synergistic activation of era by estrogen and prolactin in ... · that activates ga and gbg...

Molecular and Cellular Pathobiology

Synergistic Activation of ERa by Estrogen andProlactin in Breast Cancer Cells Requires TyrosylPhosphorylation of PAK1Peter Oladimeji, Rebekah Skerl, Courtney Rusch, and Maria Diakonova

Abstract

Serine/threonine kinase PAK1 is activated by estrogen andplays an important role in breast cancer. However, the integra-tion of PAK1 into the estrogen response is not fully understood.In this study, we investigated the mechanisms underlying thehormone-induced activation of estrogen receptor (ERa, ESR1).We show that estrogen activated PAK1 through both the ERa andGPER1 membrane receptors. Estrogen-dependent activation ofPAK1 required the phosphorylation of tyrosine residues by Etk/Bmx and protein kinase A (PKA) within an assembled signalingcomplex comprising pTyr-PAK1, Etk/Bmx, the heterotrimer G-protein subunits Gb1, Gg2, and/or Gg5, PAK-associated guaninenucleotide exchange factor (bPIX, ARHGEF7), and PKA. More-over, the PKA RIIb subunit is a direct target of PAK1, and thus in

response to estrogen, the activated pTyr-PAK1 complex recipro-cally potentiated PKA activity, suggesting a positive feedbackmechanism. We also demonstrate that PKA phosphorylatedSer305-ERa in response to estrogen, but pTyr-PAK1 phosphor-ylated Ser305-ERa in response to prolactin (PRL), implying thatmaximal ERa phosphorylation is achieved when cells areexposed to both PRL and estrogen. Furthermore, S305-ERaactivation led to enhanced phosphorylation of Ser118-ERa andpromoted cell proliferation and tumor growth. Together, thesedata strongly support a critical interplay between PRL andestrogen via PAK1 and suggest that ligand-independent activa-tion of ERa through PRL/PAK1 may impart resistance to anti-estrogen therapies. Cancer Res; 76(9); 2600–11. �2016 AACR.

IntroductionEstrogens play a crucial role in regulating the growth and

differentiation of breast cancers, with about two thirds of allbreast tumors expressing the estrogen receptor a (ERa; ref. 1).Although the involvement of estrogen in breast cancer tumori-genicity has been known for years, the exact mechanisms areunclear. Estrogen action is mediated by "classical" (initiated innucleus) and "alternative" (initiated at the plasma membrane)pathways. The "alternative" pathway promotes a fast response toestrogen and is mediated by membrane-localized ERa (mERa)that activates Ga and Gbg proteins, leading to calcium and cyclicAMP (cAMP) generation (2). Estrogen also signals through G-protein–coupled receptor 30 [GPR30 or G-protein–coupled ER 1(GPER1); ref. 3]. GPER1 activation results in the stimulation ofboth Gas and Gai/o, through which adenylyl cyclase (AC) iseither positively or negatively regulated, leading to changes incAMP level and protein kinase A (PKA) activity. Upon the acti-vation of both mER and GPER1, Ga dissociates from Gbg , andboth trigger the activation of downstream effectors (3).

The role of prolactin (PRL) in the pathogenesis of breast canceris increasingly appreciated (reviewed in refs. 4, 5, 6). PRL receptor(PRLR) is expressed in many breast cancers, including both ERaþ

and ERa� tumors (7), and PRLRmRNA is elevated in tumors (8).Augmented circulating PRL was linked to a 60% increased risk ofERþ tumors (4), and elevated circulating PRL 10 years prior todiagnosis was associated with the risk for metastatic ERþ disease(9). Circulating PRL and PRL secreted in hyperprolactinemia havebeen also associated with metastatic breast cancer (10, 11).

In addition, breast cancer tumorigenicity is amplified by PRLproduced locally by the breast tumor (12). In response to PRL,PRLR activates nonreceptor tyrosine kinase JAK2, which activatesseveral signaling cascades, including STATs (13).

Although PRL and estrogen exert independent effects on breastcancer cells, there is cross-talk between the two hormones thatleads to enhanced breast cancer cell proliferation (14). This cross-talk occurs at multiple levels and is bidirectional in that modu-lation of the ER pathway influences PRL pathways and vice versa.PRL increases expression and phosphorylation of ERa, andenhances alternative estrogen signaling, while estrogen inducestranscription of both PRL and PRLR and potentiates PRL-depen-dent STAT5 activity (reviewed in ref. 15). Defining the mechan-isms of cross-talk between estrogen and PRL has importantimplication for treatment of tumor resistance and for identifyingnew therapeutic targets in breast cancer. Here, we provide evi-dence that the serine/threonine kinase PAK1 is a commonnode inPRL and estrogen cross-talk.

The misregulation of PAK1 activity has been demonstrated inmany cancers and has been extensively studied in breast cancer(16). PAK1 directly phosphorylates ERa at Ser305 to promote thetransactivation of the receptor, leading to upregulation of cyclinD1. This transactivation may contribute to the development of

The Department of Biological Sciences, University of Toledo, Toledo,Ohio.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Maria Diakonova, University of Toledo, 2801 W. Ban-croft Street, Toledo, Ohio 43606. Phone: 419-530-7876; Fax: 419-530-7737;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1758

�2016 American Association for Cancer Research.

CancerResearch

Cancer Res; 76(9) May 1, 20162600

on February 8, 2021. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 4, 2016; DOI: 10.1158/0008-5472.CAN-15-1758

http://cancerres.aacrjournals.org/

estrogen-independent growth of breast cancer cells and tamoxifenresistance (17). However, the role of PAK1 in the respectivesignaling pathways affected is not defined.

We have discovered previously that PAK1 is a target for PRL-activated JAK2 and that JAK2 phosphorylates PAK1 on tyrosines153, 201, and 285 (18). Tyrosyl phosphorylation of PAK1 (pTyr-PAK1) enhances PAK1 kinase activity and its ability to formprotein/protein interactions. Both PAK1 activities are importantfor PRL-dependent breast cancer cell adhesion, motility, andinvasion (19, 20, 21). pTyr-PAK1 also regulates PRL-inducedcyclin D1 promoter activity (22). Similarly to PRL treatment,irradiation of lung cancer cells also leads to the phosphorylationof tyrosines 153, 201, and 285 by JAK2, resulting in increases inPAK1 stability, epithelial–mesenchymal transition, and radio-resistance (23). In addition to JAK2, the nonreceptor tyrosinekinase Etk/Bmx phosphorylates and activates PAK1, leading toincreased proliferation of MCF-7 cells (24).

In this study, we sought to elucidate the mechanism by whichestrogen and PRL coactivate ERa and a role of pTyr-PAK1 in thisprocess. We demonstrated that estrogen-activated Etk directlyphosphorylated PAK1 on Tyr153. Activated Gbg bound to PAK1in a complex with Etk and PAK1-associated bPIX to potentiatePKA activation. PKA-Ca directly phosphorylated PAK1, whilePKA-RIIb was a direct target of PAK1. We also demonstrated that,in response to estrogen, PKA phosphorylated Ser305-ERa, where-as PRL caused phosphorylation of the same site by PAK1. Ourresults identified reciprocal relationships between PAK1 and PKA,wherein each activated the other in response to estrogen. Ourfindings established PAK1 as a new target in both estrogen andprolactin signaling and provide a possible explanation of howthese two hormones synergistically regulate breast cancer cellproliferation.

Materials and MethodsPlasmids, antibodies, siRNA, reagents, and inhibitors are

described in Supplementary Material and Methods and in Sup-plementary Table S1.

