THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 268, No. 32, Issue of November 15, p p . 24210-24214, 1993

Printed in U.S.A.

Modulation of Actin Microfilament Dynamics and Fluid Phase Pinocytosis by Phosphorylation of Heat Shock Protein 27*

(Received for publication, July 27, 1993)

Josbe N. LavoieSQ, Eileen Hickeyn, Lee A. Webern, and Jacques LandrySII From the $Centre de recherche en cancerologie de 1’Universite Laual, L’H6teL-Dieu de Quebec, 11, cdte du Palais, Quebec G l R 256, Canada and the 7Department of Biology, University of Nevada, Reno, Nevada 89557

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 expo- sure to cytochalasin D (Lavoie, J. N., Gingras-Breton, G., Tanguay, R. M., and Landry, J. (1993) J. Biol. Chern. 268, 342034291. Because HSP27 is a ubiquitous target of phosphorylation upon cell stimulation with a variety of growth factors and agents that affect cellular differen- tiation, 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 mem- brane ruffles where most actin polymerization occurs. We developed Chinese hamster cell lines that constitu- tively overexpressed either human HSP27 or a non- phosphorylatable mutant form of the protein. Overex- pression of HSP27 caused an increased concentration of filamentous actin (F-actin) at the cell cortex and el- evated pinocytotic activity. In contrast, overexpression of the non-phosphorylatable mutant form of HSP27 re- duced cortical F-actin concentration and decreased pi- nocytosis activity relative to control cells. Mitogenic stimulation of fibroblasts resulted in a rapid polymeri- zation of submembranous actin filaments. HSP27 en- hanced growth factor-induced F-actin accumulation, whereas mutant HSP27 exerted a dominant negative ef- fect and inhibited this response to growth factors. Thus, HSP27 is a component of a signal transduction pathway that can regulate microfilament dynamics.

~~~~ ~

Heat shock proteins are expressed at elevated levels in re- sponse to physiological stress but are also present in significant amounts in unstressed cells where they accomplish essential functions. For example, heat shock proteins (HSP)’ 60, 70, and 90 have molecular chaperonin activities involved with protein translocation, protein folding, and signal transduction (1). The function of HSP27 is still not defined. HSP27 is an early target of phosphorylation upon stimulation by serum or a variety of mitogens, cytokines, and inducers of differentiation (2-13). In

Canada Grant MT-1088 (to J. L.) and National Institutes of Health * This study was supported in part by Medical Research Council of

Grant GM 43167 (to L. A. W. and E. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Canada. 0 Supported by a studentship from the Medical Research Council of

en canc6rologie de l’Universit6 Laval, L’HGtel-Dieu de Quebec, 11, cbte 11 To whom correspondence should be addressed: Centre de recherche

du Palais, Quebec G1R 256, Canada. Tel.: 418-691-5281; Fax: 418-691- 5439; E-mail: 2020053@saphir.ulaval.ca.

fibroblast growth factor; FITC, fluorescein isothiocyanate; M A P , mito- The abbreviations used are: HSP, heat shock protein; FGF, basic

gen-activated protein; PBS, phosphate-buffered saline.

quiescent cells stimulated by addition of serum, thrombin, or fibroblast growth factor (FGF), HSP27 phosphorylation is regu- lated by the activation of a specific serine protein kinase that is possibly homologous to the recently described MAP kinase- activated protein kinase I1 (3, 14).2 The activity of purified HSP27 kinase is highly sensitive to treatment with protein phosphatases (3) and can be activated in vitro by MAP kinase,2 suggesting that HSP27 kinase and thus HSP27 may be linked to the major signal transduction pathways involving MAP ki- nase (15). This is further supported by numerous reports that propose a role for HSP27 and its phosphorylated isoforms in events associated with growth and differentiation of various normal and cancerous cell lines (3, 4, 10, 16-22).

Fibroblastic cells deprived of serum exit the growth cycle and show a dramatic decline in F-actin content. Upon re-addition of serum or specific growth factors, microfilaments rapidly reform beneath the membrane and reorganize into stress fibers that extend through the cytoplasm (23, 24). Actin polymerization/ depolymerization occurs predominantly at the leading edge of fibroblastic cells and affects plasma membrane activities such as ruffling, pinocytosis, extension of lamellipodia, and cell mo- tility (23-27). This dynamic process is controlled in vivo by actin-binding proteins, some of which are regulated by extra- cellular signals (28, 29). Intriguingly, the avian homologue of mammalian HSP27, IAP, behaves in vitro as an actin cap- binding protein and inhibits actin polymerization (30, 31). Mi- croinjection of antibodies against HSP27 into mouse smooth muscle cells has been shown to block bombesin- and kinase C-induced sustained contraction (32). Moreover, artificially in- creasing the expression of HSP27 in cells by gene transfection resulted in increased resistance of actin stress fibers to disag- gregation by hyperthermia and cytochalasin D (33).

