可溶性纤维连接蛋白对SGC-7901细胞中PKA活性的影响

Effect of Soluble Fibronectin on Protein Kinase A Activation in SGC-7901

本研究通过向SGC-7901细胞中加入可溶性纤维连接蛋白(fibronectin,FN)探讨可溶性纤维连接蛋白对PKA活性的影响。实验采用蛋白印迹技术检测可溶性FN在不同浓度及作用时间情况下对PKA C_α亚基表达水平以及PKA作用底物血管扩张刺激磷蛋白(vasodilator-stimulated phosphoprotein,VASP)磷酸化水平的影响;采用免疫荧光技术检测可溶性FN对PKA作用底物P-VASP分布的影响。结果显示,在SGC-7901细胞中,可溶性FN在0.5～8μg/ml浓度区间内,对PKA活性存在剂量依赖性抑制效应;选取1μg/ml可溶性FN作用12h以后,可溶性FN对PKA活性存在时间依赖性抑制效应;同时,我们建立了能够稳定表达人甲状腺A激酶锚定蛋白(human thyroid AKAP,Ht31)肽段的SGC细胞,Ht31破坏PKA锚定后,FN对PKA活性的抑制作用消失;免疫荧光显示可溶性FN可以使VASP磷酸化位置聚集于细胞边缘。因此,我们认为在SGC-7901细胞中可溶性FN对PKA活性存在抑制作用,这种抑制效应与可溶性FN的浓度及作用时间呈正比且取决于PKA能否正常锚定;可溶性FN可以影响PKA活化位置。

To investigate the effect of soluble fibronectin on protein kinase A activation in gastric cancer cell line SGC-7901, we treated Gastric cancer cell line SGC-7901 with soluble fibronectin of different concentrations or different time, expression of PKA catalytic subunit and the phosphorylation level of vasodilator-stimulated phosphoprotein （VASP）, one of PKA substrates were determined by Western blot, the distribute of p-VASP was determined by immumofluorescence method. The results revealed that, FN can inhibit the PKA activation in SGC- 7901 among concentrations of 0.5-8μg/ml. As the dose and time increased, the inhibitory of FN on PKA activation of SGC-7901 was significantly increased after 12 h. FN mediate p-VASP accumlate on the edge of cell. However, these effect disapper in stably transfected SGC cells expressing the AKAP inhibitory peptide Ht31. These data suggest that PKA activation in SGC-7901 could be inhibited by FN, the inhibition determined by FN concentration, functionary time and PKA anchor. FN mediate localized activation of PKA.