CellsMCF-7 and T47D cell lines were purchased from the ATCC. The

following cell lines were kindly donated: TMX2-28 (Dr. Eisen-mann, University of Toledo, Toledo, OH); SKBR3 (Dr. Chen,Clemson University, Clemson, SC), HC-11 (Dr. Carter-Su, Uni-versity of Michigan, Ann Arbor, MI), and MDA-MB-134 andBT474 (Dr. Rae, University of Michigan). All cell lines wereauthenticated using Promega GenePrint10 System and weregrown up for fewer than 6 months following resuscitation. Theywere routinely verified to be mycoplasma-free using the MycoP-robeMycoplasmaDetection Kit (R&DSystems). All cell linesweretested and authenticated in November 2015. MCF-7 clones,SKBR3, HC-11, MDA-MB-134, and BT474 cells were maintainedin DMEM (Corning-Cellgro) supplemented with 10% FBS (Sig-ma-Aldrich). T47D and TMX2-28 clones were maintained asdescribed in refs.19, 20.

Immunoprecipitation, PAK1 in vitro kinase assay, and genesilencing were performed as described in ref. 21. Briefly, immu-noprecipitated PAK1 was subjected to an in vitro kinase assay inthe presence of 10 mCi of [g-32P]ATP and histone H4 (substrateof PAK1). For siRNA transfection, 100 nmol/L of specificsiRNAs [PAK1 and bPIX (Santa Cruz Biotechnology), Etk/Bmx,GPER1, and ERa (Cell Signaling Technology)] were transiently

transfected using Lipofectamine RNAiMAX Reagent (Invitro-gen) according to the manufacturer's instructions.

PKA kinase assayCells were lysed by 5 freeze/thaw cycles. Equal amount of

protein was subjected to an in vitro kinase assay in the presenceof 5 mCi [g-32P] ATP and kemptide (synthetic peptide substrate).Twenty microliters of the reaction volume was spotted on P81phosphocellulose paper (EMDMillipore) and subjected to liquidscintillation counting (25).

Cell proliferation assayThe cells were deprived for 24 hours in phenol red–free DMEM

supplementedwith 1% charcoal-stripped serum, treatedwith 500ng/mL PRL or 1 nmol/L 17b-estradiol (E2). Cell proliferation wasassessed in 7 days by MTT cell proliferation assay (Life Technol-ogies) according to the manufacturer's instructions (26).

Treatment with inhibitorsSerum-deprived cells were treated with E2 (1 nmol/L, 30

minutes) in the presence or absence of inhibitors: LY294002(10 mmol/L, 1 hour); UO126 (10 mmol/L, 1 hour); SB203580(10 mmol/L, 6 hours); SP600125 (30 mmol/L, 1 hour); AG879(increasing concentrations, 1 hour); PP1 or PP2, (increasingconcentrations, 2 hours); GF109203X (5 mmol/L, 2 hours); IKKinhibitor VII (5 mmol/L, 16 hours); imatinib mesylate (STI 571;increased concentrations, 24 hours); or canertinib (increasedconcentrations, 4 hours). To inhibit PKA, H89 (10 mmol/L, 1hour);myristoylated-PKI amide (14–22;myr; 20mmol/L, 1hour),Rp-cAMP (100 mmol/L, 2 hours), and 4-cyano-3methylisoquino-line (4C3MQ; 2 mmol/L, 4 hours) were used. Whole-cell lysateswere probed with indicated antibodies. For ETK and Src inhibi-tion, ETK and Src were immunoprecipitated with the aETK andaSrc, respectively, and subjected to in vitro kinase assay.

Etk in vitro kinase assayHA-PAK1wild-type (WT)/pCMV6or PAK1mutants were trans-

lated in vitro using TNT Coupled Reticulocyte Lysate Systems T7(Promega) with S35-methionine according to the manufacturer'sinstructions. Themixture of recombinant active Etk (50 ng) and invitro–translated or immunoprecipitated PAK1 was incubated inthe presence of 10 mCi of [g-32P]ATP or 20 mmol/L nonradioactiveATP.

Xenograft modelMCF-7 clones stably overexpressing vector, PAK1 WT, or PAK1

Y3F were inoculated directly into mammary fat pad of NSG(NOD/SCID/IL2Rg) female mice. The mice were supplied withdoxycycline (2 mg/mL) in drinking water and implanted withslow-release estradiol pellets. hPRL (20 mg/100 mL) was injectedsubcutaneously every other day until the end of the experiment.Tumors were measured and volume calculated as V ¼ (4/3)R12R2. Mouse experimental procedures were performed in theanimal research core of Lerner Research Institute, ClevelandClinic(Cleveland, OH; Dr. Daniel J. Lindner) and were approved by theInstitutional Animal Care and Use Committee, Cleveland Clinic.

Statistical analysisData from at least three separate experiments were pooled and

analyzed using one-way ANOVA plus Tukey honest significantdifference test. Differences were considered to be statisticallysignificant at a P < 0.05. Results are expressed as the mean � SE.

Tyrosyl-Phosphorylated PAK1 Regulates Estrogen Signaling

www.aacrjournals.org Cancer Res; 76(9) May 1, 2016 2601




Results and DiscussionEstrogen activates PAK1 in a tyrosyl phosphorylation–dependent manner

E2 is responsible for the proliferation of normal and neoplasticbreast tissue. Because PAK1 also stimulates cell proliferation (16),we tested whether PAK1 overexpression enhances the E2-depen-dent proliferation of MCF-7 and T47D breast cancer cells. Asdemonstrated previously, E2 induced proliferation of T47D andMCF-7 cells (Fig. 1A, vector). PAK1 WT overexpression did notaffect basal cell proliferation, and E2 increased cell growth in bothlines (Fig. 1A, PAK1WT cells). In contrast, overexpression of PAK1Y3F (3 JAK2 phosphorylation sites mutated) strongly decreasedE2-dependent cell proliferation relative to PAK1 WT overexpres-sion (Fig. 1A, PAK1 Y3F cells). Similar results were obtained inBT474, MDA-MB-134, and HC-11 cell lines (Supplementary Fig.S1A), indicating that this finding was not restricted to MCF-7 andT47D cells. To confirm the role of PAK1 in cell proliferation, wetreated control MCF-7 and T47D cells with PAK1 inhibitor IPA-3

(Supplementary Fig. S1B; ref. 27). IPA-3 did not inhibit prolifer-ation of vehicle-treated cells and was not toxic but it did inhibitE2-dependent cell proliferation in a concentration-dependentmanner (Supplementary Fig. S1C), confirming that PAK1 parti-cipates in E2-dependent cell proliferation.

To uncover the mechanism by which pTyr-PAK1 regulates E2-dependent cell proliferation, we assessed PAK1 kinase activity inE2-treated cells, because E2 activates PAK1 (28). As demonstratedpreviously, PRL activated PAK1 WT significantly stronger thanPAK1 Y3F confirming that tyrosines 153, 201, and 285 arerequired for full activation (Fig. 1B, left; refs.19, 20). PAK1 WTactivation by E2 or PRL was similar. Surprisingly, E2 failed toactivate PAK1 Y3F (Fig. 1B, right). In addition, E2-induced phos-phorylation of Thr423-PAK1 (a marker of PAK1 activation) wasdetected in PAK1 WT, but not in PAK1 Y3F cells (SupplementaryFig. S1D), demonstrating that PAK1 tyrosyl phosphorylation isimportant for E2-dependent PAK1 activation. To confirm thatPAK1 Y3F retains its kinase activity, we assessed the effect ofheregulin (HRG), a known activator of PAK1 (29), on PAK1

Figure 1.Estrogen activates PAK1. A,MCF-7 andT47D cells overexpressing PAK1WT orPAK1 Y3F were incubated with/without E2. Changes in cell numbers atday 7 are shown as percentages of thevehicle (Veh)-treated cell number.� , P < 0.05 comparedwith control cellsin the same condition, n ¼ 3. B, PAK1WT or Y3F MCF-7 clones were treatedwith PRL (left) or E2 (right).PAK1 was immunoprecipitated andsubjected to an in vitro kinase assaywith H4 histone as a substrate. PAK1kinase activity was normalized byimmunoprecipitated PAK1 for eachlane and plotted. C, PAK1 WT orY3F clones were treated with vehicle,PRL, E2, PRL þ E2, or HRG andassessed as in B. � , P < 0.05, n ¼ 3.;HA, hemagglutinin; vec, vector.