To test the hypothesis that HSP27 could be a signaling com- ponent of the microfilament responses to external stimuli, we developed a family of clonal Chinese hamster CCL39 cell lines that constitutively express human HSP27 or a mutant form of the human HSP27 gene in which codons for serine 15, 78, and 82 were replaced by glycine codons. These serine residues are the targets of phosphorylation upon mitogenic stimulation (2, 34). The data obtained from these cell lines on the immunolo- calization of HSP27, the organization of F-actin, pinocytotic activity, and growth factor-induced F-actin accumulation sug- gested that HSP27 has a major function in signal transduction and regulation of actin filament dynamics.

EXPERIMENTAL PROCEDURES Plasmids and Cell Lines-Plasmid pRSV-NE0 contains the Geneti-

cin resistance gene (35); pKS2711 contains the wild type human HSP27 gene (36); pKSm157882 contains a human HSP27 gene in which codons for Ser-15, Ser-78, and Ser-82 were converted to Gly by site-directed mutagenesis as described previously (2). Cell lines were developed by

H. Lambert and J. Landry, manuscript in preparation.

24210

HSP27 in Signal Dansduction to Microfilaments 24211

transfecting the Chinese hamster CCL39 cell line (American Type CUI- ture Collection) with pRSV-NE0 alone, pRSV-NE0 and pKS2711, or pRSV-NE0 and pKSm157882. After selection for 3 weeks with G418 (400 pg/d), four to six positive clones from each group were pooled to establish the CCL39-neo control, HU27, and HU27pm3 cell lines. Pools were made to eliminate possible clonal variabilities. All results were also confirmed with individual clonal cell lines from each group. The transfections were performed as previously described (33, 37) using exponentially growing cells plated 24 h before transfection at a concen- tration of 7500 celldcm2 in a 75-cm2 culture flask. CC139 cells and their transfectant derivatives were maintained at 37 "C in a 5% C02 bumidi- fied atmosphere, in Dulbecco's modified Eagle's medium (Life Technolo- gies, Inc.), containing 2.2 g/liter NaHC03, 4.5 g/liter glucose, and supplemented with 5% fetal calf serum (Life Technologies, Inc.). Pools of clonal cell lines were used at passage numbers lower than 10.

Zmmunofluorescence Microscopy-Immunolabeling of HSP27 and/or F-actin visualization was performed on cells growing on Lab-Tek poly- styrene chamber slides (Life Technologies, Inc.) or on fibronectin-coated glass slides. Cell fixation and staining for HSP27 or F-actin was per- formed as described before (33), except that the cells were permeabi- lized with 0.1% saponin to better preserve the cell architecture (25). F-actin was detected using FITC-conjugated palloidin (33.3 pg/ml) di- luted 150 in PBS (10 m~ NaH2P04, 130 m~ NaCl, 2.7 mM KCl, 1 mM MgCl,, pH 7.5) and HSP27-antigen-antibody complexes were detected with biotin-labeled anti-rabbit IgG diluted 1:50 in PBS revealed with Texas Red-conjugated streptavidine 1:50. The anti-hamster (Ha27Ab) and anti-human HSP27 (Hu27Ab) antibodies were characterized pre- viously (37). Both are species-specific and do not cross-react with any cellular structure in cells not containing the respective protein. Confo- cal microscopy was performed using a Bio-Rad MRC-600 imaging sys- tem mounted on a Nikon Diaphot-TDM, and 60x objective lens with a 1.4 numerical aperture. A630 nm filter was used for dual labeling to cut FITC emission in the Texas Red range.

