p21-activated Kinase 2 (PAK2) Inhibits TGF-� Signaling inMadin-Darby Canine Kidney (MDCK) Epithelial Cells byInterfering with the Receptor-Smad Interaction*

Received for publication, January 29, 2012, and in revised form, March 2, 2012 Published, JBC Papers in Press, March 5, 2012, DOI 10.1074/jbc.M112.346221

Xiaohua Yan‡1,2, Junyu Zhang‡1, Qinyu Sun‡, Polygena T. Tuazon§, Xiaoping Wu¶, Jolinda A. Traugh§,and Ye-Guang Chen‡3

From the ‡State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua-Peking Center for Life Sciences, Schoolof Life Sciences, Tsinghua University, Beijing 100084, China, the §Department of Biochemistry, University of California at Riverside,Riverside, California 92521, and the ¶Department of Biotechnology, Nanchang University, Nanchang 330031, China

Background: PAK2 is a mediator of TGF-� in mesenchymal cells. However, whether PAK2 could modulate TGF-� signal-ing remains elusive.Results: PAK2 associates with Smad2/3 and phosphorylates Smad2 at Ser417, thus inhibiting the TGF-�-induced Smad2-T�RIassociation and signal transduction in MDCK cells.Conclusion: PAK2 inhibits TGF-� signaling in MDCK epithelial cells.Significance: This study unravels a novel regulatory mechanism of R-Smad activity.

TGF-� (transforming growth factor �) plays a variety of cel-lular functions mainly through the Smad pathway. Phosphory-lation of the carboxyl SXSmotif inR-Smads (Smad2 andSmad3)by the type I receptor T�RI is a key step for their activation. Ithas been reported that the serine/threonine kinase PAK2 (p21-activated kinase 2) can mediate TGF-� signaling in mesenchy-mal cells. Here, we report that PAK2 restricts TGF-�-inducedSmad2/3 activation and transcriptional responsiveness inMDCK epithelial cells. Mechanistically, PAK2 associates withSmad2 and Smad3 in a kinase activity-dependent manner andblocks their activation. PAK2phosphorylates Smad2 at Ser-417,which is adjacent to the L3 loop that contributes to the T�RI-R-Smad association. Consistently, substitution of Ser-417 withglutamic acid attenuates the interaction of Smad2 with T�RI.Together, our results indicate that PAK2 negatively modulateTGF-� signaling by attenuating the receptor-Smad interactionand thus Smad activation.

Transforming growth factor-� (TGF-�)4 is a pleiotropiccytokine regulating diverse cellular processes in a cell type- andcontext-dependent manner (1–5). To achieve this, TGF-� elic-its multiple signaling pathways, and the R-Smads (receptor-

regulated Smad proteins) (Smad2 and Smad3 for TGF-� signal-ing)-mediated canonical pathway is involved in most ofTGF-�-mediated functions (6–10). TGF-� initiates signaltransduction via binding to a pair of serine/threonine kinasereceptors, the type II receptor (T�RII), and the type I receptor(T�RI), which is phosphorylated by T�RII for activation. T�RIthen activates Smad2 and Smad3 through phosphorylation atthe farmost C-terminal SXS motif, leading to their oligomeri-zation with Co-Smad (common Smad, Smad4), nuclear trans-location and transcriptional activation.R-Smad phosphorylation by T�RI serves as a key step for

Smad activation and requires the direct association between thereceptor and Smads. The L45 loop of T�RI and the L3 loop ofR-Smads were shown to determine the specificity of this inter-action (10–14). R-Smads have been reported to be phosphoryl-ated at various sites by other kinases, such asMAPKs (mitogen-activated protein kinases), CDK2/4 (cyclin-dependent kinase2/4), GSK-3� (glycogen synthase kinase 3�), and ROCK (Rho-associated protein kinase), which finely regulate the activities ofR-Smads and integrate different signal inputs (reviewed in Refs.15, 16).The serine/threonine kinase p21-activated kinase 2 (PAK2)

