whereas cells expressing GEF-H1-S885D/959D showed muchless activity to induce stress fiber formation (Fig. 4, C/D).PAR1b-induced Phosphorylation of GEF-H1 on S885 and

S959 Inhibits GEF-H1-mediated RhoA ActivationTo sub-stantiate the involvement of PAR1b in the suppression ofRhoA-GEF activity of GEF-H1 through phosphorylation,COS-7 cells were co-transfected with the T7-tagged PAR1bvector and Myc-tagged RhoA G17A vector together with theFlag-tagged GEF-H1 vector or Flag-tagged GEF-H1-S885A/S959A vector. Cell lysates were immunoprecipitated with ananti-Myc antibody and subjected to immunoblotting with ananti-Flag antibody. The results of the experiment revealed thatexpression of PAR1b reduced wild-type GEF-H1 binding affin-ity toward RhoA G17A. On the other hand, PAR1b did notaffect binding activity of GEF-H1-S885A/S959A toward RhoAG17A. Hence, PAR1b inhibits RhoA-GEF activity of GEF-H1by inducing phosphorylation at both S885 and S959 (Fig. 5A).Given this, we next wished to know the effect of PAR1b-medi-atedGEF-H1phosphorylation on stress fiber formation. To thisend, AGS cells were transfected with wild-type GEF-H1, GEF-H1-S885A or GEF-H1-S959A vector together with a wild-typePAR1b vector. As described above, expression of GEF-H1induced stress fiber formation, which was inhibited by co-ex-pression of PAR1b. In contrast, induction of stress fibers by theexpression of GEF-H1-S885A or GEF-H1-S959A mutant wasnot impaired by PAR1b co-expression (Fig. 5, B/C), consistentwith the conclusion that PAR1b-mediated phosphorylation ofboth S885 and S959 is required for inactivation of GEF-H1. Toconfirm that the change in actin stress fiber formation was due

to activation/inactivation of RhoA, we determined the levels ofactive RhoA by using a Rhotekin RBD pull-down assay. Asexpected, GEF-H1 expression increased the active form (GTP-bound form) of RhoA, which was significantly decreased uponco-expression of PAR1b. In contrast, the amount of activeRhoA in GEF-H1-S885A/S959A-expressing cells was notaffected by co-expressed PAR1b (Fig. 5D). These results collec-tively indicated that phosphorylation of GEF-H1 on both S885and S959 is necessary for PAR1b to regulate RhoA-mediatedstress fiber formation.Endogenous PAR1b Inhibits GEF-H1 through Phosphory-

lationGiven the above-described observations, we wished toknow if endogenous PAR1b regulates RhoA activity throughphosphorylation ofGEF-H1.To this end,we compared the acti-vation status of GEF-H1/RhoA signaling in AGS cells trans-fected with wild-type GEF-H1 or with GEF-H1-S885A/S959Atogether with PAR1b-specific siRNA (Fig. 6A). The RhoAG17A binding assay showed that the amount of active GEF-H1was increased when PAR1b was knocked down in wild-typeGEF-H1-expressing cells. In contrast, the amount of activeGEF-H1 in cells expressing GEF-H1-S885A/S959A was notaffected by PAR1b knockdown. In this experiment, we alsodetermined the amount of active RhoA by a Rhotekin RBDpull-down assay and found that the level of active RhoA wasincreased when PAR1b was knocked down in cells expressingwild-type GEF-H1 but not in cells expressing GEF-H1-S885A/S959A (Fig. 6B). Additionally, we compared the intensity ofstress fiber formation in AGS cells expressing wild-typeGEF-H1 and that in AGS cells expressing the GEF-H1-S885A/S959A mutant. The number of wild-type GEF-H1-expressingcells and that of GEF-H1-S885A/S959A-expressing cells show-ing stress fiber formation were not significantly different (datanot shown). However, the intensity of stress fiber formation,whichwas determined by F-actin fluorescence intensity in eachcell, was significantly greater in cells expressing GEF-H1-S885A/S959A than in cells expressing wild-type GEF-H1 (Fig.7,A/B). To know if the difference was due to differential degreeof GEF-H1 phosphorylation on S885 and S959 by endogenousPAR1b, we next compared themagnitude of stress fiber assem-bly in AGS cells expressing wild-type GEF-H1 in the presenceor absence of PAR1b-specific siRNA (Fig. 7,C/D). Hence, stressfiber formationwas substantially increased following inhibitionof endogenous PAR1b expression. From these observations, weconcluded that endogenous PAR1b-mediated phosphorylationof GEF-H1 on S885 and S959 represses RhoA-dependent stressfiber formation in cells.