Pinocytosis AnaZysis--To measure fluid phase pinocytotic activity, cells were grown exponentially on Lab-Tek polystyrene chamber slides and incubated at 37 "C for 20 min in the presence of 1 mg/ml FITC- labeled dextran-lysine (Molecular Probe). Cells were then fured in 3.7% formaldehyde and visualized by confocal microscopy. To biochemically measure pinocytosis, cells growing exponentially on plastic dishes (16 mm in diameter) were exposed to 1 mg/ml horseradish peroxidase (Sigma) for 20 min at 37 "C or at 4 "C. Cells were then rinsed exten- sively in PBS and lysed with 0.1% Triton X-100, and the amount of peroxidase inside the cells was determined enzymatically using the TMB peroxidase substrate kit (Bio-Rad).

F-actin Accumulation-Subconfluent cultures of cells plated on fibro- nectin-coated glass slides were serum-starved for 24 h. The quiescent cells were then exposed to 1 unit/ml thrombin (Sigma) or 100 ng/ml FGF (Calbiochem) at 37 "C. At varying times thereafter, the cells were fured in 3.7% formaldehyde and stained with FITC-phalloidin as described above. The relative levels of F-actin were determined by confocal mi- croscopy. The relative intensity was obtained by summing the fluores- cence intensity of successive 0.75-pm-thick optical sections made every 1 pm over the entire cell volume, using the Histogramm function of the Bio-Rad Comos software. The software displays the intensity of fluo- rescence of a selected area in terms of integrated pixel values per unit

least 75-100 cells in 3 4 distinct fields. area. At each time point, the fluorescence intensity was calculated for at

RESULTS Previous immunocytofluorescence studies of cultured cells

have shown that HSP27 is diffusely distributed throughout most of the cytoplasm (33, 38). In these studies, Triton X-100 was used to permeabilize the cells. In preliminary experiments we performed immunolocalization studies of HSP27 using sap- onin for permeabilization, an agent that better preserves plasma membrane protrusions and cortical actin structures (25). We observed, both in mouse NIH 3T3 and Chinese ham- ster CCL39 cells, that HSP27 antibodies stained predomi- nantly region of highly motile cytoplasm such as the lamelli- podia. Because lamellipodia are active sites of actin polymerization in fibroblasts (25, 26) and because HSP27 can interact with actin in vitro (30, 311, we postulated that HSP27 might affect some aspects of microfilament assembly in uiuo. Thus, overexpression of HSP27 could result in identifiable al- terations in the structure or dynamics of actin filaments. l'~,

test this hypothesis, we developed a family of clonal Chinese hamster CCL39 cell lines that constitutively express varying levels of human HSP27 or a mutant form of the human HSP27 gene in which codons for serine 15,78, and 82 were replaced by glycine codons. These 3 serine residues are the targets of phos- phorylation upon heat shock or mitogenic stimulation (2, 34). Individual clonal lines were characterized with respect to the level of expression of human HSP27. A detailed analysis of the cell lines will be presented e l~ewhere .~ Briefly, clones were selected that contained, in addition to the normal amount of 1 ng of Chinese hamster HSP27/pg of total protein, 3-6 ng of human proteidpg. This level of expression is sufficient to con- fer significant thermopr~tection~ but is still less than the amount found in heat induced thermotolerant cells (10 ng/pg). Clones containing equivalent amounts of wild type or mutant human HSP27 were pooled separately to yield the HU27 and HU27pm3 cell lines, respectively.

Two-color confocal microscopy was performed to visualize F-actin and HSP27 in the same cells using FITC-conjugated phalloidin and species-specific anti-HSP27 antibodies, respec- tively. Localization studies were performed in control CCL39- neo hamster fibroblasts, HU27 cells, and HU27pm3 cells. Each cell type was stained for both human and hamster HSP27 along with actin and also examined by phase contrast microscopy. Only three representative examples are shown in Fig. 1. In all cases, HSP27 localized preferentially with membrane ruffles and lamellipodia. In cells growing on a highly adherent fibro- nectin 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 phase-dark membrane ruffles containing enhanced concentra- tion of HSP27 (Fig. 1, E-F). In all cell lines and in several individual clones analyzed, the endogenous Chinese hamster HSP27 protein, the wild type human, and the non-phosphory- latable human protein all had an equivalent intracellular lo- calization, showing a diffise distribution throughout most of the cytoplasm but an enrichment at the leading edges of polar- ized cells.

