Schäfer C, Clapp P, Welsh M J, Benndorf R, Williams J A
Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622, USA.
Am J Physiol. 1999 Dec;277(6):C1032-43. doi: 10.1152/ajpcell.1999.277.6.C1032.
We investigated how heat shock protein 27 (HSP27) and its phosphorylation are involved in the action of cholecystokinin (CCK) on the actin cytoskeleton by genetic manipulation of Chinese hamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. This effect could be blocked with the p38 MAP kinase inhibitor SB-203580. Examination by confocal microscopy of cells stained with rhodamine phalloidin showed that CCK dose-dependently induced changes of the actin cytoskeleton, including cell shape changes, which were coincident with actin cytoskeleton fragmentation and formation of actin filament patches in the cells. To further evaluate the role of HSP27, CHO-CCK-A cells were transfected with expression vectors for either wild-type (wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27 and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen after high-dose CCK stimulation. In contrast, overexpression of nonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition of phosphorylation of HSP27 by preincubation of wt-HSP27 transfected cells with SB-203580 did not protect the actin cytoskeleton. These results suggest that phosphorylation of HSP27 is required to stabilize the actin cytoskeleton and to protect the cells from the effects of high concentrations of CCK.
我们通过对稳定转染了胆囊收缩素 A 受体（CCK-A 受体）的中国仓鼠卵巢（CHO）细胞进行基因操作，研究了热休克蛋白 27（HSP27）及其磷酸化如何参与胆囊收缩素（CCK）对肌动蛋白细胞骨架的作用。在这些细胞中，如同在大鼠腺泡中一样，CCK 激活 p38 丝裂原活化蛋白（MAP）激酶并增加 HSP27 的磷酸化。这种作用可用 p38 MAP 激酶抑制剂 SB-203580 阻断。用罗丹明鬼笔环肽染色的细胞经共聚焦显微镜检查显示，CCK 剂量依赖性地诱导肌动蛋白细胞骨架发生变化，包括细胞形态改变，这与细胞内肌动蛋白细胞骨架的碎片化和肌动蛋白丝斑块的形成同时发生。为了进一步评估 HSP27 的作用，用野生型（wt）或突变型（3A、3G 和 3D）人 HSP27 的表达载体转染 CHO-CCK-A 细胞。野生型 HSP27 和 3D-HSP27 的过表达抑制了高剂量 CCK 刺激后对肌动蛋白细胞骨架的影响。相反，不可磷酸化突变体 3A-HSP27 和 3G-HSP27 的过表达，或用 SB-203580 预孵育野生型 HSP27 转染细胞以抑制 HSP27 的磷酸化，并不能保护肌动蛋白细胞骨架。这些结果表明，HSP27 的磷酸化是稳定肌动蛋白细胞骨架以及保护细胞免受高浓度 CCK 影响所必需的。