is a member of the group I PAK family (17–19). At the N ter-minus, it contains a Cdc42/Rac1 GTPase binding domain andan auto-inhibitory domain, which binds to the C-terminalkinase domain and keeps it at an inactive state. Binding ofCdc42/Rac1 to PAK2 changes its conformation, relieves theauto-inhibition and frees the kinase domain, resulting in itsactivation. In addition, apoptotic stimuli such as DNA damagelead to caspase-mediated cleavage of PAK2, generating a con-stitutively active kinase fragment p34 and an N-terminal regu-latory fragment p27 (20, 21). Several substrates of PAK2 havebeen identified in various contexts and mediate its wide rangeof cellular events, from cytoskeletal rearrangements to survivalpromotion and cellular transformation (19, 22). Interestingly,PAK2 was reported to mediate TGF-� signaling in mesenchy-

* This work was supported by the China Postdoctoral Science Foundationand the National Natural Science Foundation of China (NSFC,31171340) (to X. Y.), NSFC (30930050, 30921004), and the 973 Program(2010CB833706, 2011CB943803) (to Y. G. C.).

1 Both authors contributed equally to this work.2 To whom correspondence may be addressed: School of Life Sciences, Tsin-

ghua University, Beijing 100084, China. Tel.: 86-10-62784794; Fax: 86-10-62794376. E-mail: yanxiaohua@tsinghua.org.cn.

3 To whom correspondence may be addressed: School of Life Sciences, Tsin-ghua University, Beijing 100084, China. Tel.: 86-10-62795184; Fax: 86-10-62794376; E-mail: ygchen@tsinghua.edu.cn.

4 The abbreviations used are: TGF-�, transforming growth factor-�; PAK2,p21-activated kinase 2; T�RI, type I TGF-� receptor; T�RII, type II TGF-�receptor; R-Smad, receptor-regulated Smad protein; WT, wild-type; ca,constitutively active; KR, kinase-deficient; IP, immunoprecipitation; IB,immunoblotting; WCL, whole cell lysates.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 17, pp. 13705–13712, April 20, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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mal cells, but not in epithelial cells (23–25). The cell type-spe-cific activation of PAK2 by TGF-� is controlled by the tumorsuppressor Merlin and an epithelial-specific protein Erbin,both of which form a complex and inactivate PAK2 throughblocking Cdc42/Rac1 binding in epithelial cells (26).Although the canonical Smad pathway was shown not to be

affected by PAK2 in fibroblasts (23), it remains unclear whetherPAK2 has any effect in other types of cells. In the present study,we report that PAK2 restricts TGF-�-elicited phosphorylationand transcriptional activities of R-Smads in madine-darbycanine kidney (MDCK) epithelial cells. PAK2 associates withR-Smads in a kinase activity-dependent manner, but the asso-ciation is not influenced by TGF-� signaling. In addition, invitro phosphorylation in combination with mass spectrometricanalysis revealed that Ser417 in Smad2 is phosphorylated byPAK2, and its phosphorylation controls the TGF-�-inducedT�RI-Smad2 association.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—MDCK cells, human embry-onic kidney (HEK) 293T cells, HEK293 cells, normal murinemammary gland (NMuMG) epithelial cells, human keratino-cyte HaCaT cells, and NIH-3T3 fibroblasts were grown inDMEM (Dulbecco’sModified EagleMedia) supplementedwith10% fetal bovine serum (FBS) at 37 °C in a humidified, 5% CO2incubator. Transfection was accomplished with calcium phos-phate for HEK293T cells, and VigoFect (Vigorous Biotechnol-ogy, Beijing, China) or Lipofectamine 2000 (Invitrogen) forothers.Plasmids and Antibodies—HA-tagged wild-type (WT) and

kinase-deficient (K278R/KR or T402A/TA) PAK2 constructswere described previously (21). The plasmid encoding GST-fused wild-type PAK1 was a gift from Dr. Zhijun Luo (BostonUniversity, School of Medicine). Constitutively active PAK2(T402E) construct was generated by PCR-based site-directedmutagenesis. Various tags (Myc-, HA-, and Flag-) were addedto the N termini of wild-type or mutant PAK2, which are thensubcloned into pcDNA3.1(�). The HA-tagged constitutivelyactive (ca-) T�RI (L193G, P194G, T204D) construct was gen-erated by site directed mutagenesis. Other plasmids are pre-served in the laboratory. Anti-Flag antibody (M2) was pur-chased from Sigma, and other antibodies were from Santa CruzBiotechnology.Reporter Assay, Immunoprecipitation, and Immunoblotting—