Initial biochemical analyses revealed no difference in actin content between HU27, HU27pm3, and control cells (data not shown). However, as shown in Fig. 2, the distribution of F-actin between cortical filaments and stress fibers differed consider- ably between HU27 and HU27pm3 cells. In HU27 cells, F-actin staining was mostly beneath the cell surface. The plasma mem- brane was often distorted by irregularities, ruffles, and blebs. By contrast, F-actin in HU27pm3 cells was found mostly in stress fibers extended through the cytoplasm and little staining is observed at the cell cortex. Control cells (data not shown) or occasional cells in cultures of HU27 or HU27pm3 cells that express only low levels of either form of the protein (Fig. 2) showed a more equal distribution of F-actin between the cortex and the stress fibers.

These results provided the first evidence that HSP27 might affect the dynamics of actin filaments. Furthermore, we ob- served by phase contrast microscopy that quiescent HU27 cells contained large cytoplasmic vacuoles whereas the HU27pm3 cells did not (data not shown). Fluid phase pinocytosis is de- pendent on the dynamics of the actin cytoskeleton (39), and increased actin polymerization associated with membrane ruf- fling is often linked to elevated pinocytic activity (23,27). Fluid phase pinocytic activity was thus measured in the three cell types (Fig. 3). Pinocytotic activity was visualized by confocal microscopy after incubating the cells for 20 min in medium containing FITC-labeled dextran-lysine (Fig. 3, A-C). By com-

preparation. J. N. Lavoie, E. Hickey, L. A. Weber, and J. Landry, manuscript in

24212 HSP27 in Signal Dansduction to Microfilaments

FIG. 1. Localization of HSP27 in lamellipodia and membrane ruffles. Two-color confocal microscopy was performed to visualize F- actin and HSP27 in the same cells using FITC-conjugated phalloidin and species-specific anti-HSP27 antibodies, respectively. Localization studies were performed in control CCL39-neo hamster fibroblasts (A and R ) , HU27pm3 cells (C and D), and HU27 cells ( E and F ) . Each cell type was stained for both human and hamster HSP27 along with actin and also examined by phase contrast microscopy. Three representative examples are shown. A and B, Chinese hamster HSP27 (B) co-localizes with cortical F-actin ( A ) in a large lamellipodium of a control CCL39- neo cell. Cells were grown on fibronectin-coated coverslips to enhance lamellipodia development. C and D, mutant human HSP27 (D) and F-actin (C) in HU27pm3 cells. E and F, Chinese hamster HSP27 is enriched in phase dark membrane ruffles in HU27 cells. Cells were grown on plastic coverslips. E , phase contrast microscopy of HU27 cells. Ruflles are visualized as dark structures. F, immunological staining for hamster HSP27 in the same cells. Scale bars are 10 pm.

parison to control cells, HU27 cells contained more numerous and more heavily stained vacuoles that were fused into large intensively stained secondary vesicles. HU27pm3 cells have fewer and more lightly stained vacuoles than controls. The effect of HSP27 content on pinocytotic activity was confirmed using another marker, horseradish peroxidase. After an incu- bation of 20 min in the presence of horseradish peroxidase a t 37 or 4 "C (a negative control), the amount of peroxidase inside the cells was determined enzymatically (Fig. 30). The uptake was enhanced approximately 3-fold in HU27 cells, whereas it was inhibited 2-fold in HU27pm3 cells, relative to control cells.

The increaseddecreased pinocytotic activity and enhanced diminished concentration of F-actin at the plasma membrane seen in HU27/HU27pm3 cells, as well as the localization of HSP27 to the lamellipodia, all suggested that HSP27 may be involved in the control of actin polymerization. Because HSP27 is rapidly phosphorylated in quiescent cells in response to growth factors, it could be a signaling component of the micro- filament responses to external stimuli. Fibroblastic cells de- prived of serum exit the growth cycle and show a dramatic decline in F-actin content. Upon re-addition of serum or specific growth factors, microfilaments rapidly reform beneath the membrane and reorganize into stress fibers that extend through the cytoplasm (23,241. Thus, we examined the effect of

FIG. 2. HSP27 and the phosphorylation mutant form of HSP27 have opposite effects on the distribution of F-actin between the cortex and the stress fibers. Growing HU27 ( A and RJ and HU27pm3 IC and D ) cells were stained to visualize in the same cell F-actin (A and C) and human HSP27 (B and D). Occasional cells not expressing or expressing little human HSP27 were purposely included in the microscopic fields in order to evaluate the effect of HSP27 ex- pression on F-actin distribution. In HU27 cells, a high level of expres- sion of HSP27 (brightly stained cells in B) favors the organization of

with an enhanced staining of F-actin with stress fibers. By contrast, a F-actin in cortical filaments (A); a low level of expression correlates

high level of expression of the phosphorylation mutant form of HSP27 in HU27pm3 cells (brightly stained cells in D ) favors fiber formation (C), whereas low expressers show an enhanced level of F-actin in the cell cortex. Scale bars are 10 pm.

