G Protein βγ Subunits Induce Stress Fiber Formation and Focal Adhesion Assembly in a Rho-dependent Manner in HeLa Cells*

In fibroblasts, the G protein α subunits Gα12 and Gα13 stimulate Rho-dependent stress fiber formation and focal adhesion assembly, whereas G protein βγ subunits instead exert a disruptive influence. We show here that the latter can, however, stimulate the formation of stress fibers and focal adhesions in epithelial-like HeLa cells. Transient expression of β1 with γ2, γ5, γ7, and γ12 in quiescent HeLa cells induced stress fiber formation and focal adhesion assembly as did expression of the constitutively active Gα12. Co-expression of βγ with Gαi2 and the C-terminal fragment of the β-adrenergic receptor kinase, both of which are known to bind and sequester free βγ, blocked βγ-induced stress fiber and focal adhesion formation. Inhibition was also noted with co-expression of a dominant negative mutant of Rho. Botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho, and a Rho-associated protein kinase inhibitor, Y-27632, similarly inhibited βγ-induced stress fiber and focal adhesion assembly. These results indicate that G protein βγ subunits regulate Rho-dependent actin polymerization in HeLa cells.

Rho family small GTP-binding proteins play a major role in regulating the actin polymerization necessary for cytoskeleton formation, determination of cell shape, and regulatory responses including chemotaxis and mitogenesis (1). In fibroblasts, the formation of stress fibers generally parallels the assembly of focal adhesions. For instance, some stimuli involving lysophosphatidic acid (LPA) 1 , thrombin, and bombesin induce both. G proteins of the G 12 subfamily have been shown to be involved in Rho-dependent actin stress fiber formation and focal adhesion assembly stimulated by G protein-coupled receptors (2,3). Recent reports have indicated that G␣ 12 and G␣ 13 are directly associated with p115 (4) and PDZ (5) guanine nucleotide exchange factors (GEF) for Rho, G␣ 13 stimulating the guanine nucleotide exchange activity of p115-RhoGEF (4). RhoGEFs then activate Rho. In contrast to G␣ 12 and G␣ 13 , microinjection of constitutively active forms of G␣ i2 , G␣ q , and G␣ 11 into fibroblasts does not induce stress fiber formation (2,3). Furthermore, pertussis toxin does not inhibit LPA-induced stress fiber formation, suggesting uncoupling of this signal pathway to G proteins of the G i/o subfamily (6).
G protein ␤␥ subunits regulate the c-Jun N-terminal kinase cascade through Rho family GTP-binding proteins such as Rac, Cdc42, and Rho (7,8). This suggests the possibility that ␤␥ regulates Rho family GTP-binding proteins, leading to production of stress fibers and focal adhesions. However, microinjection of ␤␥ into quiescent Swiss 3T3 fibroblasts did not induce stress fiber formation (2). Furthermore, co-transfection of ␤ 1 with ␥ 2 , ␥ 5 , or ␥ 7 into CV-1 (9) and NIH 3T3 fibroblasts (10) did not remarkably change the stress fibers in medium containing serum, although a slight decrease was observed. In contrast to these slight effects of ␤␥ subunits, co-transfection of ␥ 12 with ␤ 1 into NIH 3T3 cells induced cell rounding, disruption of stress fibers, and enhancement of cell migration associated with specific phosphorylation of ␥ 12 by protein kinase C (10,11).
In this study, we transfected ␤␥ subunits into epithelial-like HeLa cells. Contrary to the results with fibroblasts, ␤␥ as well as G␣ 12 induced stress fiber formation and focal adhesion assembly. We show here that ␤␥ subunits regulate Rho-dependent actin polymerization in HeLa cells.