Reporter assay, immunoblotting, and immunoprecipitation wereperformed as described previously (27). Reporter assays were per-formed in triplicate, and the data are presented as means � S.D.after normalization to Renilla activity.In Vitro Phosphorylation and Mass Spectrometric Analysis—

In vitro phosphorylation was carried out using 2 �g of purifiedHis-Smad2(Linker�MH2, 185–467aa) or 1 �g of purified GST-Smad2 (full-length) proteins in the presence of recombinantcaspase 3 in a final volume of 20 �l of phosphorylation reactionbuffer (20mMTris-HCl (pH7.5), 10mMMgCl2, 1 mMDTT, 1 �M

[�-32P]ATP (2,000 cpm/pmol)) for 30 min at room temperature.PhosphorylatedproteinswereanalyzedbySDS-PAGEfollowedbyautoradiography. Then the phosphorylated proteins were excisedfrom the gel and subjected to in-gel digestion with diphenylcar-

bamoyl chloride-treated trypsin, and finally the MALDI-TOFspectrometric analysis as described (28).RNA Isolation and Quantitative RT-PCR—Total RNA was

prepared with Trizol reagent (Invitrogen) and cDNA was syn-thesized from 1 �g of RNAwith Revertra Ace (Toyobo). Quan-titative RT-PCR was performed by the SYBR Green detectionmethod with the Mx3000p Quantitative PCR system (Strat-agene). The primers used were as follows: for dog GAPDH, 5�-GGTCATCATCTCTGCTC CTTC-3� and 5�-GATGCCTGC-TTCACTACCTTC-3�; for dog Smad7, 5�-CCAACTGCAGA-CTGTCCAGA-3� and 5�-TTCTCCTCCCAGTATGCCAC-3�;for dog c-myc, 5�-AGAAGTGACCCAGCCATCGTT-3� and5�-CTCTTCATAGTCCAACACCTG-3�; and for dog Bim, 5�-GCTGTCTCGATCCTCCAGTG-3� and 5�-ATTAAATTCG-TCTCCAATACG-3�. Gene expression was normalized againstGAPDHmRNA. Each sample wasmeasured in triplicates. Datawere analyzed using Microsoft Excel.

RESULTS

PAK2 Inhibits TGF-�-driven Transcriptional Responses inMDCK Epithelial Cells—To investigate whether PAK2 couldaffect TGF-�-driven transcription in epithelial cells, we exam-ined the effect of PAK2on the expression of theTGF-�-respon-sive reporters ARE-luciferase and CAGA-luciferase. As shownin Fig. 1A, TGF-�1 treatment ofMDCK epithelial cells inducedARE-luciferase expression, and overexpression of wild-type(WT)PAK2 attenuated this induction, implicating that PAK2 isan antagonist for TGF-�-driven transcription. Expression ofconstitutively active PAK2mutant (T402E,TE) showed a stron-ger inhibition while the kinase-deficient PAK2mutant (K278R,KR) had no effect, suggesting that this inhibitory effect of PAK2requires its kinase activity. Similar results were obtained withCAGA-luciferase (Fig. 1B). PAK2 also inhibited the constitu-tively active TGF-� type I receptor (ca-T�RI)-driven expres-sion of ARE-luciferase and CAGA-luciferase (data not shown).Next we consolidated these findings by testing the effect of