growth factors that induce phosphorylation of HSP27 on the polymerization and reorganization of F-actin in resting control, HU27, and HU27pm3 cells.

Thrombin and FGF are mitogenic for CCL39 cells (5), stimu- late HSP27 kinase activity, and induce phosphorylation of HSP27 (3). In HU27 cells, both the human and the endogenous proteins were phosphorylated in response to thrombin and FGF. In HU27pm3 cells, the presence of the non-phosphorylat- able HSP27 had no effect of the phosphorylation of the endog- enous protein (data not shown). Addition of thrombin or FGF to serum-starved control CCL39-neo cells caused a rapid accumu- lation of F-actin as measured by quantification of total phalloi- din-FITC fluorescence (Fig. 4A) and visualized by confocal mi- croscopy (Fig. 4B, shown for FGF only). Total F-actin in HU27 cells increased to approximately double the amount induced in control cells in response to the mitogens. The enhanced re- sponse in HU27 cells relative to control cells caused prominent F-actin accumulation under membrane ruffles, cell rounding, and formation of surface blebbing (Fig. 4,A and C). By contrast the non-phosphorylatable form of HSP27 expressed in HU27pm3 cells appeared to act as a dominant negative mu- tant, inhibiting both thrombin and FGF induction of F-actin accumulation and formation of ruffles (Fig. 4, A and D). Little increase in F-actin content is detected in these cells within

HSP27 in Signal Dansduction to Microfilaments

I 37 OC

L 4 O C

Flc. 3 . HSP27 enhances whereas mu tan t HSP27 reduces fluid phase pinocytotic activity. A-C, pinocytotic activity was visualized by confocal microscopy after incubating exponentially growing control CCL39-neo cells (A), HU27 cells ( B ) , or HU27pm3 cells (C) for 20 min in medium containing FITC-labeled dextran. By comparison to control cells, HU27 cells contains more numerous and more heavily stained vacuoles that are fused into large intensively stained secondary vesicles. HU27pm3 cells have fewer and more lightly stained vacuoles than controls. The scale bar is 10 pm. D, fluid phase pinocytosis of horseradish peroxidase (HRP) in control CCL39-neo (CON), HU27, and HU27pm3 cells. After an incubation of 20 min in the presence of 1 mg/ml horseradish peroxidase at 37 “C ( top) or at 4 “C (a negative control, bottom), the amount of peroxidase inside the cells was deter- mined enzymatically.

0.5-1 h following addition of either thrombin or FGF. These results imply that HSP27 is a component of the signaling path- way between mitogens and actin polymerization at the cell membrane.

DISCUSSION

In summary, we have found that overexpression of HSP27 in fibroblastic cells caused increased accumulation of cortical F- actin relative to stress fiber actin, increased fluid phase pino- cytic activity, and promoted F-actin accumulation in quiescent cells in response to growth factors. Most strikingly, we found that overexpression of a non-phosphorylatable form of the pro- tein had the opposite effect on F-actin organization, pinocyto- sis, and growth factor-stimulated F-actin accumulation, sug- gesting that the mutant protein can interfere with a normal function of the endogenous protein in these processes. Because HSP27 is rapidly phosphorylated in response to growth factors, localizes in lamellipodia (a site of active actin polymerization in fibroblastic cells; Fig. 11, and can interfere in vitro with actin polymerization, we propose that HSP27 exerts a crucial func- tion at the end of a signaling pathway between mitogens and actin polymerization at the plasma membrane.

