Abstract
The in vivo activity of the 27-kDa heat shock protein, a barbed-end microfilament capping protein, may be localized to the plasma membrane. To investigate this putative association, bovine endothelial cells expressing the human wild type or a mutant nonphosphorylatable 27-kDa heat shock protein were subjected to subcellular fractionation and immunoblot analysis. The 25-kDa endogenous bovine homolog and both exogenous gene products partitioned with cytosolic or plasma membrane components, indicating that phosphorylation is not required for membrane association. Phorbol ester treatment resulted in phosphorylation of only membrane-associated 25-kDa and wild type 27-kDa heat shock protein and did not induce redistribution. In a second fractionation protocol, streptavidin-agarose precipitation of extracts prepared from cells biotinylated at either the apical or basal surface localized membrane 25- and 27-kDa heat shock protein exclusively to the basolateral surface. Stimulation of transfectants expressing the wild type 27-kDa heat shock protein resulted in its phosphorylation and a doubling in the amount of membrane-associated F-actin precipitated, whereas the mutant protein decreased the amount of F-actin precipitated. These data suggest that membrane-associated 25- and 27-kDa heat shock proteins inhibit the generation of basolateral microfilaments and that phosphorylation releases this inhibition.
Highlights
Endothelial cell F-actin exists as a dynamic and responsive microfilament cytoskeleton tightly regulated by a number of spatial and temporally regulated microfilament modulating proteins [1,2,3,4]
We demonstrate that a significant portion of cellular HSP25/27 fractionates with plasma membrane components, that phosphorylation is not required for this localization, and that this subpopulation of HSP25/27 is a substrate for kinase activity induced by brief treatment of the cells with phorbol ester
Using a second fractionation protocol, we further demonstrate that a portion of the membrane-associated HSP25/27 localizes to the basolateral membrane where it is substrate for HSP27 kinase activity
Summary
Endothelial cell F-actin exists as a dynamic and responsive microfilament cytoskeleton tightly regulated by a number of spatial and temporally regulated microfilament modulating proteins [1,2,3,4]. We demonstrate that a significant portion of cellular HSP25/27 fractionates with plasma membrane components, that phosphorylation is not required for this localization, and that this subpopulation of HSP25/27 is a substrate for kinase activity induced by brief treatment of the cells with phorbol ester.
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