细胞黏附对血管扩张刺激磷蛋白磷酸化的影响

Effect of cell adhesion on vasodilator-stimulated phosphoprotein phosphorylation

目的研究HeLa细胞黏附状态的改变对血管扩张刺激磷蛋白(VASP)磷酸化的影响,探讨VASP在肿瘤细胞黏附与去黏附过程中的作用。方法HeLa细胞分为4组,即贴壁细胞组、悬浮细胞组、再黏附细胞组及阳性对照组(Forskolin处理)。倒置相差显微镜观察细胞形态,W estern b lot检测VASP磷酸化水平,试剂盒检测蛋白激酶A(PKA)活性。结果HeLa细胞处于黏附状态时,PKA活性较低,VASP主要以去磷酸化形式(基态水平)存在;细胞处于悬浮状态时,PKA激活,VASP主要以磷酸化形式存在;细胞再黏附于I型胶原的过程中,VASP逐渐发生磷酸化,待细胞伸展后,VASP磷酸化又回复到基态水平。结论在HeLa细胞黏附/去黏附的交替过程中,VASP呈现磷酸化与去磷酸化的动态改变,这种改变与PKA信号通路有关。

Objective To study the changes of Vasodilator-stimulated Phosphoprotein phosphorylation which was dynamically regulated by cell adhesion to extracellular matrix, and to investigate the roles of VASP in the process of tumor cell adhesion and de-adhesion. Methods HeLa cells were divided into four groups： stably adherent cells, cells held in suspension, cells replated onto dishes coated with collagen I and positive control （cells treated with Forskolin）. Changes in cell morphology were observed in inverted phase contrast microscope. Phosphorylation of VASP was detected by Western blot, and PKA activity was measured by PKA assay kit. Results In untreated, stably adherent cells, VASP was predominantly in the unphosphorylated form; Detachment of cells stimulated PKA activity and induced PKA-dependent phosphorylation of VASP; VASP was phosphorylated gradually following reattachment but returned to base level of phosphorylation during active cell spreading. Conclusion During the process of HeLa cell adhesion and de-adhesion, dynamical changes of VASP from the form of phosphorylation to dephosphorylation via PKA have been observed.