Oladimeji et al.

Cancer Res; 76(9) May 1, 2016 Cancer Research2602




activation. HRG-induced PAK1 kinase activity was similar in cellsexpressing PAK1 WT and PAK1 Y3F (Fig. 1C), confirming thatPAK1 Y3F is functionally active.

Etk kinase directly phosphorylates and activates PAK1 inresponse to estrogen

To understand how E2 mediated PAK1 tyrosyl phosphoryla-tion and activation, we tested whether E2 activates JAK2. Asexpected, PRL, but not E2, strongly activated JAK2 (Supplemen-tary Fig. S2A). We hypothesized that kinase(s) other than JAK2tyrosyl phosphorylated PAK1 in response to E2 and tested Etk/Bmx, Src, c-Abl, and ErbB because they have been previouslyimplicated in tyrosyl phosphorylation of either PAK1 or PAK2(24, 30, 31, 32). Inhibition of c-Abl by STI571 and ErbB bycanertinib did not affect E2-dependent PAK1 tyrosyl phosphor-ylation, although Erk 1/2 activation was eliminated by both

inhibitors (Supplementary Fig. S2B). Similarly, Src inhibitors PP1and PP2 did not affect E2-dependnet PAK1 tyrosyl phosphory-lation but blocked Src activation (Supplementary Fig. S2C).

AG879 inhibition of Etk resulted in a concentration-dependentdecrease in pTyr-PAK1 (Fig. 2A, left), suggesting that Etk isinvolved in E2-dependent PAK1 phosphorylation. Tyrosines153, 201, and 285 are implicated in this E2-dependent phos-phorylation because Y3F was not phosphorylated in response toneither PRL nor E2 (Fig. 2A, right). More importantly, Etk inhi-bition attenuated E2-dependent PAK1 kinase activity (Fig. 2B). Tosupport this finding, we knocked downEtk by siRNA and assessedE2-dependent PAK1 activity. Indeed, Etk silencing decreased E2-dependent PAK1 activity (Fig. 2C). We recovered E2-dependentPAK1 activation by reintroducing WT, but not kinase-dead Etk(33), in the Etk-depleted cells (Fig. 2C). These data stronglysuggest that Etk tyrosyl phosphorylates and activates PAK1 in

Figure 2.Etk directly phosphorylates andactivates PAK1 in response toestrogen. A, PAK1 WT cells weretreated with Etk inhibitor AG879(AG) before E2 treatment andimmunoprecipitated (IP) PAK1, orwhole-cell lysates (WCL)were probedwith antibodies (left). PAK1 or JAK2were immunoprecipitated from PAK1WT and Y3F clones treated with E2 orPRL and immunoblotted (right). B,cells were treated with AG879. PAK1was immunoprecipitated andsubjected to an in vitro kinase assay asin Fig. 1. C, cells were transfected withcontrol (scr) or Etk siRNA. In siRNArescue experiments, 24 hours after EtksiRNA transfection, cells weretransfected with either Etk WT or Etkkinase-dead mutant (KD). PAK1 wasimmunoprecipitated and subjected toin vitro kinase assay as in Fig. 1.D, PAK1 WT and Y3F wereimmunoprecipitated and assessed inthe in vitro kinase assay in thepresence of recombinant active Etk.The levels of P32 incorporation in PAK1were plotted. E, PAK1 WT andindicated tyrosine-deficient PAK1mutants were translated in vitro andsubjected to the in vitro kinase assay inthe presence of recombinant Etk andnonradioactive ATP. F, WT or kinase-dead Etk were immunoprecipitatedfrom the cells treated with/without E2and assessed in the in vitro kinaseassay in the presence of either PAK1WT or Y153F as in E. Each figurerepresents the same blot reblottedwith the indicated antibodies. All blotsare representative of at least threeexperiments.�, P < 0.05, n ¼ 3.HA, hemagglutinin; vec, vector;veh, vehicle.






response to E2. To mechanistically link Etk and PAK1, we immu-noprecipitated PAK1 WT or Y3F, performed the in vitro kinaseassay with active recombinant Etk, and demonstrated that Etkphosphorylates PAK1 WT, but not Y3F (Fig. 2D). To map PAK1phosphorylation site(s), we performed in vitro kinase assays usingrecombinant Etk and in vitro–translated PAK1 WT, Y3F, Y153F,Y201F, Y285F, or Y55FPAK1mutants. Etk failed to phosphorylateboth PAK1 Y3F and Y153F (Fig. 2E). To show that Etk phosphor-ylates Tyr-153 of PAK1 in response to E2, we immunoprecipitatedoverexpressed EtkWTor kinase-dead Etk fromMCF-7 cells treatedwith/without E2 and assessed kinase activity in vitro with in vitro–translated PAK1 WT and Y153F. As expected, E2-activated Etkdirectly phosphorylated PAK1 WT, but not Y153F (Fig. 2F). Our

data strongly suggest that E2-activated Etk directly phosphorylatesPAK1 WT on Tyr 153, causing PAK1 activation.

Although themechanisms bywhich estrogen receptors regulateEtk kinase need to be further investigated, we speculate that Etkmay be an effector of Gbg subunits in response to E2 (see below).

E2 promotes PAK1 activation through both ERa and GPER1plasma membrane receptors

The rapid activation of PAK1 by E2 (Fig. 1B) indicatesinvolvement of either mER and/or GPER1. These two estro-gen-responsive receptors are structurally different and can trig-ger different signaling pathways in the same cell, includingMCF-7 cells (34, 35, 36).

Figure 3.Both GPER1 and mERa are involved inestrogen-dependent PAK1 activation. Aand B, MCF-7 and TMX2-28 cells weretreated with E2 and either whole-celllysates were probed with indicatedantibodies (A) or in vitro kinase assaywas performed (B). C and D, MCF-7 andSKBR3 cells were treated with E2 andeither whole-cell lysates were probedwith the indicated antibodies (C) or invitro kinase assaywas performed (D). E,MCF-7 PAK1 WT cells were transfectedwith control (scr), ERa, GPER1 (GPR), orERaþGPER1 (ER/G) siRNAs. HA-PAK1was immunoprecipitated and subjectedto in vitro kinase assay. F, TMX2-28PAK1 WT cells were transfected cDNAsencoding GPER1 (GPR), ERa, orERaþGPER1 (ER/G). HA-PAK1 wasimmunoprecipitated and subjected tothe in vitro kinase assay. HA,hemagglutinin; vec, vector; veh,vehicle. � , P < 0.05, n ¼ 3.

Oladimeji et al.