DISCUSSION

In this work, we found that the polarity-regulating serine/threonine kinase PAR1b induces phosphorylation of RhoAactivator GEF-H1 on S885 and S959. This PAR1b-inducedGEF-H1 phosphorylation inhibits the RhoA-specific nucleo-tide exchange activity of GEF-H1, which results in the suppres-sion of actin stress fiber formation by RhoA.The molecular basis for the establishment and maintenance

of cell polarity remains largely unknown. Nevertheless, it is rea-sonable to assume that the organization of actin and microtu-bule cytoskeletons and the direction of vesicular transport

FIGURE 6. Endogenous PAR1b suppresses RhoA activity through GEF-H1phosphorylation. A, RhoA-GEF activity was analyzed by RhoA G17A affinitybinding assay. AGS cells co-transfected with PAR1b siRNA and indicatedGEF-H1 vectors were lysed, and Total cell lysates (TCLs) were pulled downwith Myc-RhoA G17A. Precipitates and TCLs were immunoblotted (IB) withindicated antibodies. B, RhoA activity was determined by a GST-RBD-Rho-tekin pull down assay. AGS cells co-transfected with PAR1b siRNA and theindicated GEF-H1 vector were lysed and subjected to precipitation with GST-RBD-Rhotekin beads. Precipitates and TCLs were immunoblotted (IB) withindicated antibodies.

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pathways are key events in this process. Our results support arole of PAR1b in the regulation of actin cytoskeleton via theGEF-H1/RhoA signaling pathway. Given that other PAR1members also interact with GEF-H1 (22, 23), it is likely that allof the PAR1 isoforms redundantly regulate GEF-H1. However,the observation that treatment of cells with PAR1b-specificsiRNA potentiates stress fiber assembly suggests that PAR1b isa major regulator of GEF-H1 among the PAR1 members inepithelial cells. The present study revealed that PAR1b phos-phorylates GEF-H1 to restrict RhoA activity below a certainthreshold, thereby ensuring an adequate level of stress fiberformation.GEF-H1 activity is controlled by two distinct mechanisms

(21). First, binding with microtubules in the cytoplasm inhibitsGEF-H1 activity. Second, phosphorylation regulates the nucle-otide exchange activity of GEF-H1. Surprisingly, GEF-H1 canundergo phosphorylation at 36 residues (including serine, thre-onine and tyrosine residues by PhosphoSite), indicating com-plicated regulationofGEF-H1activity bydifferential phosphor-ylation (27, 30). For instance, PAK-1 phosphorylatesGEF-H1 atS885 and this phosphorylation induces recruitment of GEF-H1to microtubules through binding to 14-3-3, which may alsoinhibit RhoA GEF activity of GEF-H1 (27). Early in mitosis,

AuroraA/B kinase and cyclin B1/Cdk1 phosphorylate S885 andS959, respectively, onGEF-H1 and thereby inhibit GEF-H1 cat-alytic activity (30). Dephosphorylation of GEF-H1 during telo-phase triggers RhoA activation, which provokes cleavage fur-row formation and ingression during cytokinesis. Theseobservations together with results of the present work suggestthat S885 and S959 behave as inhibitory switches of GEF-H1,which is targeted by multiple distinct kinases in a context-de-pendent manner. In contrast, ERK1 and ERK2 phosphorylateGEF-H1 at Thr-678 and this phosphorylation stimulates theability of GEF-H1 to activate RhoA (31).How isGEF-H1 inhibited by PAR1b? In the present study, we

obtained evidence that S885 is phosphorylated by PAR1b bydemonstrating that co-expression with PAR1b induced inter-action of GEF-H1 with 14-3-3, which specifically binds toGEF-H1 that has been phosphorylated on S885. Because 14-3-3binding causes conformational change of its target protein,modulates the activity of its target protein, and changes subcel-lular localization of its target protein (32), it is possible that thereduction of GEF-H1 catalytic activity by PAR1b may involve14-3-3 binding induced by S885 phosphorylation. However, asshown in Fig. 4, we found that a phosphomimic mutant ofGEF-H1 (S885D/S959D), which should not bind 14-3-3, lost

