| Abstract: | 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 Chinesehamster 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. Thiseffect could be blocked with the p38 MAP kinase inhibitor SB-203580.Examination by confocal microscopy of cells stained with rhodaminephalloidin showed that CCK dose-dependently induced changes of theactin cytoskeleton, including cell shape changes, which were coincidentwith actin cytoskeleton fragmentation and formation of actin filamentpatches in the cells. To further evaluate the role of HSP27, CHO-CCK-Acells were transfected with expression vectors for either wild-type(wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen afterhigh-dose CCK stimulation. In contrast, overexpression ofnonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition ofphosphorylation of HSP27 by preincubation of wt-HSP27 transfected cellswith SB-203580 did not protect the actin cytoskeleton. These resultssuggest that phosphorylation of HSP27 is required to stabilize theactin cytoskeleton and to protect the cells from the effects of highconcentrations of CCK. |