Rac1, which is an upstream activator of PAK2, on TGF-�-in-duced expression of ARE-luciferase inMDCK cells. Like PAK2,wild-type Rac1 attenuated the reporter expression to someextent, and the constitutively active Rac1(V12) decreased it fur-ther, alone or in cooperation with PAK2 (Fig. 1C), whereas thedominant-negative Rac1(N17) enhanced the reporter expres-sion. Furthermore, IPA-3, an allosteric PAK inhibitor (29),enhanced the reporter expression in a dose-dependentmanner.Since Rac1 and IPA-3 could also act on PAK1, which is closelyrelated to PAK2,we examinedwhether PAK1 could affectTGF-�-induced transcriptional response. As shown in Fig. 1D, bothPAK2 and Smad7 were able to attenuate TGF-�-driven expres-sion of CAGA-luciferase reporter, whereas PAK1 had littleeffect, suggesting a specific inhibitory effect of PAK2. Theseresults altogether suggested that PAK2 negatively regulatesTGF-� signaling inMDCK cells, and this inhibition depends onits kinase activity.To examine whether PAK2 has a more general inhibitory

effect on TGF-�-driven transcription, we carried out CAGA-luciferase reporter assays in other cell lines. PAK2 attenuatedthe reporter expression in HEK293, NMuMG, andHaCaT cells

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in varying degrees, but showed no effect in NIH3T3 fibroblasts(Fig. 1E).PAK2 Inhibits TGF-�-induced R-Smads Activation and

Signaling—Having showed the inhibitory effect of PAK2 onTGF-�-induced transcription in MDCK cells, we continued toinvestigatewhether PAK2 affects TGF-�-induced Smad2 phos-phorylation, using an antibody specifically recognizing thephosphorylated carboxyl S465MS467 motif of Smad2. As shownin Fig. 2A, both of wild-type and TE mutant PAK2 decreasedTGF-�-induced phospho-Smad2 levels, but PAK2(KR) had lit-tle effect. Consistently, IPA-3 treatment enhanced the phos-pho-Smad2 level (Fig. 2B).Next we testedwhether PAK2 regulates the R-Smads/Smad4

interaction. Constructs encoding GST-fusion Smad4, Flag-tagged Smad2, HA-tagged ca-T�RI, and Myc-tagged PAK2

(WT, TE and KR) were transfected into HEK293T cells as indi-cated in Fig. 2C. At 40 h post-transfection, the cells were har-vested for GST pulldown followed by anti-Flag immunoblot-ting (IB). Both WT- and TE-PAK2, but not KR-PAK2,attenuated the Smad2-Smad4 interaction. In addition, animmunoprecipitation-immunoblotting assay showed thatPAK2 inhibited Smad3-Smad4 oligomerization dependentlyon the kinase activity (Fig. 2D). These results demonstrated thatPAK2 interferes with Smad2 activation and attenuates TGF-�signaling in MDCK epithelial cells.PAK2 Associates with Smad2/3 in a Kinase Activity-depen-

dent Manner—As PAK2 attenuated TGF-�-induced Smad2phosphorylation and subsequent Smad2-Smad4 complex for-mation, it is likely to function at or upstream of the Smad level.To explore the underlying mechanism, we attempted to test

