Rapid Estrogen-Induced Phosphorylation of the SRC-3 Coactivator Occurs in an Extranuclear Complex Containing Estrogen Receptor

SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 is a primary transcriptionalcoregulator for estrogen receptor (ER). Six SRC-3 phosphorylationsites have been identified, and these can be induced by steroids,cytokines, and growth factors, involving multiple kinase signalingpathways. Using phosphospecific antibodies for six phosphorylationsites, we investigated the mechanisms involved in estradiol(E2)-induced SRC-3 phosphorylation and found that this occursonly when either activated estrogen receptor ${alpha}$ (ER ${alpha}$ ) or activatedERß is present. Both the activation function 1 andthe ligand binding domains of ER ${alpha}$ are required for maximal induction.Mutations in the coactivator binding groove of the ER ${alpha}$ ligandbinding domain inhibit E2-stimulated SRC-3 phosphorylation,as do mutations in the nuclear receptor-interacting domain ofSRC-3, suggesting that ER ${alpha}$ must directly contact SRC-3 for thisposttranslational modification to take place. A transcriptionallyinactive ER ${alpha}$ mutant which localizes to the cytoplasm supportsE2-induced SRC-3 phosphorylation. Mutations of the ER ${alpha}$ DNA bindingdomain did not block this rapid E2-dependent SRC-3 phosphorylation.Together these data demonstrate that E2-induced SRC-3 phosphorylationis dependent on a direct interaction between SRC-3 and ER ${alpha}$ andcan occur outside of the nucleus. Our results provide evidencefor an early nongenomic action of ER on SRC-3 that supportsthe well-established downstream genomic roles of estrogen andcoactivators.

INTRODUCTION

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Introduction

Steroid receptor coactivator 3 (SRC-3, also known as AIB1/ACTR/RAC3/pCIP/TRAM-1)is a member of the SRC/p160 family of transcription coactivatorsfor nuclear receptors and other transcription factors (2, 9,32, 36, 42, 58, 64, 65, 67, 77). Loss of SRC-3 expression incells severely impairs the transcriptional output from nuclearreceptors (71, 76). SRC-3 is overexpressed in a significantpercentage of breast cancers, and in its role as an oncogene,it is involved in the development and maintenance of breastand prostate cancers (2, 19, 27, 68, 79).

The activities of the SRC coactivators are affected by posttranslationalmodifications such as phosphorylation, acetylation, sumoylation,and ubiquitination (6, 10, 17, 22, 34, 74, 75). We and othershave shown that SRC-3 activity is regulated by phosphorylation,which dramatically affects its association with nuclear receptorsand other coregulators and transcription factors and its coactivatorfunctions, subcellular localization, and oncogenic activities(53, 68, 74, 75). This phosphorylation can be induced by differentstimuli including steroid hormones, growth factors, and cytokinesand involves a wide range of kinases including p42/p44 mitogen-activatedprotein kinase (MAPK), c-Jun N-terminal kinase, p38 MAPK, andI ${kappa}$ B kinases (IKKs) (17, 50, 75). Six in vivo SRC-3 phosphorylationsites have been identified (Fig. 1A), and phosphorylation state-specificantibodies against each site have been generated and validated(75). Different stimuli induce distinct patterns of SRC-3 phosphorylation,and mutations at different phosphorylation sites have differentdownstream effects. For example, 17ß-estradiol (E2)induces SRC-3 phosphorylation at all six sites, while tumornecrosis factor alpha (TNF- ${alpha}$ ) induces phosphorylation of allbut the serine-860 (S860) site (75). Consistent with these data,mutation of any of the six phosphorylation sites to an alanineresidue impairs the ability of the mutant SRC-3 to coactivateE2-induced estrogen receptor ${alpha}$ (ER ${alpha}$ ) target gene expression inreporter assays, while all but S860 mutations affect TNF- ${alpha}$ -inducedNF- ${kappa}$ B target gene activation. Likewise, mutation of the threonine-24(T24), S543, S857, and S867 sites, but not the S505 and S860sites, negatively influences the expression of the SRC-3 targetgene interleukin-6, while the tumorigenic activity of SRC-3,demonstrated via its ability to potentiate cellular transformationby Ras^V12, is affected by mutation of all the phosphorylationsites except S505 (75). These observations likely result fromthe different affinities of various SRC-3 phosphorylation sitemutants for other coregulators and transcription factors suchas CBP, CARM1, ER ${alpha}$ , and NF- ${kappa}$ B (75).