To test whether mERa is required for E2-triggered PAK1activation, we used TMX2-28 cells, an ER� variant of MCF-7cells (37). As expected, E2 caused phosphorylation of Ser118-ERa and Ser305-ERa (markers of ER activation) in MCF-7cells, but not in TMX2-28 cells (Fig. 3A). Furthermore, TMX2-28 cells are also GPER1 deficient (Fig. 3A). E2 activated PAK1in MCF-7 cells, but not in TMX2-28 cells, whereas PRL acti-vated PAK1 similarly in both lines (Fig. 3B), suggesting thateither mERa or GPER1 are required for E2-dependent PAK1activation.

To determine whether E2 activates PAK1 via GPER1, weemployed ER�/GPER1þ SKBR3 cells. E2 activated GPER1 inboth MCF-7 and SKBR3 cells as demonstrated by Erk1/2phosphorylation, whereas E2 activated ERa only in MCF-7cells (Fig. 3C). E2 activated PAK1 in both lines, suggesting thatGPER1 participates in PAK1 activation (Fig. 3D). To confirmthis observation, we performed siRNA knockdown of eitherGPER1, ERa, or both receptors in MCF-7 cells. Silencing ofeither ERa or GPER1 significantly decreased E2-triggered PAK1activation; however, only downregulation of the ERa andGPER1 together completely abolished E2-dependent PAK1activation (Fig. 3E). We also overexpressed GPER1, ERa, orboth receptors in TMX2-28 cells and showed that E2 causedmaximal PAK1 activation when both GPER1 and ERa werepresent (Fig. 3F). These data suggest that E2 promotes PAK1activation through both mERa and GPER1 plasma membranereceptors.

Gbg subunits of G-protein activate PAK1 in response toestrogen

mERa and GPER1 activate heterotrimeric G-proteins (3). TheGbg dimer directly binds to both PAK1 (38) and E2-activatedmERa (39). To determine which combination of b and g subunitsparticipates in E2-dependent PAK1 activation, we overexpressedb1 or b2 together with g2, g5, or g13 in different combinationsand assessed E2-dependent PAK1 activity (Fig. 4A). As expected,E2 activated PAK1 (lane 2 vs. lane 1). b1g2 and b1g5 overexpres-sion strongly enhanced E2-dependent PAK1 activity (lanes 4 and6). Interestingly,b2g2 (lane 8),b2g5 (lane 10), orb1g13 (lane 12)overexpression inhibited E2-dependent PAK1 activity, whereasb2g13 (lane 14) overexpression had no effect compared withcontrol (lane 2). To test whether PAK1 tyrosines play a role inactivation, we overexpressed b1g2 and b1g5 subunits in PAK1WTand Y3F clones and assessed E2-dependent PAK1 activation. b1g5overexpression increased both basal and E2-stimulated PAK1WTactivity (Fig. 4B, lanes 5–6), whereas b1g2 overexpressionenhanced PAK1 WT activity only in response to E2 (lanes 3–4).b1g2 and b1g5 overexpression did not activate PAK1 Y3F (lanes9–12), whereas HRG strongly activated PAK1 WT and Y3F (lanes13–14). To assess PAK1 binding to b1g2 and b1g5, PAK1 WT orY3F were immunoprecipitated and assessed for b1g2, b1g5 (Fig.4C), and b1g13 (Supplementary Fig. S2D). PAK1 WT bound toE2-activated b1g2 and to b1g5 independent of E2 (Fig. 4C andSupplementary Fig. S2D, lanes 3–6). We considered PAK1 WTbinding to b1g2without E2 (Fig. 4C and Supplementary Fig. S2D,

Figure 4.Gbg subunits activate PAK1 inresponse to estrogen. A and B,combinations of b1, b2, g2, g5, and g13subunits were overexpressed. E2-dependent PAK1 activation wasassessed in the in vitro kinase assay.Both gray bars in B represent the cellstreated with HRG. C, HA-PAK1 wasimmunoprecipitated (IP) from PAK1WT or Y3F clones overexpressing b1g2or b1g5 subunits and immunoblotted.HA, hemagglutinin; vec, vector; veh,vehicle; WCL, whole-cell lysates.� , P < 0.05, n ¼ 3.






lane 3) to b1g13 (Supplementary Fig. S2D, lanes 1–2) and Y3F tob1g13, b1g2, and b1g5 (Fig. 4C and S2D) as negligible. Endog-enous Etk was associated with E2-activated PAK1 WT (aEtk blot,lanes 2, 4, and 6 in Fig. 4C and Supplementary Fig. S2D), whilePAK1 Y3F failed to bind to Etk (lanes 7–12). These data suggestthat E2 treatment promotes the formation of Gb1g2,5/PAK1/Etkcomplex.

PAK1 can be activated by b1g2 or b1g5 subunits via the PI3Kand Akt pathways independently of Rac1 or Cdc42 (40). In thatstudy, EGF, PMA, LPA, and carbachol did not augment PAK1activity beyond that induced by b1g2 or b1g5 (40). Our datademonstrated additive effect of E2 underlying a high degree ofspecificity for this pathway.

bPIX facilitates E2-dependent PAK1 activationIn response to chemoattractants, Gbg binds directly to PAK1,

which simultaneously associates with aPIX (PAK-associated gua-nine nucleotide exchange factor) to form a Gbg/PAK1/aPIX/Cdc42 complex, leading to PAK1 activation (38). Because wehavepreviously shown that PRL increases associationofbPIXwith

PAK1WT but not Y3F and Y285Fmutants (21), we reasoned thatformation of the Gbg/PAK1/bPIX complex plays a role in E2-dependent PAK1 activation. We immunoprecipitated PAK1 fromE2-treated PAK1 WT and Y3F cells and probed for endogenousbPIX. E2 increased bPIX association with PAK1 WT but not withY3F (Fig. 5A), demonstrating that tyrosyl phosphorylation ofPAK1 facilitates E2-dependent binding with bPIX. We next over-expressed bPIX in WT and Y3F clones and assess E2-dependentPAK1 kinase activity. bPIX significantly activated PAK1WT versusY3F in response to E2 (Fig. 5B). bPIX silencing abolished E2-dependent PAK1activation (Fig. 5C, lane3),while reexpressionofbPIX WT rescued PAK1 activation (lane 4). Reexpression of bPIXDHm(L238R/L239S), lacking guanine nucleotide exchange(GEF) activity, partially rescued PAK1 kinase activity (lane 5),and reexpression of bPIX SH3m(W43K), which lacks PAK1 bind-ing activity, did not (lane 6). These data suggest that bPIX bindingto PAK1 is required for its activation and that bPIX GEF activity issufficient for E2-dependent PAK1 activation. Furthermore, PAK1tyrosyl phosphorylation is also required for E2-dependent bPIXbinding.

Figure 5.bPIX facilitates E2-dependent PAK1activation. A, immunoprecipitated (IP)PAK1 fromWT and Y3F clones treatedwith E2 was assessed for endogenousbPIX (endobPIX). B, bPIX wasoverexpressed in WT and Y3F clonesand PAK1 kinase activity wasassessed. Asterisk (�) indicates longerexposure with abPIX revealingendogenous bPIX (bottom doublearrow). Overexpressed bPIX(overbPIX) is indicated by the topdouble arrow. C, the cells weretransfected with control (scr; lanes1–2) or bPIX siRNA (lanes 3–4). InsiRNA rescue experiments, bPIX DHm(L238R/L239S; lane 5) or SH3m(W43K; lane 6) mutants werereexpressed and PAK1 activation wasassessed in the in vitro kinase. co-IP,coimmunoprecipitation; HA,hemagglutinin; IB, immunoblot; vec,vector; veh, vehicle. � , P < 0.05, n ¼ 3.

Oladimeji et al.