FIGURE 1. PAK2 inhibits TGF-�-mediated transcriptional induction in MDCK epithelial cells. A and B, PAK2 inhibits TGF-�-induced expression of reportergenes. MDCK cells transfected with constructs encoding reporters ARE-luciferase (ARE-Luc, 200 ng)/FAST2 (150 ng) or CAGA-luciferase (CAGA-Luc, 200 ng)together with Renilla-luciferase (20 ng), wild-type (WT), constitutively active (T402E, TE), or kinase-deficient (K278R, KR) PAK2 (100 ng) were treated with orwithout 100 pM TGF-�1 for 20 h, and then harvested for luciferase activity measurements. C, effects of Rac1 and IPA-3 on TGF-�-mediated expression ofARE-luciferase reporter. Plasmids encoding ARE-luciferase/FAST2, Renilla-luciferase, PAK2 (100 ng) and wild-type or mutant Rac1 (50 ng) were transfected intoMDCK cells. At 20 h post-transfection, the cells were treated with or without 100 pM TGF-�1 and IPA-3 as indicated and harvested for luciferase activitydeterminations. D, specificity of PAK2 in inhibiting TGF-�-induced expression of CAGA-luciferase reporter. Plasmids encoding PAK1 (100 ng), PAK2 (100 ng), orSmad7 (50 ng) were transfected into MDCK cells for the CAGA-luciferase reporter assay. E, cell type specificity of PAK2 in antagonizing TGF-�-inducedtranscriptional activities. HEK293, NMuMG, HaCaT, or NIH3T3 cells were transfected with plasmids encoding CAGA-luciferase (200 ng), Renilla-luciferase (20ng), PAK2 (50 or 100 ng), and Smad7 (50 ng) as indicated above. At 20 h post-transfection, the cells were treated for another 20 h with 100 pM TGF-�1 andharvested for determination of luciferase activity. * indicates p value is lower than 0.05. In all of the reporter assays, empty vectors were used to equalize the totalamounts of plasmids in each sample. Each experiment was performed in triplicate, and the data are presented as means � S.D. after normalization to Renillaactivity.

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whether PAK2 associates with components of the TGF-�/Smad pathway. It was showed that PAK2 does not associatewith TGF-� receptors T�RI or T�RII (23), thus we examined ifPAK2 interacts with Smad proteins. GST pulldown assay inHEK293T cells revealed that only Smad2 and Smad3 interactedwith PAK2 (Fig. 3A). This interaction was further confirmed atthe endogenous protein level inMDCK cells (Fig. 3B). Blockingof PAK2 kinase activity with IPA-3 completely abolished theirinteraction, while TGF-� treatment had no effect, suggestingthe interaction is regulated by the PAK2 kinase but not byTGF-�.GST pulldown assay also showed the kinase-active

PAK2(TE) had a stronger interaction with Smad2 in compari-son to wild-type PAK2, while the kinase-deficient mutantsK278R (KR) and T402A (TA) dramatically decreased the inter-action. Co-expression of constitutively active Rac1(V12) alsostrengthened the PAK2-Smad2 interaction, whereas domi-nant-negative Rac1(N17) and IPA-3 diminished their binding(Fig. 3D), indicating the kinase activity of PAK2 is essential forits binding to Smad2. A similar experiment showed the kinaseactivity was also required for PAK2 to associate with Smad3(Fig. 3E).The effect of TGF-� signaling on the PAK2-Smad2/3 inter-

action was then examined by co-immunoprecipitation experi-ments in HEK293T cells. As shown in Fig. 3F, the interactionbetween PAK2 and Smad2 or Smad3 were at the similar levelsin the presence of the constitutively active (CA) and wild-typeT�RI, which is consistent with the above observation that

TGF-� had no effect on this interaction. These results togethersupported the notion that the PAK2-Smad2/3 interactions areinduced by the kinase activity of PAK2, but not affected byTGF-� signaling.PAK2 Phosphorylates Smad2 at Ser417 and the Phosphoryla-

tion Blocks TGF-�-induced Smad2 Activation—The notionthat PAK2 associates with R-Smads in a kinase activity-depen-dent manner suggests a kinase-substrate relationship. To studywhether PAK2 could phosphorylate Smad2 directly, His-Smad2 (Linker-MH2, 185–467aa) and GST-Smad2 (full-length, FL) were expressed and purified from Escherichia colicells and used in an in vitro kinase assay as substrates for PAK2,in the presence of recombinant caspase 3, which is an activatorfor PAK2 (20). As shown in Fig. 4A, PAK2 could phosphorylateSmad2 and this phosphorylation was greatly enhanced bycaspase 3. To identify the phosphorylation sites, PAK2 phos-phorylated Smad2 proteins were subjected to MALDI-TOFspectrometric analysis. Identified peptides corresponded tophophorylated CS464S465MS467 (with one or two phosphates)and MS417FVK (Fig. 4B).