The replacement of Ser-15, Ser-78, and Ser-82 by Gly in HSP27 caused no gross alteration in the structure of the mu- tant protein. The mutant HSP27 retains the capacity described for wild type HSP27 to assemble into large multimeric aggre- gates:’ and to localize in membrane protrusions in unstressed cells (Fig. 1) and in nuclei after heat shock.“ Furthermore, overexpression of the protein (either wild type or mutant hu- man HSP27) at the levels used in this study, did not modify the intracellular distribution of the endogenous protein (Fig. l),

0.5

0 o I O 20 3n

MINUTES FIG. 4. HSP27 enhances whereas mutant HSP27 inhibits

growth factor induced F-actin accumulation in serum-starved ce1ls.A. the kinetics of accumulation of F-actin following stimulation by FGF ( top) or thrombin (bottom) was measured in CCL39-neo cells (O) , HU27 cells (m), and HU27pm3 cells (A). B-D, FITC-phalloidin stained

control CCL39-neo ( R ) , HU27 ( C ) , and HU27pm3 (Dl cells. The scale F-actin was visualized 30 min after addition of FGF to serum-starved

bar is 10 pm.

nor induce a stress response as judged from monitoring the level of the inducible HSP70 (data not shown). Finally, the endogenous HSP27 is phosphorylated normally in response to agonists or heat shock in both HU27pm3 and HU27 cells (data not shown), indicating that inhibition of the function of the endogenous hamster HSP27 by the non-phosphorylatable mu- tant human protein does not result from a competition with an upstream activator ( e g . HSP27 kinase).

It appears most likely that competition between the non- phosphorylatable mutant and the endogenous wild type protein occurs downstream, close to the site of HSP27 action. A first model consistent with the available data is that the described actin capping activity of HSP27 (30, 31) is regulated by phos- phorylation. Phosphorylation of HSP27, as i t occurs during stimulation by growth factors, may cause a change in the con- formation of the protein, resulting in the dissociation of HSP27 from the barbed (+) end of actin filaments. Local phosphoryla- tion of cortical HSP27 could thus regulate the spatial organi- zation of F-actin by freeing barbed ends of microfilaments for addition of monomers. A higher concentration of HSP27 in cells would increase the proportion of HSP27 relative to other actin- capping proteins, thus enhancing polymerization in response to growth factors. The presence of the non-phosphorylatable form of HSP27 in high amount, would then increase the life-time of HSP27 at the barbed end and reduce the microfilament re- sponse to growth factors. A second model is based on the re- cently reported chaperonin-like activity of purified HSP27 (40, 41). HSP27 might facilitate attachment of other actin-capping proteins at the barbed end of microfilaments. Such a catalytic process could involve cycles of HSP27 phosphorylation and de- phosphorylation. The non-phosphorylatable HSP27 would then compete with the wild type protein and directly inhibit the assembly process.

The phenotype of HU27pm3 cells is intriguingly similar to that recently described for cells expressing the dominant nega-

242 14 HSP27 in Signal Dansduction to Microfilaments

tive mutant form of racl GTP binding protein (23, 24). As we found here for mutant HSP27, accumulation of a dominant inhibitory mutant Racl protein prevents cortical microfilament assembly, membrane ruffling, and pinocytosis in growth factor- stimulated cells. Racl and HSP27 may thus function in the same pathway that transduces signals to cortical actin.

Acknowledgment-We thank Claude Chamberland for technical as- sistance with confocal microscopy.

REFERENCES 1. Gething, M. J., and Sambrook, J. (1992) Nature 356,3345 2. Landry, J., Lambert, H., Zhou, M., Lavoie, J. N., Hickey, E., Weber, L. A., and

Anderson, C. W. (1992) J. Biol. Chem. 267,794-803

4. Welch, W. J. (1985) J. Biol. Chem. 2e0,3058-3062 3. Zhou, M., Lambert, H., and Landry, J. (1993) J. Biol. Chem. 268,3543

5. Chambard, J.-C., Franchi, A,, Le Cam, A,, and PouyssBgUr, J. (1983) J. Biol.

6. Guesdon, F., and Saklatvala, J. (1991) J. Immunol. 147, 34023407 7. Arrigo, A. P. (1990) Mol. Cell. Biol. 10, 12761280 8. Robaye, B., Hepburn, A., Lecocq, R., Fiers, W., Boeynaems, J . M., and Dumont,

9. Gmse, J. H., Caron, L., Lebel, M., and Landry, J . (1991) Adu. Prostaglandin

10. Shibanuma, M., Kuroki, T., and Nose, K. (1991) Cell Growth Diffez 2,583-591 11. Saklatvala, J., Kaur, P., and Guesdon, F. (1991) Biochem. J. 277, 635442 12. Michishita, M., Satoh, M., Yamaguchi, M., Hirayoshi, K., Okuma, M., and

Nagata, K. (1991) Biochem. Biophys. Res. Commun. 176,979-984 13. Darbon, J. M., Issandou, M., 'lburnier, J. E , and Bayard, F. (1990) Biochem.