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FIG. 1. Estradiol induction of SRC-3 phosphorylation. (A) Schematic diagram of SRC-3. The known functional domains of SRC-3 are shown on top: bHLH/PAS, basic helix-loop-helix/Per-Arnt-Sim domain; RID, receptor-interacting domain; CID, CBP/p300-interacting domain; HAT, histone acetyltransferase domain. The identity and position of the six identified phosphorylated amino acids of SRC-3 are shown below. (B) Estradiol induced an ER-dependent change in electrophoretic mobility of SRC-3. HEK293T cells were transfected with plasmids for either Flag-tagged wild-type SRC-3 (SRC-3) or a Flag-SRC-3 mutant (A6) in which alanines were substituted for all six phosphorylation sites (S/T to A), and either ER ${alpha}$ or ERß, or empty vectors (V). Forty-eight hours posttransfection, 0.01% ethanol (–) or 10 nM estradiol (E2; +) was added for 1 h. Cells were then lysed and analyzed by Western blotting using an anti-SRC-3 antibody.

How E2 induces phosphorylation of SRC-3 is poorly understood.The major biological functions of E2 are mediated through ER ${alpha}$ and ERß, which are members of the nuclear receptorsuperfamily of ligand-dependent transcription factors (46).Estradiol can induce cellular responses through direct "genomic"ER-dependent activation of gene transcription at target promotersas well as by "nongenomic" actions, the latter including rapidactivation of various protein kinase cascades independent ofprior gene transcription (3). Nongenomic E2-activated pathwaysmay be ER dependent. For example, E2 can induce a rapid andtransient activation of the Src/Erk phosphorylation cascadethrough the association between cytoplasm-localized ER ${alpha}$ and MNAR(modulator of nongenomic activity of ER) (73). Alternatively,in an ER-independent fashion, E2 has been shown to activatethe MAPK pathway through the membrane-bound G protein-coupledreceptor GPR30 (16, 37). Whether E2-induced SRC-3 phosphorylationis mediated through a "genomic" or "nongenomic" activity ofE2 and its dependence on ER is unclear at present.

Estrogen receptors regulate the differentiation and maintenanceof many tissues including reproductive, neural, skeletal, andcardiovascular tissues (26) and possess several domains essentialfor their functions (46). The transcriptional activity of ERis dependent on two activation function (AF) domains, AF-1 atthe N terminus and AF-2 in the C-terminal ligand binding domain(LBD). The AF-1 domain can exert its activity in a ligand-independentmanner (15, 44, 69), while AF-2 activity is dependent on agonistbinding to the LBD (28). Both AF-1 and AF-2 activities are associatedwith their abilities to bind to coregulators. The DNA bindingdomain (DBD) contains two zinc finger motifs, which bind tospecific estrogen response elements (EREs) within the promotersof ER target genes (25). The first zinc finger contains a Pbox directly contacting DNA, and the latter contains a D boxresponsible for ER dimerization (57). The hinge region whichis located between DBD and LBD contains most of the nuclearlocalization signal (NLS) (51). Estrogen receptor shuttles betweennucleus and cytoplasm, although under steady-state conditionsit is mainly detected in the nucleus (14, 39). In the absenceof ligand, 5 to 10% of green fluorescent protein (GFP)-ER ${alpha}$ islocalized in the cytoplasm (39, 78). Estradiol increases ERnuclear localization. It also has been shown that E2 inducesa translocalization of SRC-3 from the cytoplasm to the nucleus(53, 71, 74). Up to now, the "classical" mode of activationof coactivators and their interaction with transcription factorswere believed to occur on nuclear promoters in response to differentstimuli (77).

The ligand-dependent interaction between nuclear receptors andSRC-3 is direct and is mediated through the LBD domains andthe LXXLL motifs, respectively (9, 21, 67). Upon binding toan agonist, ER ${alpha}$ undergoes a conformational change and presentsa coactivator-binding groove on the surface of its LBD capableof binding to the LXXLL motifs (43, 59). This coactivator-bindinggroove involves residues (e.g., K362, V376, and L539) from severalhelices, H3 to H5 and H12 (38). After coactivator binding, ER ${alpha}$ LBD undergoes a further conformational change, resulting ina more stable receptor (66). There are three LXXLL motifs locatedin the receptor-interacting domain of SRC-3 (9, 21, 67). Mutationsthat disrupt the formation of the coactivator-binding grooveof ER ${alpha}$ or mutations in the LXXLL motifs of SRC-3 abolish interactionbetween SRC-3 and ER ${alpha}$ and thus coactivation of ER ${alpha}$ (38).