It is intriguing that PAK2might phosphorylate serines in theCS464S465MS467motif of Smad2, of which the Ser465/467 resi-dues are known to be phosphorylated by T�RI (30, 31). How-ever, overexpression of WT- or TE-PAK2 could not phos-phorylate these two serine residues in MDCK cells (Fig. 2A). Inaddition, mimicking phosphorylation of Ser464 by mutating itto glutamic acid (E) did not affect Smad2 C-terminal phos-phorylation by T�RI (data not shown).

FIGURE 2. PAK2 inhibits TGF-�-induced R-Smad phosphorylation and signal transduction. A, PAK2 attenuates TGF-�-induced Smad2 phosphorylation.MDCK cells expressing ectopic PAK2 (WT, TE, KR) proteins were treated as indicated with 100 pM TGF-�1 for 1 h, and then harvested for immunoblotting (IB)examination. B, effect of IPA-3 on TGF-�-induced Smad2 activation. MDCK cells with a confluence of over 80% were treated with or without 100 pM TGF-�1 andIPA-3 for 1 h and harvested for immunoblotting. C, PAK2 inhibits the Smad2-Smad4 interaction in a kinase activity-dependent manner. HEK293T cellstransfected with plasmids encoding GST-Smad4, Flag-Smad2, ca-T�RI-HA and WT-, TE-, or KR-PAK2 were harvested at 40 h post-transfection and subjected forGST pulldown using glutathione-Sepharose beads followed by anti-Flag immunoblotting (IB). D, PAK2 inhibits Smad3-Smad4 association in HEK293T cells.HEK293T cells expressing ectopic proteins as indicated were treated with 100 pM TGF-�1 for 2 h before being harvested and subjected to anti-HA immuno-precipitation (IP) followed by anti-Myc immunoblotting (IB). In GST pulldown or co-IP experiments, protein expression in whole cell lysates (WCL) wasexamined by immunoblotting. Band intensity (Fold Change) was analyzed by BandScan 5.0.

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To study the function of Smad2 Ser417 (Smad3 Ser375) phos-phorylation, this serinewasmutated to alanine (A) or E tomakeit resistant to phosphorylation or tomimic phosphorylation. Asshown in Fig. 4C, ca-T�RI induced the carboxyl phosphoryla-tion ofWT-Smad2 and S417Amutant to a similar extent, whileS417E mutation completely abolished this phosphorylation.S417E mutation also decreased the ca-T�RI-induced Smad2-Smad4 interaction to the basal level in HEK293T cells (Fig. 4D),and eliminated Smad2-mediated transcriptional induction inMDCK cells (Fig. 4E). Finally, we tested the expression of sev-eral TGF-� target genes, including Smad7, c-Myc, and Bim(32–36). As shown in Fig. 4F, wild-type Smad2/3 could stimu-late the expression of Smad7 and Bim and repress Myc expres-sion in cooperation with Smad4, but Smad2(S417E) andSmad3(S375E) lost the stimulatory ability in MDCK cells.Together, these results demonstrated Ser417 phosphorylationof Smad2 is critical for PAK2 to antagonize TGF-�/Smadsignaling.PAK2-mediated Ser417 Phosphorylation of Smad2 Interferes

with the T�RI-Smad2 Interaction—It is notable that Ser417locates just at the juncture of �10 sheet and the L3 loop inSmad2 (Fig. 5A), and the L3 loop contributes to and determinesthe specificity of the T�RI-Smad2 association (11). Thus wereasoned that phosphorylation of Ser417 may alter the confor-mation of the L3 loop, thereby affecting T�RI-Smad2 associa-tion. To test this hypothesis, HEK293T cell lysates containingoverexpressed kinase-deficient (K232R) T�RI-His and Flag-Smad2 were subjected to precipitation with nickel beads fol-lowed by anti-Flag immunoblotting, and T�RII was co-ex-pressed to activate T�RI and induce its interactionwith Smad2.As shown in Fig. 5B, co-expression of WT- or TE-PAK2, butnot KR-PAK2, decreased the T�RI-Smad2 interaction.

