Lack of Correlation between Changes in Polyphosphoinositide Levels and Actin / Gelsolin Complexes in A 431 Cells Treated with Epidermal Growth Factor

The polyphosphoinositides, PIP and PIP2, have been proposed to regulate actin polymerization in vivo because they dissociate actin/gelsolin complexes in vitro. We tested this hypothesis by comparing the ability of EGF and bradykinin to affect PI metabolism and the actin cytoskeleton in A431 cells. EGF, but not bradykinin, was found to induce ruffling and dissociation of actin/gelsolin complexes in these cells. However, both EGF and bradykinin stimulated the accumulation of inositol phosphates in [3H]inositol-labeled cells indicating that stimulation of PI turnover is not sufficient for the induction of changes in actin/gelsolin complex levels. EGF stimulated a twofold increase in the level of PIP in A431 cells. Other phosphoinositide levels were not markedly altered. Treatment of the cells with cholera toxin abrogated the EGF-induced rise in PIP levels without altering the dissociation of actin from gelsolin. These data indicate that increases in PIP and/or PIP2 are not necessary for dissociation of actin/gelsolin complexes. Overall, these experiments suggest that in A431 cells, the effects of EGF on the actin cytoskeleton are unlikely to be mediated through changes in PIP or PIP2 levels. T HE stimulation of phosphoinositide turnover has been shown to be a central event in the transduction of signals via receptors for a variety of agents. Hormonestimulated cleavage of the polyphosphoinositide, PIP2, yields two intracellular second messengers, diacylglycerol and inositol 1,4,5,-trisphosphate (29). Diacylglycerol activates protein kinase C (21) while inositol 1,4,5-triphosphate induces the release of calcium from intracellular stores (3). For many hormones, the production of these two second messengers is thought to be the principal mechanism used by the hormone to bring about changes in the functions of intracellular proteins and enzymes. Recently, several reports have appeared that have contradicted the current dogma that the only metabolic effects of phosphoinositides are due to their role as substrates for breakdown by phospholipase C. For example, the activity of the sarcoplasmic reticulum Ca2+-ATPase appears to be stimulated directly by binding of PIP or PIP2 (15). The function of two cytoskeletal proteins; profilin and gelsolin, also appears to be regulated by polyphosphoinositides. Profilin forms a 1:1 complex with actin monomers inhibiting polymerization (32). Polyphosphoinositides dissociate the profilin/actin complexes, promoting actin polymerization (25, 27). Gelsolin is an 87-kD protein that severs, caps, and nucleates actin filaments in vitro in a calcium-dependent C. Y. Dadabay's present address is University of Washington, Department of Biochemistry, SJ-70, Seattle, WA 98195. fashion. Each of these processes results in gelsolin bound to the barbed end of an actin filament (32). This actin/gelsolin complex is stable to EGTA but is dissociated by polyphosphoinositides (6, 7, 20, 23). The effects of polyphosphoinositides on profilin and gelsolin in vitro may serve to regulate actin polymerization in vivo. Treatment ofplatelets with thrombin results in the stimulation of PI turnover (33, 44) and is associated with changes in actin filament concentration and distribution (16, 27). Lind et al. (27) demonstrated that in thrombin-stimulated platelets gelsolin and profilin are rapidly converted from a free form to one in which they are bound to actin in high affinity 1:1 complexes. After several minutes, these complexes dissociate and the gelsolin and profilin are once again free in the cytosol. More recent work by Hartwig et al. (17) suggests that thrombin may induce the association of gelsolin with the cell membrane, positioning it in proximity to the regulatory polyphosphoinositides. Based on these findings, a model has been proposed to explain the thrombin-induced changes in the actin cytoskeleton of platelets (27, 28, 38). In this model, gelsolin is first activated via the rapid increase in intraceUular calcium resulting from the breakdown of PIP2 and the generation of inositol 1,4,5-trisphosphate. Gelsolin then caps, nucleates, or severs actin filaments resulting in the formation of actin/gelsolin complexes in which gelsolin is bound to the barbed end of actin filaments. A subsequent rise in polyphosphoinositide levels due to resynthesis of metabolized lipids releases the © The Rockefeller University Press, 0021-9525/91/03/1151/6 $2.00 The Journal of Cell Biology, Volume 112, Number 6, March 1991 1151-1156 1151 on D ecem er 9, 2017 jcb.rress. org D ow nladed fom gelsolin from the actin filaments, permitting actin polymerization at the newly created free barbed ends. Consistent with this model is the observation that the membranes of neutrophils exhibit an increased ability to nucleate actin polymerization after treatment with a chemotactic peptide (8). EGF induces membrane ruffling (12) and a change in the distribution of the actin-based cytoskeleton in A431 cells (35), a human epidermoid carcinoma cell line. EGF also stimulates turnover of phosphoinositides, activates a PI kinase, and induces a rise in the levels of PIP (31, 43). In light of these observations, we tested the hypothesis that EGFinduced changes in polyphosphoinositide levels affect the actin cytoskeleton and cell motility by altering actin/gelsolin and actin/profilin interactions. We report here that although EGF induces an increase in PIP levels and a concomitant decrease in actin/gelsolin complexes, the two effects of the growth factor can be completely dissociated in cholera toxin-treated cells. In addition, bradykinin stimulates more extensive PI turnover than EGF but does not cause dissociation of actin/gelsolin complexes indicating that breakdown of polyphosphoinositides is not always associated with changes in the actin cytoskeleton. Materials and Methods