Abstract
We recently reported that overexpression of heat shock protein 27 (HSP27) in rodent fibroblasts increases the stability of stress fibers during hyperthermia and partially prevents actin depolymerization during exposure to cytochalasin D (Lavoie, J.N., Gingras-Breton, G., Tanguay, R. M., and Landry, J. (1993) J. Biol. Chem. 268, 3420-3429). Because HSP27 is a ubiquitous target of phosphorylation upon cell stimulation with a variety of growth factors and agents that affect cellular differentiation, we examined the role of HSP27 phosphorylation in regulating actin filament dynamics. Here we show that HSP27 is enriched at the leading edge of polarized fibroblasts. HSP27 is localized in lamellipodia and membrane ruffles where most actin polymerization occurs. We developed Chinese hamster cell lines that constitutively overexpressed either human HSP27 or a nonphosphorylatable mutant form of the protein. Overexpression of HSP27 caused an increased concentration of filamentous actin (F-actin) at the cell cortex and elevated pinocytotic activity. In contrast, overexpression of the non-phosphorylatable mutant form of HSP27 reduced cortical F-actin concentration and decreased pinocytosis activity relative to control cells. Mitogenic stimulation of fibroblasts resulted in a rapid polymerization of submembranous actin filaments. HSP27 enhanced growth factor-induced F-actin accumulation, whereas mutant HSP27 exerted a dominant negative effect and inhibited this response to growth factors. Thus, HSP27 is a component of a signal transduction pathway that can regulate microfilament dynamics.
Highlights
We developed Chinese hamstercell lines that constitu- Fibroblasticcells deprived of serum exit the growctyhcle and tively overexpressed either humanHSP27or anon- show a dramatic decline in filamentous actin (F-actin) contenUt.pon re-addition of phosphorylatable mutant form of the protein
Thisdynamic process is controlled invivo by stimulation of fibroblasts resulted in a rapid polymeri- actin-binding proteins, some of which are regulated by extrazation ofsubmembranous actin filaments
A first fibroblastic cells caused increased accumulation of cortical F- model consistent with the available data is that the described actin relative to stress fiber actin, increased fluid phase pino- actin capping activityof HSP27 [30, 31] is regulated by phoscytic activity, and promoted F-actin accumulation in quiescent phorylation.Phosphorylation of HSP27, as it occurs during cells in response to growth factors
Summary
Centrede recherche en canc6rologie de l’Universit6Laval, L’HGtel-Dieude Quebec, 11,cbte du Palais, Quebec G1R 256, Canada. In addition to the normal amounotf 1 ng of Chinese hamster HSP27/pg of total protein, 3-6 ng of ofhuman proteidpg This level of expression is sufficient to conclonal cell lines were useadt passage numberslower than 10. F-actin visualizationwas performed on cells growing on Lab-Tek poly- Clones containing equivalent amounts of wild type or mutant styrene chamber slides (Life Technologies, Inc.) or on fibronectin-coahtuedman HSP27 were pooled separately to yield the HU27 and glass slides. In cells growing on a highly adherent fibronectin coated surface, HSP27 co-localized with cortical actin in well extended lamellipodia (Fig. 1,A-Dl. On a less adherent plastic substratum, the lamellipodia retracted, forming thin and incubated at 37 "C for 20 min in the presence of 1 mg/ml FITC- phase-dark membrane ruffles containing enhanced concentralabeled dextran-lysine (Molecular Probe). Control cells (data not shown)or least 75-100 cells in 3 4 distinct fields
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