To study whether this effect was mediated by Smad2 Ser417phosphorylation, we compared the interaction between T�RIand wild-type or mutant Smad2. Accordantly, S417E mutationdiminished the interaction, while S417Amutation had no effect

(Fig. 5C). These data indicated that PAK2 interferes with theT�RI-Smad2 interaction by phosphorylating Smad2 at Ser417.

DISCUSSION

TGF-� regulates various aspects of cellular events, and thusplaying pivotal roles in different pathophysiological processes(1–3). Although the canonical Smad-mediated signaling is rel-atively simple, it is precisely controlled at different levels fromthe availability and activation of extracellular ligands, the activ-ity and stability of membrane receptors to the activity, stabilityand localization of Smad proteins in the cytoplasm or in thenucleus (15, 35, 37–39).PAK2 has been shown to be a mediator of TGF-� in a set of

mesenchymal cells, but not in epithelial cells (23, 24). More-over, TGF-�-induced activation of PI3K and fibroblasticresponses (such as enhanced proliferation and morphologicchanges) are mediated by PAK2 (40, 41), and it was alsoreported that TGF-�-activated PAK2 is required for Ras-de-pendent ERK phosphorylation and Elk-1-driven transcription(42). The fibroblast-specific activation of PAK2 by TGF-�could be due to the absence of an epithelial cell specific proteinErbin, which traps PAK2 in the Erbin-Merlin-PAK2 complex(26). However, whether PAK2 couldmodulate TGF-� signalingstays an open question.In this study, we provided evidence showing that PAK2

antagonizes TGF-�-elicited R-Smad activation, R-Smad-Smad4 oligomerization and transcriptional induction inMDCK epithelial cells. In addition, the inhibitory effect ofPAK2 was also observed in HEK293T, NMuMG and HaCaTcells in varying degrees, but not inNIH3T3 fibroblasts, suggest-ing this effect is context-dependent. Intriguingly, the inhibitoryeffect of PAK2 depends on its kinase activity, as overexpressionof a kinase-deficient (KR) mutant had little effect. Accordantly,blocking of endogenous PAK2 kinase activity through IPA-3 oroverexpression of a dominant-negative Rac1 mutant (N17)enhanced TGF-� signaling.

FIGURE 3. PAK2 associates with Smad2/3 in a kinase activity-dependent manner. A, PAK2 associates with Smad2/3. HEK293T cells overexpressing GSTfused-PAK2 and Flag-tagged Smad proteins were subjected to GST pulldown assay using glutathione-Sepharose beads followed by anti-Flag immunoblotting.B, endogenous PAK2-Smad2 association in MDCK cells. MDCK cells of over 80% confluence were treated with 100 pM TGF-�1 and 30 �M IPA-3 as indicated andharvested for co-IP assay. To shift the IgG(H) band, the protein A Sepharose-IgG isolated immuno-complex were diluted in DTT-free loading buffer andsubjected to SDS-PAGE gel analysis after moderate vortex without boiling. C–E, protein associations in HEK293T cells were assessed by GST pulldown assays asdescribed above. F, TGF-� signaling does not affect PAK2-Smad2/3 associations. Co-IP assay was carried out as described above.

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PAK2 associates with both Smad2 and Smad3 in a kinaseactivity-dependentmanner. In vitro kinase assay suggested thatPAK2 phosphorylates Smad2 at Ser417 or serine residues in theC-terminal SSXS region. However, overexpression of wild-typeor constitutively active PAK2 in MDCK cells could not phos-phorylate the C-terminal Ser465/467, and mimicking phos-phorylation of Ser464 did not affect Smad2 activation either.Instead, substitution of Ser417 with Glu completely abolished

TGF-�-induced Smad2 activation and signaling. In addition,Glu substitution of the corresponding serine (Ser375) in Smad3inhibited TGF-�-induced Smad3 phosphorylation and down-stream signaling (data not shown), and co-expression of bothSmad2/3 mutants destroyed Smad2/3/4-mediated target genetranscription, together demonstrating that Ser417/375 inSmad2/3 could be functionally targeted by PAK2 to modulateTGF-� signaling.

