Najar Mohd Altaf, Arefian Mohammad, Sidransky David, Gowda Harsha, Prasad T S Keshava, Modi Prashant Kumar, Chatterjee Aditi
Center for Systems Biology and Molecular Medicine, Yenepoya (Deemed to be University), Mangalore, India.
Department of Oncology and Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, United States.
Front Genet. 2022 May 13;13:854764. doi: 10.3389/fgene.2022.854764. eCollection 2022.
Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.
钙/钙调蛋白依赖性蛋白激酶激酶2（CAMKK2）是一种丝氨酸/苏氨酸蛋白激酶，它与钙调蛋白依赖性蛋白激酶1（CAMK1）、钙调蛋白依赖性蛋白激酶4（CAMK4）和腺苷酸活化蛋白激酶α（AMPKα）作为直接下游底物，共同参与钙触发的信号级联反应。据报道，CAMKK2在胃癌中过表达；然而，其信号传导机制尚不清楚。我们进行了无标记定量酪氨酸磷酸化蛋白质组学研究，以探讨胃癌细胞中与CAMKK2相关的酪氨酸介导的分子信号。使用高分辨率的Orbitrap Fusion Tribrid傅里叶变换质谱仪，我们鉴定出350个磷酸化酪氨酸位点，这些位点对应于157种蛋白质。在抑制CAMKK2后，我们观察到对应于63种蛋白质的81个磷酸肽发生了显著变化，其中16个肽段对应于13种蛋白质发生了超磷酸化，65个肽段对应于51种蛋白质发生了低磷酸化。我们在此报告，抑制CAMKK2会导致几种酪氨酸激酶的磷酸化发生变化，如盘状结构域蛋白2（PKP2）、非受体酪氨酸蛋白激酶2（PTK2）、红细胞生成素产生肝细胞受体A1（EPHA1）、红细胞生成素产生肝细胞受体A2（EPHA2）、蛋白激酶Cδ（PRKCD）、丝裂原活化蛋白激酶12（MAPK12）等。通路分析显示，响应CAMKK2抑制而差异磷酸化的蛋白质参与了粘着斑、肌动蛋白细胞骨架、轴突导向和血管内皮生长因子（VEGF）信号传导。对CAMKK2进行抑制和/或沉默后的蛋白质免疫印迹分析显示，PTK2在Y925位点、c-JUN在S73位点以及信号转导和转录激活因子3（STAT3）在Y705位点的磷酸化水平降低，这与质谱数据一致。该研究表明，抑制CAMKK2在胃癌细胞中具有抗癌作用，可通过PTK2/c-JUN调节STAT3的磷酸化。