Biophys. Res. Commun. 168,527-536 14. Stokoe, D., Engel, K., Campbell, D. G., Cohen, P., and Gaestel, M. (1992) FEES

Lett. 313,307-313 15. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556 16. Gaestel, M., Gross, B., Benndorf, R., Strauss, M., Schunk, W. H., KraR, R.,

Otto, A., Bohm, H., Stahl, J., Drabsch, H., and Bielka, H. (1989) Eul: J . Biochern. 179,20%213

17. Shibanuma, M., Kuroki, T., and Nose, K. (1992) Biochern. Biophys. Res. Corn. mun. 187, 1418-1425

Chem. 268,1706-1713

J. E. (1989) Biochem. Biophys. Res. Commun. 163,301-308

Thromboxane Leukotriene Res. 21& 145-148

18. Mendelsohn, M. E., Zhu, Y., and O'Neill, S . (1991) Proc. Nutl. Acud. Sei. U. S . A. SS, 11212-11216

19. Spector, N. L., Samson, W., Ryan, C., Gribben, J., Urba, W., Welch, W. J., and Nadler, L. M. (1992) J. Immunol. 148, 1668-1673

20. Strahler, J. R., Kuick, R., and Hanash, S . M. (1991) Biochem. Biophys. Res. Comrnun. 176, 134-142

21. Shakoori, A. R., Oberdorf, A. M., Owen, T. A,, Weber, L. A,, Hickey, E., Stein, J. L., Lian, J. B., and Stein, G. S. (1992) J. Cell Biochem. 48,277-287

22. Zantema, A,, de Jong, E., Lardenoije, R., and van der Eb, A. J. (1989) J. Virol.

23. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A.

25. Symons, M. H., and Mitchison, T. J. (1991) J. Cell Biol. 114, 503-513 24. Ridley, A. J., and Hall, A. (1992) Cell 70, 389-399

26. Theriot, J. A,, and Mitchison, T. J. (1991) Nature 362, 126131

28. Stossel, T. P. (1989) J. Bid. Chem. 264, 18261-18264 27. Bar-Sagi, D., and Feramisco, J. R. (1986) Science 233,1061-1068

30. Miron, T., Wilchek, M., and Geiger, B. (1988) Eur. J . Biochem. 178,543-553 29. Luna, E. J., and Hitt, A. L. (1992) Science 268, 955-963

31. Miron, T., Vancompernolle, K., Vandekerckhove, J., Wilchek, M., and Geiger, B.

32. Bitar, K. N., Kaminski, M. S., Hailat, N., Cease, K B., and Strahler, J. R.

33. Lavoie, J. N., Gingras-Breton, G., Tanguay, R. M., and Landly, J. (1993) J.

34. Gaestel, M., Schroder, W., Benndorf, R., Lippmann, C., Buchner, K., Hucho, F.,

35. Gorman, C., Padmanabhan, R., and Howara, B. H. (1983) Science 221,551-

36. Hickey, E., Brandon, S . E., Potter, R., Stein, G., Stein, J., and Weber, L. A.

37. Landry, J., Chretien, P., Lambert, H., Hickey, E., and Weber, L. A. (1989) J. Cell

38. Arrigo, A. P., Suhan, J. P., and Welch, W. J. (1988) Mol. Cell. Biol. 8,5059-5071 39. Gottlieb, T. A,, Ivanov, I. E., Adesnik, M., and Sabatini, D. D. (1993) J. Cell

Biol. 120, 695-710 40. Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) J. Biol. C k m . 288,

1517-1520 41. Merck, K. B., Groenen, P. J., Voorter, C. E., de Haard-Hoekman, W.A., Horwitz,

J., Bloemendal, H., andde Jong, W. W. (1993)J. Biol. Chem. 268,10461052

63,336~375

(1992) Cell 70,401-410

(1991) J. Cell Biol. 114, 255-261

(1991) Biochem. Biophys. Res. Commun. 181, 1192-1200

Biol. Chem. 268,3420-3429

Erdmann, V. A,, and Bielka, H. (1991) J. Biol. Chem. 266,14721-14724

553

(1986) Nucleic Acids Res. 14,4127-4145

Biol. 109, 7-15