EXPERIMENTAL PROCEDURES
Materials-cDNAs of several ␥ isoforms were prepared using synthetic polymerase chain reaction primers. cDNAs of bovine ␤ 1 and ␥ 2 were generously provided by M. I. Simon (California Institute of Technology) and T. Nukada (Tokyo Institute of Psychiatry), respectively, and subcloned into pCMV as described previously (12). cDNAs of RhoA and Rac1 were kindly provided by K. Kaibuchi (Nara Institute of Science and Technology) and Cdc42Hs cDNA by R. A. Cerione (Cornell University). All cDNAs of G protein subunits, FLAG-tagged dominant negative mutants of small GTP-binding proteins, and a C-terminal fragment (amino acids 495-689) of the ␤-adrenergic receptor kinase (13) were subcloned into the pCMV5 vector as described previously (8,12). The plasmid for the C3 exoenzyme was kindly provided by S. Narumiya (Kyoto University) (14), and that for the ␦-opioid receptor was provided by C. Evans (UCLA) (15). Antibodies against the ␤ and G␣ i2 subunits, generated by ourselves, have been described previously (16). Antibodies against G␣ 12 , G␣ s , G␣ q/11 , and phosphotyrosine were obtained from Santa Cruz Biotechnology. Phospho-specific stress-activated protein kinase/c-Jun N-terminal kinase antibody was obtained from New England Biolabs, Inc. Mouse monoclonal antibody against vinculin was purchased from Sigma. Y-27632 (17) was supplied by Yoshitomi Pharmaceutical Industries.
Transfection and Staining-HeLa cells were grown in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum. Transient transfection was performed using LipofectAMINE Plus according to the manufacturer's instructions (Life Technologies, Inc.). The medium was replaced 24 h after transfection, and the cells were starved in serum-free medium for 24 h and then fixed in 4% paraformaldehyde in phosphate-buffered saline. Before fixation, transfected cells were treated with 20 M Y-27632 for 2 h (17), with 150 M tyrophostin A25, 10 M tyrophostin AG 1478, 10 M PP2, 30 g/ml genistein, 1 M Ro31-8220, 100 nM wortmannin or 50 M LY-294002 for 3 h, or with 1 * This work was partly supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and by CREST. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
M phorbol 12-myristate 13-acetate for 24 h. Fixed cells were immunostained using antibodies against ␣, ␤, and vinculin followed by secondary antibodies, fluorescein isothiocyanate-conjugated goat anti-rabbit IgG, and tetramethylrhodamine isothiocyanate-conjugated goat antimouse IgG, as described earlier (18). The cells were also stained for F-actin with tetramethylrhodamine isothiocyanate phalloidin (Sigma). After applying coverslips, slides were examined under a laser scanning microscope (FLUOVIEW Olympus) equipped for fluorescence. All experiments were performed at least three times with similar results. Fig. 1 and 2 illustrate the effects of transient co-expression of ␤ 1 with various ␥ subunits, including ␥ 2 , ␥ 5 , ␥ 7 , and ␥ 12 in quiescent HeLa cells. Combinations of ␤ 1 with all individual ␥ subunits stimulated the formation of thick stress fibers (Fig. 1) as well as focal adhesion assembly, as assessed by localization of vinculin at the leading edges and middle body in cells (Fig.  2). Such stress fibers and focal adhesions were not observed in expression-negative cells, which were seen among surrounding transfected cells (Figs. 1 and 2). Expression of the GTPasedeficient G␣ 12 subunit (G␣ 12 Q229L) also induced actin stress fiber and focal adhesion assembly (Fig. 3, A, B, I, and J) as previously observed in Swiss 3T3 cells (2). However, the other GTPase-deficient ␣ subunits, G␣ i2 (G␣ i2 Q205L), G␣ 11 , (G␣ 11 Q209L), and G␣ s (G␣ s Q227L), were without such effects (Fig. 3, C-H), although G␣ 11 Q209L appeared to induce the formation of thin actin fibers because of bright staining of transfected cells with rhodamine phalloidin (Fig. 3, E and F). Activated G␣ i2 and G␣ 11 were also ineffective in Swiss 3T3 cells (2,3).