To test whether bPIX is a member of Gb1g2,5/PAK1/Etk com-plex, we immunoprecipitated either WT or Y3F from cells treatedwith/without E2 and blotted for bPIX. bPIX was associated withE2-activated PAK1 WT (Fig. 4C and Supplementary Fig. S2D,abPIX blot, lanes 2, 4, and 6). PAK1 Y3F complexed with b1g5only, and independently of E2 (lanes 11–12). E2 also promotesformation of this Etk/PAK1/bPIX complex in T47D, BT474,MDA-MB-134, and HC-11 cells (Supplementary Fig. S3A). These datasuggest that E2 induces amultiprotein signaling complex contain-ing pTyr-PAK1, Etk, b1/g2, or b1/g5 subunits and bPIX. It appearsthat three tyrosines of PAK1 are required for the formation of thiscomplex, which facilitates PAK1 activation.

E2-dependent PKA activity is reciprocally regulated by activatedpTyr-PAK1

E2 rapidly activates multiple signaling pathways, includingMAPKs, PI3K, PKC, PKA, NFkB, and Src (2). To determine whichof these pathways are implicated in E2-triggered PAK1 activation,we treated MCF-7 cells with E2 and selective inhibitors. Inhibitor

specificity was confirmed (Supplementary Fig. S4A), and allinhibitors had no effect on PAK1 activity in vehicle-treated cells,demonstrating that they did not independently effect PAK1activity (Supplementary Fig. S3B). Only PKA inhibition by H89inhibited E2-dependent PAK1 kinase activity (Fig. 6A). To con-firm that the effect of H89 was specific to estrogen, we treatedPAK1 WT cells with PRL þ H89 and showed that PAK1 kinaseactivity was unaffected (Supplementary Fig. S3C). In addition toH89, other selective PKA inhibitors, myristoylated-PKI amide(14–22; myr), Rp-cAMP, and 4-cyano-3 methylisoquinoline(4C3MQ; Supplementary Fig. S4B) also inhibited E2-activatedPAK1 (Fig. 6B). Furthermore, PAK1 activity was enhanced byforskolin, which triggers general cAMP elevation and PKA activa-tion, and abolished by Rp-cAMP (Fig. 6B).

In response to E2, bothmERa andGPER1 activate Gas, leadingto the stimulation of AC, cAMP, and the subsequent activation ofPKA (2, 3). PKA is a PAK1 regulator in anchorage-dependentsignal transduction (41). PKA phosphorylates bPIX, leading toactivation of PAK1 effector, Cdc42 (42). Furthermore, PKA

Figure 6.Reciprocal regulation of PAK1 and PKA in E2signaling. A, cells were treated with inhibitorsUO126 (UO), SB203580 (SB), SP600125 (SP),LY294002 (LY), IKK inhibitor (IKKinh),GF109203X (GF), and H89, then with E2, andPAK1 activation was assessed in the in vitrokinase assay. B, PAK1 activation was assessed asin A in cells treated with indicated different PKAinhibitors. � , P < 0.05 compared with cellsexpressing PAK1 WT and treated with veh þ E2.C, cell lysates were assessed for PKA activity inthe in vitro kinase assay. D, combinations of b1g2subunits, Etk, and bPIX were overexpressed inMCF-7 PAK1 WT cells. E2-dependent PKAactivation was assessed in the in vitro kinaseassay. E, mEGFP-tagged cDNAs encodingdifferent PKA subunits (RIa, RIb, RIIa, RIIb, andCa) were overexpressed in MCF-7 PAK1 WTclone and treatedwith/without E2. HA-PAK1wasimmunoprecipitated (IP) with aHA andimmunoblotted. F, in vitro–translated PAK1 wasincubated with immunoprecipitated PKA RIIb inthe in vitro kinase assay. H4 histone was used asa PAK1 substrate (lane 4). G, in vitro–translatedPAK1 was incubated with recombinant activePKA Ca (catalytic subunit) in the in vitro kinaseassay. Incorporation of P32 in PAK1 (top arrow)and in PKA Ca (bottom arrows) is indicated.Fors, forskolin; HA, hemagglutinin; vec, vector;veh, vehicle.






phosphorylates PAK1 and the PKA catalytic subunit phosphor-ylates and complexes with PAK1/4 (41, 43, 44). Hence, wehypothesized that activated PAK1 may in turn modulate PKAactivity. PKA in vitro kinase activity was significantly elevated inE2-treated, but not in RPL-treated, PAK1 WT cells (Fig. 6C).Interestingly, PAK1 Y3F inhibited E2-triggered PKA activation(Fig. 6C). To study whether the formation of Gb1g2/Etk/PAK1/bPIX complex facilitates E2-dependent PKA activation, we over-expressed these proteins in PAK1 WT cells and assessed endog-enous PKA in vitro kinase activity. PKA activity wasmaximal whenb1g2, Etk, PAK1, and bPIX were present and E2 was added (Fig.6D). To determine which PKA subunit complexed with PAK1,different PKA subunits were overexpressed in PAK1 WT cells,treated with/without E2. PKA-RIIb subunit interacted with PAK1WT in E2-independent manner (Fig. 6E). To demonstrate thatPKA RIIb is a direct substrate of PAK1, we assessed in vitrotranslated HA-PAK1 with immunoprecipitated PKA-RIIb in thein vitro kinase assay. Translated PAK1was active as judgedby the invitro phosphorylation of H4 histone (PAK1 substrate, Fig. 6F, lane4) and PAK1 directly phosphorylated RIIb (Fig. 6F, lane 5). Inagreement with published data (41), in vitro–translated PAK1wasdirectly phosphorylated by recombinant active PKA-Ca (catalyticsubunit) in the in vitro kinase assay (Fig. 6G). These data suggestthat PKA-Ca directly phosphorylates PAK1, while PKA-RIIb is thedirect target of PAK1. In response to E2, activated pTyr-PAK1complexes with b1g2/Etk/bPIX to potentiate PKA activation. Atthe same time, PKA is required for full E2-dependent PAK1activation, demonstrating positive feedback between these twokinases.

PKA phosphorylates Ser305-ERa in response to E2 and PAK1phosphorylates Ser305-ERa in response to PRL

Ser305-ERa can be phosphorylated by PAK1 and PKA (45, 46).Constitutively active PAK1 phosphorylates Ser305-ERa, resultingin subsequent phosphorylation of Ser118-ERa, ligand-indepen-dent ER activation, and tamoxifen resistance (45, 47). PAK1overexpression increased phosphorylation of both Ser305-ERaand Ser118-ERa upon E2 treatment (Fig. 7A, lanes 3 vs. 7, whiteand black bars), whereas Y3F overexpression inhibited pSer118-ERa (Fig. 7A, lanes 11 vs. 3). PRL treatment induced Ser305-ERa,but not Ser118-ERa phosphorylation, in WT PAK1 cells but hadno effect in Y3F cells (Fig. 7A, lanes 2, 6 and 10).

Next, we aimed to determine whether PAK1 or PKA participatein E2- and PRL-triggered ERa phosphorylation. PAK1 siRNAsilencing abolished PRL-dependent Ser305-ERa phosphoryla-tion, but not E2-dependent Ser305-ERa phosphorylation (Fig.7B, lanes 2 vs. 4). To confirm that PAK1 phosphorylates Ser305-ERa in response to PRL, we reintroduced PAK1 into PAK1-silenced cells and recovered PRL-dependent Ser305-ERa phos-phorylation (Fig. 7C). To test whether PKA phosphorylatesSer305-ERa in response to E2, we inhibited PKA by Rp-cAMPsand observed that pSer305-ERa signal was strongly decreased inthe cells treatedwith E2or forskolin (Fig. 7D, lanes 3 vs. 8 and5 vs.10), but not in the cells treated with PRL or PRL þ E2 (Fig. 7D,lanes 2 vs. 7 and 4 vs. 9).

