Differential modulation of the phosphoproteome by the MAP Kinases isoforms p38α and p38β

The stress-activated p38 mitogen activated protein kinases (MAPK) mediate responses to osmotic shock, radiation, immune stimuli, inflammatory cues, and many other stresses. These kinases are activated rapidly, within seconds of stress induction, yet, their continuous activation, as commonly happens in inflammations and cancer, induce different signaling cascades than transient activation. This study aimed to follow the specific signaling cascades induced by two of these p38 MAPKs activated by strong and rapid stress, or by continuous (chronic) activations. The analysis was based on large-scale proteomics and phosphoproteomics analyses using SILAC labeling of mouse embryonic fibroblasts (MEF) deficient in each of these p38 kinases, expressing constitutively expressed wildtype p38α or p38β, or their intrinsically active variants. In addition, the effects of anisomycin, inducing strong and rapid stress response, were followed in wildtype MEF or in MEF knocked-out for these p38α or p38β. The signaling events, induced the each p38 during the chronic responses, indicated adaptations of the cells expressing the intrinsically active p38 mutants. The continuous activities of the individual p38 induced feedback loops that inactivated the other p38s. Furthermore, a number of new phosphorylation sites on proteins relevant to stress responses and cancer, such as p53 and p27, were revealed in this study.

应激激活型p38丝裂原活化蛋白激酶（mitogen activated protein kinases，MAPK）可介导细胞对渗透胁迫、辐射、免疫刺激、炎症信号及多种其他应激源的应答反应。这类激酶可在应激诱导后的数秒内快速激活，但与瞬时激活不同，持续激活（常见于炎症与癌症进程中）会触发截然不同的信号级联反应。本研究旨在探究两种p38 MAPK分别在强快速应激或持续（慢性）激活状态下所诱导的特异性信号级联反应。本研究的分析基于大规模蛋白质组学与磷酸化蛋白质组学实验，采用细胞培养中氨基酸稳定同位素标记（Stable Isotope Labeling with Amino acids in Cell culture，SILAC）技术，针对两类细胞开展研究：其一为分别缺失目标p38激酶、同时表达组成型野生型p38α或p38β，或是其固有活性突变体的小鼠胚胎成纤维细胞（mouse embryonic fibroblasts，MEF）；其二为野生型MEF，或是敲除p38α或p38β的MEF，用于观测茴香霉素（anisomycin）诱导的强快速应激反应相关效应。慢性激活状态下，各p38激酶所介导的信号事件提示，表达固有活性p38突变体的细胞发生了适应性改变。单种p38的持续激活会诱导出可使其他p38失活的负反馈环路。此外，本研究还发现了一批与应激反应及癌症相关的蛋白质（如p53与p27）的全新磷酸化位点。