RESULTS AND DISCUSSION
In contrast with G␣ 12 Q229L subunits, it was reported that microinjection of ␤␥ into Swiss 3T3 cells (2) and transfection of ␤␥ into NIH 3T3 cells (10) did not stimulate stress fiber forma- tion. In addition, NIH 3T3 cells, which were transfected with various ␤␥ subunits and cultured in the same conditions as HeLa cells in this study, did not induce stress fiber formation (data not shown). Therefore, the different responses to ␤␥ observed in HeLa cells and fibroblasts seem to be cell type differences rather than experimental differences.
Expression of ␤ 1 or ␥ 2 alone failed to stimulate the formation of actin stress fibers (data not shown), suggesting that ␤␥ complexes are necessary for this purpose. To test whether the ␤␥ complex is indeed involved in induction of stress fiber formation and focal adhesion assembly, we co-expressed G␣ i2 and the C-terminal fragment of the ␤-adrenergic receptor kinase, both of which are expected to bind and sequester free ␤␥, and showed this to prevent the ␤ 1 ␥ 2 -induced actin stress fiber formation (Fig. 4, C and E) and focal adhesion assembly (Fig. 4, D and F). In contrast, G␣ i2 and the C-terminal fragment of the ␤-adrenergic receptor kinase did not prevent G␣ 12 Q229L-induced stress fiber formation and focal adhesion assembly (data not shown).
It is well known that actin stress fiber formation and focal adhesion assembly are induced by activation of Rho in several cells and tissues (1). To determine whether the effects of ␤␥ in HeLa cells were Rho-dependent, cells were co-transfected with ␤ 1 ␥ 2 and dominant negative mutants of Rho family GTP-binding proteins. As shown in Fig. 5 (A and B), co-transfection of RhoT19N completely inhibited ␤␥-induced stress fiber formation and focal adhesion assembly. In contrast, co-transfection of dominant negative mutants of other Rho family GTP-binding proteins, Rac (RacT17N) and Cdc42 (Cdc42T17N), was without effect (Fig. 5, C-F). These dominant negative mutants seemed to be functional, because RacT17N and Cdc42T17N diminished the phosphorylation of c-Jun N-terminal kinase induced by LPA in HeLa cells (19) when the phosphorylation was determined by immunoblotting with phospho-specific c-Jun N-terminal kinase antibody (data not shown). G␣ 12 Q229L-induced stress fiber formation and assembly of focal adhesion was also inhibited by co-transfection of RhoT19N (data not shown). To confirm the Rho dependence, cells were co-transfected with ␤ 1 ␥ 2 and the botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho, or ␤ 1 ␥ 2 -transfected cells were treated with Y-27632, a Rho-associated protein kinase (p160ROCK) inhibitor (17). In both cases, actin stress fiber formation and focal adhesion assembly were prevented (Fig. 5, G-J). These results clearly demonstrated that ␤␥ subunits regulate stress fiber formation and assembly of focal adhesion in a Rho and p160ROCK-dependent manner. The stimulation by expression of G␣ 12 Q229L was also prevented by C3 exoenzyme co-transfection and Y-27632 treatment (data not shown).
A previous study showed the tyrosine kinase inhibitor tyrophostin A25 to inhibit the formation of stress fibers stimulated by LPA but not by constitutively active Rho in quiescent Swiss 3T3 cells, indicating the existence of a protein-tyrosine kinase acting in the LPA pathway upstream of Rho (20). Another study showed that tyrophostin A25 and tyrophostin AG 1478 inhibit the formation of stress fibers stimulated by constitutively active G␣ 13 Q226L but not by G␣ 12 Q229L (3). Therefore, we examined the effects of several kinds of tyrosine kinase inhibitors on ␤ 1 ␥ 2 -transfected cells. Tyrophostin A25 (Fig. 6, C and D) and tyrophostin AG 1478 (data not shown) did not significantly influence the stress fiber formation and focal adhesion assembly induced by ␤␥ or G␣ 12 Q229L. Similarly, a selective inhibitor of the Src family of protein tyrosine kinases PP2 did not inhibit ␤␥-induced stress fiber formation (data not shown). Although these tyrosine kinase inhibitors did not influence stress fiber formation, they effectively inhibited tyro- sine kinases in the cells; tyrophostin A25 and PP2 decreased tyrosine phosphorylation of focal adhesion kinase-like protein (about 125 kDa) stimulated by G␣ 12 Q229L when phosphorylation was determined by antibody against phosphotyrosine (data not shown). Unlike these tyrosine kinase inhibitors, genistein blocked the formation of actin stress fibers stimulated by both ␤␥ and G␣ 12 Q229L (Fig. 6, E and F). These results suggested that genistein-sensitive but not tyrophostin-sensitive or Src-like tyrosine kinases are involved in the signaling with ␤␥ and G␣ 12 Q229L-induced Rho-dependent stress fiber formation.