These data suggest that PKA phosphorylates Ser305-ERa inresponse to E2, while PAK1 phosphorylates Ser305-ERa inresponse to PRL. PAK1 augments PKA-dependent Ser305-ERaphosphorylation, as PAK1 overexpression significantly increasedE2-dependent Ser305-ERa phosphorylation (Fig. 7A, lines 7 vs.3). Furthermore, PAK1 tyrosines 153, 201, and 285 participate in

both signaling pathways because Y3F overexpression inhibitedSer305-ERa phosphorylation after both PRL and E2 treatments(Fig. 7A, lanes 10–12). As PAK1 WT, but not Y3F, significantlyenhanced E2-dependent PKA activation (Fig. 6D), our data sug-gest that although PAK1 does not phosphorylate Ser305-ERaupon E2 treatment, it augments PKA activation and subsequentSer305-ERa phosphorylation, indirectly increasing estrogenresponsiveness.

Synergistic effects of PRL and E2 on ERa activation, cellproliferation, and tumor growth

If both PKA and PAK1 phosphorylate the same site on ERa, wehypothesized that PRLþ E2 treatmentwould have additive effectson Ser305-ERa phosphorylation and subsequent Ser118-ERaphosphorylation. Indeed, we observed amplified pSer305-ERaandpSer118-ERawhenPAK1WT cells were treatedwith PRLþE2versus either with PRL or E2 alone (Fig. 7A, lanes 8 vs. 7 and 6).S305-ERa phosphorylation by PAK1 promotes ERa transactiva-tion, leading to upregulation of cyclin D1 and hormone-inde-pendence (48). To determine whether enhanced ERa activationimpacted breast cancer cells, we evaluated the effect of PRL þ E2on proliferation. In agreement with published studies demon-strating that PRL enhances E2-induced proliferation (14, 49, 50,51,52), PRL þ E2 induced proliferation of control MCF-7 cells(Fig. 7E, vector). PAK1 overexpression further enhanced cellproliferation in response to E2 þ PRL as compared with cellstreated with either PRL or E2 (Fig. 7E, PAK1WT cells). In contrast,Y3F inhibited E2 þ PRL–dependent proliferation (PAK1 Y3Fcells), implicating these tyrosines in PAK1 regulation. To dem-onstrate that the role of PAK1 in synergistic PRL þ E2–mediatedsignaling is not limited to MCF-7 cells, we assessed proliferationof different human breast cancer cell lines endogenously expres-sing PRLR and ER (T47D, BT474, and MDA-MB-134), as well asmouse mammary HC-11 cells. In all lines, PAK1 overexpressionenhanced cell proliferation in response to E2 þ PRL, comparedwith the cells treated with either PRL or E2 (Supplementary Fig.S5). Finally, PRL þ E2–activated pTyr-PAK1 enhances breasttumor growth in a xenograft mouse model (Fig. 7F).

We have demonstrated here that tyrosines 153, 201, and 285play a role in PAK1 activation by E2 (but not HRG), ability ofPAK1 to complex with Gb1g2 and 5, Etk, and bPIX, activate PKA,phosphorylate S305-ERa, and, finally, enhance PRLþ E2–depen-dent proliferation and tumor growth. Kim and colleagues haveproposed that tyrosyl phosphorylation of these sites may disruptthe inhibitory conformation of PAK1 and/or maintain PAK1active conformation because Tyr153 resides nearby Ser144 andSer149, Tyr201 locates near Ser199 and Ser204 (autophosphor-ylation sites), and Tyr285 lies in the kinase domain and maystabilize the interaction between K299 and ATP molecules (23).Although it should be confirmed by structural studies, this prop-osition is very attractive. Another possibility is that pTyr-PAK1may create additional docking sites to recruit SH2 domain–con-taining proteins to facilitate PAK1 functions. Either way, thesetyrosine phosphorylation events are critical for PAK1 functions.

On the basis of our finding and previous studies, we propose amodel for PAK1 in estrogen- and PRL-triggered intracellularsignaling, leading to a synergistic effect of PRL and E2 onSer305-ERa phosphorylation, cell proliferation, and tumorgrowth (Fig. 7G). E2 activates mERa and GPER1, leading to G-protein activation that dissociates into a and bg subunits, both ofwhich activate additional effectors. Gas activates PKA, which can

Oladimeji et al.





phosphorylatemany substrates, including PAK1 and Ser305-ERa.In addition, b1g2 and b1g5 are released and bind to PAK1, whichis directly phosphorylated by Etk on Tyr153. pTyr-PAK1 recruitsand binds to bPIX, an association that further activates PAK1.

Although PKA is the main kinase phosphorylating Ser305-ERa inresponse to E2, PKA is reciprocally regulated by activated PAK1 asa component of b1g2/Etk/bPIX/pTyr-PAK1 complex. If cells areexposed to both PRL and E2, Ser305-ERa is phosphorylated by

Figure 7.Ser305-ERa is phosphorylated by PKA in response to E2 and by PAK1 in response to PRL. A, control, PAK1 WT, or PAK1 Y3F clones of MCF-7 cells were treated withvehicle, E2, PRL, or PRLþE2 andwhole-cell lysateswere immunoblotted. B, cellswere transfectedwith control (scr) or PAK1 siRNA, treatedwith PRL, E2, or PRLþE2,and whole-cell lysates were immunoblotted. C, in siRNA rescue experiments, 24 hours after PAK1 siRNA transfection, cells were transfected with controlor PAK1WT, treatedwith PRL, andwhole-cell lysateswere immunoblotted. D, cells were incubatedwith either vehicle or Rp-cAMP, treatedwith vehicle, PRL, E2, PRLþ E2, or forskolin. E, MCF-7-overexpressing vector, PAK1 WT, or PAK1 Y3F were incubated with vehicle, PRL, E2, or PRL þ E2 and assessed for cell proliferation asin Fig. 1. F, pTyr-PAK1 accelerates tumor growth in vivo. MCF-7 clones were injected into the mammary pad of female NSG mice. Mice were treated with PRL þ E2.� , P < 0.05 compared with control (vector) mice, n ¼ 8. G, proposed mechanisms for the role of PAK1 in synergetic effects of PRL and E2 on ERa activation.E2 activates mERa and GPER1, leading to G-protein and Etk kinase activation. E2-activated Etk binds to and directly phosphorylates PAK1 on Tyr153.Gbg subunits also bind to pTyr-PAK1, facilitating recruitment and activation of bPIX, which in turn binds and further activates PAK1 and establishes a self-amplifyingcascade. Gas subunit activates PKA, which binds to PAK1 via PKA-RIIb, while PKA-Ca directly phosphorylates PAK1. Gbg/Etk/pTyr-PAK1/bPIX complexreciprocally activates PKA in response to E2 and activates PKA phosphorylates Ser305-ERa, leading to enhanced S118-ERa phosphorylation. PRL-activated JAK2phosphorylates and activates PAK1, which also activates ERa by phosphorylation of S305-ERa. Thus, in response to E2 þ PRL, pS305-ERa is phosphorylatedby both PAK1 and PKA, which leads to enhanced cell proliferation. fskl, forskolin; vec, vector; veh, vehicle.






both PKA and pTyr-PAK1, resulting in maximal signal. Further-more, S305-ERa activation enhances Ser118-ER phosphoryla-tion, presumably due to an intramolecular conformationalchange of ERa (46, 47), and finally induces cell proliferation.E2-dependent Etk and PRL-dependent JAK2 PAK1 tyrosyl phos-phorylation are important, as PAK1 Y3F fails to establish this self-amplifying cascade and promote cell proliferation and tumorgrowth.

