Parnell Euan, Koschinski Andreas, Zaccolo Manuela, Cameron Ryan T, Baillie George S, Baillie Gemma L, Porter Alison, McElroy Stuart P, Yarwood Stephen J
Institute of Molecular, Cellular and Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.
Biochim Biophys Acta. 2015 Jul;1853(7):1749-58. doi: 10.1016/j.bbamcr.2015.04.009. Epub 2015 Apr 23.
Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T-EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2'-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T-EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP.
最近的研究表明，肌动蛋白结合蛋白埃兹蛋白（ezrin）和环磷酸腺苷（cAMP）传感器EPAC1协同作用，以响应细胞内cAMP升高诱导细胞铺展。为了研究这些效应背后的机制，我们基于将EPAC1稳定转染到HEK293T细胞（HEK293T-EPAC1）中，构建了一个EPAC1依赖性细胞铺展模型。我们发现，用EPAC选择性类似物8-pCPT-2'-O-Me-cAMP（007）直接激活EPAC1可促进这些细胞的铺展。此外，发现用腺苷酸环化酶激活剂福斯高林（forskolin）和cAMP磷酸二酯酶抑制剂咯利普兰（rolipram）联合激活EPAC1和蛋白激酶A（PKA）可协同增强细胞铺展，这与皮质肌动蛋白束集以及埃兹蛋白向质膜的动员有关。PKA激活还与埃兹蛋白在苏氨酸567位点的磷酸化有关，这在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）过程中通过电泳条带迁移率变化检测到。抑制PKA活性可阻断埃兹蛋白磷酸化，并将细胞对cAMP升高的铺展反应降低至仅由EPAC1激活所诱导的水平。用缺乏关键PKA磷酸化位点的抑制性埃兹蛋白突变体（埃兹蛋白-苏氨酸567丙氨酸）或缺乏与肌动蛋白结合能力的埃兹蛋白突变体（埃兹蛋白-精氨酸579丙氨酸）转染HEK293T-EPAC1细胞，可促进细胞分支形成，并阻断EPAC1和PKA进一步促进细胞铺展的能力。埃兹蛋白的PKA磷酸模拟突变体（埃兹蛋白-苏氨酸567天冬氨酸）对EPAC1驱动的细胞铺展没有影响。我们的结果表明，埃兹蛋白与肌动蛋白细胞骨架的结合以及苏氨酸567位点的磷酸化是PKA和EPAC1在细胞内cAMP升高后协同促进细胞铺展所必需的，但并不充分。