Because ␤␥ subunits can stimulate phospholipase C-␤, which results in up-regulation of protein kinase C (21,22), we examined whether protein kinase C activation was required for the formation of actin stress fibers by ␤␥. However, down-regulation of endogenous protein kinase C by a 24-h exposure of cells to 1 M phorbol 12-myristate 13-acetate or treatment of cells with protein kinase C inhibitor Ro31-8220 for 3 h before fixation did not significantly inhibit ␤␥-induced stress fiber formation (Fig. 6, G and H).
There have been several reports that G protein-coupled receptors, including LPA and muscarinic receptors, are linked with phosphatidylinositol 3-kinase in a ␤␥-dependent fashion in cells (23,24). To examine whether activation of phosphatidylinositol 3-kinase was required for ␤␥-induced stress fiber formation, the cells were treated with the phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002 for 3 h before fixation. No inhibition was observed (Fig. 6, I and J). To verify that these inhibitors for protein kinase C and phosphatidylinositol 3-kinase and protein kinase C down-regulation actually inhibited the respective pathways, we determined effects of these treatments on the phosphorylation of c-Jun N-terminal kinase induced by LPA (25,26). All inhibitors and protein kinase C depletion diminished the phosphorylation of c-Jun N-terminal kinase (data not shown), indicating that these treatments effectively inhibited these signaling pathways.
It is generally accepted that the ␤␥-mediated signal pathway mainly acts through G i/o -coupled receptors, and when cells were transiently transfected with cDNA encoding the ␦-opioid receptor and stimulated by (D-Ala 2 ,D-Leu 5 )enkephalin, stress fiber formation and focal adhesion assembly were induced (data not shown). However, co-transfection with the C-terminal fragment of the ␤-adrenergic receptor kinase or treatment with pertussis toxin did not significantly block these effects. Thus ␦-opioid receptors may couple not only with G i/o but also with G 12 and G 13 , for whose ␣ subunits more effectively stimulate Rho than ␤␥ subunits in HeLa cells.
The present study clearly demonstrated that ␤␥ subunits, like G␣ 12 , regulate Rho-dependent actin polymerization, resulting in stress fiber formation and focal adhesion assembly in HeLa cells. Some recent reports have indicated that G␣ 12 and G␣ 13 are able to bind directly to p115-RhoGEF (4) or PDZ-RhoGEF (5) and that G␣ 13 but not G␣ 12 stimulates the GDP-GTP exchange reaction of p115-RhoGEF (4). Therefore, it is possible that ␤␥ subunits also directly interact with RhoGEF. In the budding yeast Saccharomyces cerevisiae, the ␤␥ complex has been shown to associate with Cdc24, a GEF for Cdc42, suggesting a cascade from ␤␥ to actin organization via Cdc42 (27,28). The large number of RhoGEFs so far found share Dbl and pleckstrin homology domains (29). Some members of the family also have Src homology 2, Src homology 3, GTPaseactivating protein, RasGEF ,and/or serine/threonine kinase domains, suggesting that they may interact with various molecules. In addition, analyses of the expression of RhoGEF family members have revealed that most are subject to varying degrees of tissue restriction (29). The different cell-specific responses to ␤␥ subunits observed in HeLa and NIH 3T3 cells may thus result from differential expression of the RhoGEF regulated by ␤␥ in these cells.