Another notable finding is that Ser305-ERa is phosphorylatedby PAK1 in response of PRL in the absence of estrogen. Ser305-ERaphosphorylationwas implicated in tamoxifen resistance, anda correlationbetweenhigh level of PAK1and tamoxifen sensitivityin breast cancer patients is described in refs. 47, 53. Our datapredict that patients with augmented levels of PRL will likelydevelop tamoxifen resistance due to Ser305-ERa phosphoryla-tion by PRL-activated PAK1. Furthermore, the synergistic effects ofestrogen and PRL signaling described here are physiologically andtherapeutically relevant as breast cells are constantly exposed toboth hormones, but anti-estrogen therapy targets only the estro-gen component. We introduce PAK1 as a common node forestrogen- and PRL-dependent pathways, making it an attractivetarget for anticancer therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. DiakonovaDevelopment of methodology: P. OladimejiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Oladimeji, R. Skerl, C. RuschAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Oladimeji, M. DiakonovaWriting, review, and/or revision of themanuscript:P.Oladimeji,M.DiakonovaStudy supervision: M. Diakonova

AcknowledgmentsThe authors thank Drs. Sorokin (Medical College of Wisconsin, WI) and

Chen (Institute of Biological Chemistry, Taiwan) for providing cDNAs encodingbPIX WT, bPIX mutants, Etk WT, and kinase-dead Etk mutant, respectively. Wethank Drs. Eisenmann (University of Toledo, OH), Chen (Clemson University,SC), Carter-Su (University of Michigan, MI), and Rae (University of Michigan,MI) for providing cells.

Grant SupportThis work was supported by a grant from the NIHR01 DK88127

(M. Diakonova).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 6, 2015; revised January 18, 2016; accepted February 6, 2016;published OnlineFirst March 4, 2016.

References1. Barone I, Brusco L, Fuqua SA. Estrogen receptor mutations and changes in

downstream gene expression and signaling. Clin Cancer Res 2010;16:2702–8.

2. Levin ER. Plasma membrane estrogen receptors. Trends Endocrinol Metab2009;20:477–82.

3. Nilsson BO, Olde B, Leeb-Lundberg LM. G protein-coupled oestrogenreceptor 1 (GPER1)/GPR30: a new player in cardiovascular and metabolicoestrogenic signalling. Br J Pharmacol 2011;163:1131–9.

4. Tworoger SS, Hankinson SE. Prolactin and breast cancer etiology: anepidemiologic perspective. J Mammary Gland Biol Neoplasia 2008;13:41–53.

5. Fernandez I, Touraine P, Goffin V. Prolactin and human tumourogenesis.J Neuroendocrinol 2010;22:771–7.

6. O'Leary KA, Shea MP, Schuler LA. Modeling prolactin actions in breastcancer in vivo: insights from the NRL-PRL mouse. Adv Exp Med Biol2015;846:201–20.

7. Swaminathan G, Varghese B, Fuchs SY. Regulation of prolactin receptorlevels and activity in breast cancer. J Mammary Gland Biol Neoplasia2008;13:81–91.

8. Touraine P, Martini JF, Zafrani B, Durand JC, Labaille F, Malet C, et al.Increased expression of prolactin receptor gene assessed by quantitativepolymerase chain reaction in human breast tumors versus normal breasttissues. J Clin Endocrinol Metab 1998;83:667–74.

9. Tworoger SS, Eliassen AH, Zhang X,Qian J, Sluss PM, Rosner BA, et al. A 20-year prospective study of plasma prolactin as a risk marker of breast cancerdevelopment. Cancer Res 2013;73:4810–9.

10. Mujagic Z, Mujagic H. Importance of serum prolactin determination inmetastatic breast cancer patients. Croat Med J 2004;45:176–80.

11. Mujagic Z,Mujagic H, Prnjavorac B. Circulating levels of prolactin in breastcancer patients. Med Arh 2005;59:33–5.

12. Clevenger CV, Furth PA, Hankinson SE, Schuler LA. The role of prolactin inmammary carcinoma. Endocr Rev 2003;24:1–27.

13. Clevenger CV,Gadd SL, Zheng J. Newmechanisms for PRLr action in breastcancer. Trends Endocrinol Metab 2009;20:223–9.

14. Gutzman JH, Nikolai SE, Rugowski DE, Watters JJ, Schuler LA. Prolactinand estrogen enhance the activity of activating protein 1 in breast cancercells: role of extracellularly regulated kinase 1/2-mediated signals to c-fos.Mol Endocrinol 2005;19:1765–78.

15. Carver KC, Arendt LM, Schuler LA. Complex prolactin crosstalk in breastcancer: new therapeutic implications. Mol Cell Endocrinol 2009;307:1–7.

16. Eswaran J, Li DQ, Shah A, Kumar R. Molecular pathways: targeting p21-activated kinase 1 signaling in cancer–opportunities, challenges, andlimitations. Clin Cancer Res 2012;18:3743–9.

17. Rayala SK, Kumar R. Sliding p21-activated kinase 1 to nucleus impactstamoxifen sensitivity. Biomed Pharmacother 2007;61:408–11.

18. Rider L, Shatrova A, Feener EP,Webb L,DiakonovaM. JAK2 tyrosine kinasephosphorylates PAK1 and regulates PAK1 activity and functions. J BiolChem 2007;282:30985–96.

19. Rider L, Oladimeji P, Diakonova M. PAK1 regulates breast cancer cellinvasion through secretion of matrix metalloproteinases in response toprolactin and three-dimensional collagen IV. Mol Endocrinol 2013;27:1048–64.

20. Hammer A, Rider L, Oladimeji P, Cook L, Li Q, Mattingly RR, et al. Tyrosylphosphorylated PAK1 regulates breast cancer cell motility in response toprolactin through filamin A. Mol Endocrinol 2013;27:455–65.

21. Hammer A, Oladimeji P, De Las Casas LE, Diakonova M. Phosphorylationof tyrosine 285 of PAK1 facilitates bPIX/GIT1 binding and adhesionturnover. FASEB J 2015;29:943–59.

22. Tao J, Oladimeji P, Rider L, Diakonova M. PAK1-Nck regulates cyclin D1promoter activity in response to prolactin. Mol Endocrinol 2011;25:1565–78.

23. Kim E, Youn H, Kwon T, Son B, Kang J, Yang HJ, et al. PAK1 tyrosinephosphorylation is required to induce epithelial-mesenchymal transitionand radioresistance in lung cancer cells. Cancer Res 2014;74:5520–31.

24. Bagheri-Yarmand R, Mandal M, Taludker AH, Wang RA, Vadlamudi RK,et al. Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicityof breast cancer cells. J Biol Chem 2001;276:29403–9.

25. Corbin JD, Reimann EM. Assay of cyclic AMP-dependent protein kinases.Methods Enzymol 1974;38:287–90.

26. Oladimeji P, Kubohara Y, Kikuchi H, Oshima Y, Rusch C, Skerl R,et al. A derivative of differentiation-inducing factor-3 inhibits PAK1activity and breast cancer cell proliferation. Int J Cancer Clin Res2015;2:1–6.

