Comprehensive proteomic and transcriptomic characterization of hepatic expression signatures affected in p14 liver conditional knockout mice

Scaffold proteins regulate intracellular MAP kinase signaling by providing critical spatial and temporal specificity. We have shown previously that the scaffold protein MEK1 partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in livers of mice we analysed protein and transcript signatures in tissue samples. Further biological network analysis predicted that the differentially expressed transcripts and proteins are involved in cell cycle progression and regulation of cellular proliferation. Although some of the here identified signatures were previously linked to phospho-ERK activity, most of them were novel targets of late endosomal p14/MP1/MEK/ERK signaling module. Finally, the proliferation defect was confirmed in a chemically induced liver regeneration model in p14 liver knock-out mice. Conditional liver p14 KO mice were generated by mating p14 f/f mice with mice expressing Cre recombinase under control of Alb promoter and alpha-fetoprotein enhancer. The obtained p14 floxed/floxed;AlfpCre (p14 liver KO) mice were used in experiments as KO mice. To prevent the influence of AlfpCre transgene we used p14 +/+;AlfpCre mice as a control. All mice were derived in a C57BL/6 background and were six weeks old. Expression profiles of KO mouse samples were compared to expression profiles of corresponding control mice and differentially expressed genes were identified.支架蛋白（scaffold protein）通过赋予关键的时空特异性，调控细胞内丝裂原活化蛋白激酶（MAP kinase）信号通路。本团队此前已证实，支架蛋白MEK1结合蛋白（MP1）可通过衔接蛋白（adaptor protein）p14锚定至晚期内体（late endosomes）。本研究通过在小鼠肝脏中实施p14的条件性基因敲除（conditional gene disruption），对组织样本中的蛋白质与转录特征进行了分析。进一步的生物网络分析（biological network analysis）预测，差异表达的转录本与蛋白质参与细胞周期进程（cell cycle progression）与细胞增殖（cellular proliferation）的调控。尽管本研究中鉴定的部分特征此前已与磷酸化ERK活性（phospho-ERK activity）相关联，但绝大多数均为晚期内体p14/MP1/MEK/ERK信号模块（late endosomal p14/MP1/MEK/ERK signaling module）的全新靶点。最后，研究团队在p14肝脏敲除小鼠的化学诱导肝再生模型（chemically induced liver regeneration model）中验证了增殖缺陷表型。本研究通过将p14 flox/flox小鼠与在白蛋白启动子（Alb promoter）及甲胎蛋白增强子（alpha-fetoprotein enhancer）调控下表达Cre重组酶（Cre recombinase）的小鼠交配，构建了条件性肝脏p14敲除小鼠。所得p14 floxed/floxed;AlfpCre（即p14肝脏敲除）小鼠被用作敲除组进行实验。为排除AlfpCre转基因的干扰，本研究同时以p14 +/+;AlfpCre小鼠作为对照。所有实验小鼠均饲养于C57BL/6背景中，且周龄均为6周。将敲除组小鼠样本的表达谱（expression profiles）与对应对照组小鼠的表达谱进行比对，最终鉴定出差异表达基因（differentially expressed genes）。