Zhang Zhiwei, Isaji Tomoya, Oyama Yoshiyuki, Liu Jianwei, Xu Zhiwei, Sun Yuhan, Fukuda Tomohiko, Lu Haojie, Gu Jianguo
Division of Regulatory Glycobiology, Graduate School of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Sendai 980-0845, Miyagi, Japan.
Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Miyagi, Japan.
Biomolecules. 2024 Dec 10;14(12):1577. doi: 10.3390/biom14121577.
Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase pivotal in cellular signal transduction, regulating cell adhesion, migration, growth, and survival. However, the regulatory mechanisms of FAK during tumorigenesis and progression still need to be fully understood. Our previous study demonstrated that -GlcNAcylation regulates integrin-mediated cell adhesion. To further elucidate the underlying molecular mechanism, we focused on FAK in this study and purified it from 293T cells. Using liquid chromatography-mass spectrometry (LC-MS/MS), we identified the -GlcNAcylation of FAK at Ser708, Thr739, and Ser886. Compared with wild-type FAK expressed in -knockout 293T cells, the FAK mutant, in which Ser708, Thr739, and Ser886 were replaced with Ala, exhibited lower phosphorylation levels of Tyr397 and AKT. Cell proliferation and migration, assessed through MTT and wound healing assays, were significantly suppressed in the FAK mutant cells compared to the wild-type FAK cells. Additionally, the interaction among FAK, paxillin, and talin was enhanced, and cell adhesion was increased in the mutant cells. These data indicate that specific -GlcNAcylation of FAK plays a critical regulatory role in integrin-mediated cell adhesion and migration. This further supports the idea that -GlcNAcylation is essential for tumorigenesis and progression and that targeting the -GlcNAcylation of FAK could offer a promising therapeutic strategy for cancer treatment.
粘着斑激酶（FAK）是一种非受体酪氨酸激酶，在细胞信号转导中起关键作用，调节细胞粘附、迁移、生长和存活。然而，FAK在肿瘤发生和发展过程中的调控机制仍有待充分了解。我们之前的研究表明，O-连接的N-乙酰葡糖胺糖基化（O-GlcNAcylation）调节整合素介导的细胞粘附。为了进一步阐明潜在的分子机制，我们在本研究中聚焦于FAK，并从293T细胞中纯化了它。使用液相色谱-质谱联用（LC-MS/MS），我们鉴定出FAK在Ser708、Thr739和Ser886位点发生了O-GlcNAc糖基化。与在O-GlcNAc敲除的293T细胞中表达的野生型FAK相比，将Ser708、Thr739和Ser886替换为Ala的FAK突变体，其Tyr397和AKT的磷酸化水平较低。通过MTT和伤口愈合试验评估，与野生型FAK细胞相比，FAK突变体细胞的增殖和迁移受到显著抑制。此外，突变体细胞中FAK、桩蛋白和踝蛋白之间的相互作用增强，细胞粘附增加。这些数据表明，FAK特定的O-GlcNAc糖基化在整合素介导的细胞粘附和迁移中起关键调节作用。这进一步支持了O-GlcNAc糖基化对肿瘤发生和发展至关重要的观点，并且靶向FAK的O-GlcNAc糖基化可能为癌症治疗提供一种有前景的治疗策略。