27. Deacon SW, Beeser A, Fukui JA, Rennefahrt UE, Myers C, Chernoff J, et al.An isoform-selective, small-molecule inhibitor targets the autoregulatorymechanism of p21-activated kinase. Chem Biol 2008;15:322–31.


Oladimeji et al.




28. Mazumdar A, Kumar R. Estrogen regulation of Pak1 and FKHRpathways inbreast cancer cells. FEBS Lett 2003;535:6–10.

29. Adam L, Vadlamudi R, Kondapaka SB, Chernoff J, Mendelsohn J, Kumar R.Heregulin regulates cytoskeletal reorganization and cell migration throughthe p21-activated kinase-1 via phosphatidylinositol-3 kinase. J Biol Chem1998;273:28238–46.

30. Renkema GH, Pulkkinen K, Saksela K. Cdc42/Rac1-mediated activationprimes PAK2 for superactivation by tyrosine phosphorylation. Mol CellBiol 2002;22:6719–25.

31. Roig J, Tuazon PT, Zipfel PA, Pendergast AM, Traugh JA. Functionalinteraction between c-Abl and the p21-activated protein kinase g-PAK.Proc Natl Acad Sci U S A 2000;97:14346–51.

32. McManus MJ, Boerner JL, Danielsen AJ, Wang Z, Matsumura F, Maihle NJ.An oncogenic epidermal growth factor receptor signals via a p21-activatedkinase-caldesmon-myosin phosphotyrosine complex. J Biol Chem 2000;275:35328–34.

33. Qiu Y, Robinson D, Pretlow TG, Kung HJ. Etk/Bmx, a tyrosine kinasewith a pleckstrin-homology domain, is an effector of phosphatidyli-nositol 30-kinase and is involved in interleukin 6-induced neuroendo-crine differentiation of prostate cancer cells. Proc Natl Acad Sci U S A1998;95:3644–9.

34. Ariazi EA, Brailoiu E, Yerrum S, Shupp HA, Slifker MJ, Cunliffe HE, et al.The G protein-coupled receptor GPR30 inhibits proliferation of estrogenreceptor-positive breast cancer cells. Cancer Res 2010;70:1184–94.

35. Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr. Estrogen-induced acti-vation of Erk-1 and Erk-2 requires the G protein-coupled receptor homo-log, GPR30, and occurs via trans-activation of the epidermal growth factorreceptor through release of HB-EGF. Mol Endocrinol 2000;14:1649–60.

36. Carmeci C, ThompsonDA, RingHZ, FranckeU,Weigel RJ. Identification ofa gene (GPR30) with homology to the G-protein-coupled receptor super-family associated with estrogen receptor expression in breast cancer.Genomics 1997;45:607–17.

37. FascoMJ, Amin A, Pentecost BT, Yang Y, Gierthy JF. Phenotypic changes inMCF-7 cells during prolonged exposure to tamoxifen. Mol Cell Endocrinol2003;206:33–47.

38. Li Z, Hannigan M, Mo Z, Liu B, Lu W, Wu Y, et al. Directional sensingrequires Gbg-mediated PAK1 and PIX a-dependent activation of Cdc42.Cell 2003;114:215–27.

39. Kumar P, Wu Q, Chambliss KL, Yuhanna IS, Mumby SM, Mineo C, et al.Direct interactions with Gai and Gbg mediate nongenomic signaling byestrogen receptor a. Mol Endocrinol 2007;21:1370–80.

40. Menard RE, Mattingly RR. Gbg subunits stimulate p21-activated kinase 1(PAK1) through activation of PI3-kinase and Akt but act independently ofRac1/Cdc42. FEBS Lett 2004;556:187–92.

41. Howe AK, Juliano RL. Regulation of anchorage-dependent signal trans-duction by protein kinase A and p21-activated kinase. Nat Cell Biol 2000;2:593–600.

42. Chahdi A, Miller B, Sorokin A. Endothelin 1 induces beta 1Pix transloca-tion and Cdc42 activation via protein kinase A-dependent pathway. J BiolChem 2005;280:578–84.

43. Bachmann VA, Riml A, Huber RG, Baillie GS, Liedl KR, Valovka T, et al.Reciprocal regulation of PKA and Rac signaling. Proc Natl Acad Sci U S A2013;110:8531–6.

44. Park MH, Lee HS, Lee CS, You ST, Kim DJ, Park BH, et al. p21-Activatedkinase 4 promotes prostate cancer progression through CREB. Oncogene2013;32:2475–82.

45. Wang RA, Mazumdar A, Vadlamudi RK, Kumar R. P21-activated kinase-1phosphorylates and transactivates estrogen receptor-a and promoteshyperplasia in mammary epithelium. Embo J 2002;21:5437–47.

46. Michalides R, Griekspoor A, Balkenende A, VerwoerdD, Janssen L, Jalink K,et al. Tamoxifen resistance by a conformational arrest of the estrogenreceptor a after PKA activation in breast cancer. Cancer Cell 2004;5:597–605.

47. Rayala SK, Talukder AH, Balasenthil S, Tharakan R, Barnes CJ, Wang RA,et al. P21-activated kinase 1 regulation of estrogen receptor-a activationinvolves serine 305 activation linked with serine 118 phosphorylation.Cancer Res 2006;66:1694–701.

48. Balasenthil S, Barnes CJ, Rayala SK, Kumar R. Estrogen receptor activationat serine 305 is sufficient to upregulate cyclinD1 in breast cancer cells. FEBSLett 2004;567:243–7.

49. Gonz�alez L, Zambrano A, Lazaro-Trueba I, Lop�ez E, Gonz�alez JJ, Martin-Per�ez J, et al. Activation of the unliganded estrogen receptor by prolactin inbreast cancer cells. Oncogene 2009;28:1298–308.

50. Rasmussen LM, Frederiksen KS, Din N, Galsgaard E, Christensen L,Berchtold MW, et al. Prolactin and oestrogen synergistically regulategene expression and proliferation of breast cancer cells. Endocr RelatCancer 2010;17:809–22.

51. Chen Y, Huang K, Chen KE, Walker AM. Prolactin and estradiol utilizedistinct mechanisms to increase serine-118 phosphorylation and decreaselevels of estrogen receptor alpha in T47D breast cancer cells. Breast CancerRes Treat 2010;120:369–77.

52. Sato T, Tran TH, Peck AR, Liu C, Ertel A, Lin J, et al. Global profiling ofprolactin-modulated transcripts in breast cancer in vivo. Mol Cancer2013;12:59.

53. Holm C, Kok M, Michalides R, Fles R, Koornstra RH, Wesseling J, et al.Phosphorylation of the oestrogen receptor a at serine 305 and pre-diction of tamoxifen resistance in breast cancer. J Pathol 2009;217:372–9.






2016;76:2600-2611. Published OnlineFirst March 4, 2016.Cancer Res Peter Oladimeji, Rebekah Skerl, Courtney Rusch, et al. Cancer Cells Requires Tyrosyl Phosphorylation of PAK1

by Estrogen and Prolactin in BreastαSynergistic Activation of ER

Updated version

10.1158/0008-5472.CAN-15-1758doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2016/03/04/0008-5472.CAN-15-1758.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/76/9/2600.full#ref-list-1

This article cites 53 articles, 16 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/76/9/2600.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/76/9/2600To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-15-1758

http://cancerres.aacrjournals.org/content/suppl/2016/03/04/0008-5472.CAN-15-1758.DC1

http://cancerres.aacrjournals.org/content/76/9/2600.full#ref-list-1

http://cancerres.aacrjournals.org/content/76/9/2600.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/76/9/2600


synergistic activation of era by estrogen and prolactin in ... · that activates ga and gbg...

Documents