FIGURE 7. Endogenous PAR1b suppresses RhoA-mediated stress fiber formation via GEF-H1. AGS cells were transfected with the indicated vectors.A, confocal x-y plane views of F-actin and GEF-H1 staining. Arrows indicate CFP- or GEF-H1-positive cell. The boxed regions are shown at higher magnificationin the insets. Scale bar, 10 m. B, stress fiber formation was quantified by measuring F-actin fluorescence intensity per cell. Error bars represent mean S.D., n 3. **, p 0.01; *, p 0.05, Students t test. C, confocal x-y plane views of F-actin and GEF-H1 staining. Arrows indicate CFP-positive cells or GEF-H1-positive cells.The boxed regions are shown at higher magnification in the insets. Scale bar, 10 m. D, stress fiber formation was quantified by measuring F-actin fluorescenceintensity per cell. Error bars represent mean S.D., n 3. *, p 0.05, Students t test.

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RhoA-GEF activity. It therefore seems that S885/S959 phos-phorylation on its own is sufficient to inactivate GEF-H1.Whereas PAR1b can directly phosphorylate GEF-H1 on S885,the kinase that phosphorylates S959 upon PAR1b expression isnot known. At present, the possibility that PAR1b directlyphosphorylates S959 as well remains.The PAR1b-aPKC system is not only required for the estab-

lishment/maintenance of apical-basal polarity but also involvedin front-rear polarity (33, 34). However, the actual role ofPAR1b in front-rear polarity has yet to be elucidated. Our find-ing that PAR1b inhibits GEF-H1-mediated RhoA activationmay provide clues to this question. Given that RhoA is tran-siently activated during rear retraction (35), PAR1b-mediatedregulation of the GEF-H1/RhoA signaling pathway may indeedcontribute to the regulation of rear retraction. Notably, we pre-viously reported that H. pylori virulence factor CagA, which isdelivered into gastric epithelial cells, causes deregulation ofSHP2 phosphatase and at the same time inhibits the kinaseactivity of PAR1b, thereby inducing an extremely elongated cellshape termed the hummingbird phenotype. Given that thehummingbird phenotype is characterized by a rear retractiondefect (16, 36), themorphological changemay be due at least inpart to the loss of front-rear polarity as a result of PAR1binhibition byCagA.This notion is supported by the observationthat induction of the hummingbird phenotype by CagA iscounteracted by elevated PAR1b (16, 24).While this report was in preparation, Yoshimura and Miki

reported that PAR1b phosphorylates GEF-H1 on S143, S172,and S186 (37). This phosphorylation causes dissociation ofGEF-H1 from microtubules, which could activate GEF-H1.Their observation is inconsistent with the results of the presentstudy. However, we speculate that PAR1b has two distinct rolesin the regulation of GEF-H1 activity: it alters subcellular local-ization of GEF-H1 via N-terminal phosphorylation (S143/S172/S186) and at the same time it regulates nucleotideexchange activity of RhoA-specific GEF-H1 via C-terminalphosphorylation (S885 and S959). As a result, phosphorylationof GEF-H1 at multiple sites by PAR1b has both positive andnegative effects on GEF-H1 activity. We suspect that dissocia-tion of GEF-H1 from microtubules, which is caused via itsN-terminal phosphorylation by PAR1b, is required but not suf-ficient for the activation of GEF-H1 as long as both S885 andS959 are phosphorylated. In this scenario, stimulation of RhoAGEF activity of GEF-H1 additionally requires dephosphory-lation of GEF-H1 on S885 or S959 by cellular phosphatases.Our findings reveal a dual role of PAR1b in regulation of the

microtubule-based cytoskeletal system and the actin-basedcytoskeletal system. In particular, spatiotemporal regulation ofPAR1b/GEF-H1/RhoA signaling may play an important role inthe coordination of cell polarity, cell morphology, and cellmovement.

AcknowledgmentsWe thank Dr. Shigeo Ohno for providing anti-PAR1b antibody. We also thank Dr. Yukiko Gotoh for providing14-3-3 vector.

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Inhibition of GEF-H1/RhoA Signaling by PAR1b

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Murata-Kamiya and Masanori HatakeyamaYukie Yamahashi, Yasuhiro Saito, Naoko ReorganizationRhoA-dependent Actin CytoskeletalExchange Factor H1 (GEF-H1) to Regulate Phosphorylates Guanine NucleotidePartitioning-defective 1b (PAR1b) Polarity-regulating KinaseCell Biology:

doi: 10.1074/jbc.M111.267021 originally published online November 9, 20112011, 286:44576-44584.J. Biol. Chem.

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