FIGURE 4. PAK2 phosphorylates Smad2 at Ser417 and blocks TGF-�-induced Smad2 activation and signaling. A, PAK2 phosphorylates Smad2 in vitro.His-Smad2 (Linker�MH2, 185– 467aa) and GST-Smad2 (full-length, FL) were purified from E. coli cells and subjected to an in vitro phosphorylation reaction inthe presence of recombinant PAK2, caspase 3 and [�-32P]ATP. The products were analyzed by running SDS-PAGE gel and the phosphorylated proteins wereidentified by autoradiography. B, 32P-labeled Smad2 proteins excised from the gel above were subjected to digestion with trypsin, followed by mass spectro-metric analysis. The phosphopeptides corresponding to the expected molecular weights are indicated. C, mimicking phosphorylation of Ser417 with glutamicacid blocks Smad2 activation by TGF-�. Ectopic Flag-Smad2 (WT, S417A, S417E) were immunoprecipitated from HEK293T cells using anti-Flag antibody andtested by anti-pSmad2 (Ser465/467) immunoblotting. D, Glu substitution of Ser417 inhibits the interaction of Smad2 with Smad4. HEK293T cells overexpressingFlag-Smad2 (WT, S417A, S417E), GST-fused Smad4, GST protein, and ca-T�RI-HA as indicated were subjected to GST pulldown assay followed by anti-Flagimmunoblotting. E, S417E mutation diminished the transcriptional induction ability of Smad2. Wild-type or mutant Smad2 (50 ng) together with Smad4 (50 ng)were transfected into MDCK cells for an ARE-luciferase reporter assay. F, Glu substitution of Ser417 of Smad2 and Ser375 of Smad3 results in loss of their abilitiesto regulate the expression of Smad7, c-Myc, and Bim. MDCK cells transfected with constructs as indicated were subjected to RNA isolation and quantitativeRT-PCR.

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Biochemical studies revealed that the interaction betweenthe L3 loop of R-Smads and the L45 loop of type I receptorsdetermines the specificity of the receptor-Smad interaction (11,12, 43). As Ser417 is adjacent to the L3 loop in Smad2, we rea-soned that its phosphorylation would affect the T�RI-Smad2association. Indeed, mimicking phosphorylation of Ser417 withGlu substitution abolished this interaction, coinciding with thenotion that PAK2 inhibition of the Smad2-T�RI interactionrequires its kinase activity.Besides T�RI and PAK2, R-Smads are also targeted by other

kinases to finely control their activities and TGF-� signaling.For instance, CDK2/4 were shown to phosphorylate Smad3 atThr8, Thr178, and Ser212 to control Smad3 activity in transcrip-tional induction and cell cycle regulation (44); GSK-3�-medi-ated phosphorylation of Smad3 modulates its stability at thebasal level (45), and GRK2 (G-coupled receptor kinase 2) phos-phorylates Smad2/3 to control their activities (46). Moreover,MAPKs-mediated R-Smad phosphorylation in the linkerregions has been extensively studied, to promote or inhibitTGF-� signaling depending on cell types (reviewed in Refs. 15,16). Other kinases reported include PKC, ROCK, CaMKII, andCK1�2. Phosphorylation of R-Smads by these kinases mayoccur independently or sequentially. Therefore, it will be inter-esting to study whether and how PAK2-mediated phosphory-lation of R-Smads integrates with phosphorylation induced byother kinases.

Acknowledgments—We thank Drs. Zhijun Luo and Wei Wu forreagents.

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Traugh and Ye-Guang ChenXiaohua Yan, Junyu Zhang, Qinyu Sun, Polygena T. Tuazon, Xiaoping Wu, Jolinda A.

InteractionKidney (MDCK) Epithelial Cells by Interfering with the Receptor-Smad

Signaling in Madin-Darby Canineβp21-activated Kinase 2 (PAK2) Inhibits TGF-

doi: 10.1074/jbc.M112.346221 originally published online March 5, 20122012, 287:13705-13712.J. Biol. Chem. 

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