Protein phosphatase 2A (PP2A) complex comprises an extended family of intracellular protein serine/threonine phosphatases, that participate in different signaling transduction pathways. Different functions of PP2As are determined by the variety of regulatory subunits. In this study, CRISPR/Cas9-mediated loss-of-function screen revealed that PPP2R2A downregulation suppressed cell growth in NSCLC cells. AMOTL2 was identified and confirmed as a novel binding partner of PPP2R2A in NSCLC cells by mass spectrometry, CO-IP, GST pull-down and immunofluorescence. Upregulation of AMOTL2 also led to cell proliferation delay in human and mouse lung tumor cells. The proto-oncogene JUN is a key subunit of activator protein-1 (AP-1) transcription factor which plays crucial role in regulating tumorigenesis and its activity is negatively regulated by the phosphorylation at T239. Our results showed that either AMOTL2 upregulation or PPP2R2A downregulation led to great increase in JUN T239 phosphorylation. AMOTL2 bound PPP2R2A in cytoplasm, which reduced nuclear localization of PPP2R2A. In conclusion, AMOTL2 and PPP2R2A act respectively as negative and positive regulator of cell growth in NSCLC cells and function in the AMOTL2-PPP2R2A-JUN axis, in which AMOTL2 inhibits the entry of PPP2R2A into the nucleus to dephosphorylate JUN at T239.

蛋白磷酸酶2A（PP2A）复合物包含细胞内蛋白丝氨酸/苏氨酸磷酸酶的扩展家族，它们参与不同的信号转导途径。PP2A的不同功能取决于各种调节亚基。在这项研究中，CRISPR / Cas9介导的功能丧失筛选显示PPP2R2A下调抑制了NSCLC细胞中的细胞生长。通过质谱，CO-IP，GST下拉和免疫荧光法鉴定并确认AMOTL2是NSCLC细胞中PPP2R2A的新型结合伴侣。AMOTL2的上调也导致人和小鼠肺肿瘤细胞中的细胞增殖延迟。原癌基因JUN是激活蛋白-1（AP-1）转录因子的关键亚基，在调节肿瘤发生中起关键作用，其活性受到T239磷酸化的负调控。我们的结果表明，要么AMOTL2上调，要么PPP2R2A下调导致JUN T239磷酸化大大增加。AMOTL2在细胞质中结合PPP2R2A，从而减少了PPP2R2A的核定位。总之，AMOTL2和PPP2R2A分别充当NSCLC细胞中细胞生长的负调节剂和正调节剂，并在AMOTL2-PPP2R2A-JUN轴中起作用，其中AMOTL2抑制PPP2R2A进入细胞核，从而在T239处使JUN磷酸化。这减少了PPP2R2A的核定位。总之，AMOTL2和PPP2R2A分别充当NSCLC细胞中细胞生长的负调节剂和正调节剂，并在AMOTL2-PPP2R2A-JUN轴中起作用，其中AMOTL2抑制PPP2R2A进入细胞核，从而在T239处使JUN脱磷酸化。这减少了PPP2R2A的核定位。总之，AMOTL2和PPP2R2A分别充当NSCLC细胞中细胞生长的负调节剂和正调节剂，并在AMOTL2-PPP2R2A-JUN轴中起作用，其中AMOTL2抑制PPP2R2A进入细胞核，从而在T239处使JUN磷酸化。