在 YAP 激活中的作用。ATP8A1 是一种 PS 翻转酶，可将 PS 浓缩在 RE 膜的胞质小叶中，并正向调节 YAP 信号传导。在使用细胞裂解物的亚细胞分级分离实验中，对照细胞中的 PPP1R12A 仅在微粒体级分中回收。相反，ATP8A1 耗尽的细胞中的 PPP1R12A 部分在胞质部分中回收。癌症基因组图谱提供的队列数据显示，PPP1R12A、编码肌球蛋白磷酸酶复合物催化亚基的 PP1B 或ATP8A1的高表达与乳腺癌患者的不良预后相关。这些结果表明 RE 中的“ATP8A1-PS-YAP 磷酸酶”轴促进 YAP 激活，从而促进细胞增殖。