Here we show that E2-induced SRC-3 phosphorylation requiresa direct interaction between SRC-3 and ER ${alpha}$ but does not requireDNA binding and nuclear localization of ER ${alpha}$ , indicating a "nongenomic"action of this receptor. Both ER ${alpha}$ AF-1 and AF-2 domains are requiredfor a maximal induction of coactivator phosphorylation. Theseresults indicate that, in addition to "genomic" and transcription-dependentactivities of ER ${alpha}$ , "nongenomic" activities of ER ${alpha}$ can significantlyinfluence SRC-3 functions, suggesting a multilevel functionalcooperation between coactivators and nuclear receptors on andoff their target genes.

MATERIALS AND METHODS

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Materials and Methods

Chemicals and reagents. 17ß-Estradiol (E2), progesterone (P), 4-hydroxytamoxifen(4HT), and raloxifene (Ral) were obtained from Sigma-Aldrich(St. Louis, MO). ICI 182,780 was from Tocris Cookson (Ellisville,MO). Phosphorylation state-specific antibodies against SRC-3were characterized previously (75). Antibody against SRC-3 wasfrom BD Biosciences (San Jose, CA) when used for Western blottingor from Santa Cruz Biotechnology (Santa Cruz, CA) when usedfor immunoprecipitation. The EZview red anti-Flag M2 affinitygel used for immunoprecipitating Flag-tagged proteins was fromSigma-Aldrich. Anti-ER ${alpha}$ mouse monoclonal antibody was from CellSignaling Technology (Beverly, MA), and anti-ER ${alpha}$ rabbit polyclonalantibody was from Santa Cruz Biotechnology. Anti-ERßantibody was from GeneTex, Inc. (San Antonio, TX). Antibodyagainst progesterone receptor B (PR-B) was described previously(1). The small interfering RNA (siRNA) against human ER ${alpha}$ (siGENOMESMARTpool reagent) was purchased from Dharmacon (Lafayette,CO). The siRNA against luciferase was purchased from Ambion(Austin, TX). Transfection reagents (TransIT-LT1 and TransIT-TKO)were from Mirus Bio Corporation (Madison, WI).

Plasmids. The expression plasmid for human Flag-tagged SRC-3 (pCMV-Flag-SRC-3)was generated as described previously (75). The wild-type full-lengthcDNA of SRC-3 was subcloned from the plasmid pCMX-F.RAC3 (32).pCMV-Flag-SRC3-A6 is an expression plasmid for human Flag-taggedSRC-3 mutant (A6) in which all six identified phosphorylationsites (Fig. 1A) have been mutated to alanine (75). The expressionplasmid pCMV-Flag-SRC3-AAA encodes a Flag-tagged SRC-3 mutant(AAA) in which all three LXXLL motifs have been mutated to LXXAAand was constructed by replacing the AflII-XbaI fragment ofpCMV-Flag-SRC3 with the corresponding region of pCMX-ACTRAAA(36) containing the desired mutations. The expression plasmidsfor human ER ${alpha}$ (pCR3.1-hER ${alpha}$ [45], pCMV₅-hER ${alpha}$ , pCMV₅-hER ${alpha}$ phosphorylationmutant [S104A/S106A/S118A as 3A] [30], pRST₇hER ${alpha}$ , pRST₇hER ${alpha}$ -N282G[amino acids (aa) 1 to 282], and pRST₇hER ${alpha}$ -179C [aa 179 to 595][70]), human ERß (pCXN₂-hERß [47] and pCR3.1hERß-143C[aa 143 to 530] [11]), and human PR-B (pCR3.1-hPRB [1]) weredescribed previously.

The expression plasmids (pCR3.1-hER ${alpha}$ -K362D, pCR3.1-hER ${alpha}$ -V376D,or pCR3.1-hER ${alpha}$ -L539A) for full-length hER ${alpha}$ with single point mutations(K362D, V376D, or L539A) were described previously (34). Theexpression plasmid (pCR3.1-hER ${alpha}$ KV) for the hER ${alpha}$ double mutant(K362D/V376D) was constructed as follows. PCR amplificationwas carried out using the forward primer 5'-TACGGCCCCGGGTCTGAGGCT-3'containing an XmaI site (underlined) and the reverse primer5'-ACATTCTAGAAGGTGGTCCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACCCTATCCGCCCA-3'containing the K362D and V376D mutations (italics) and an XbaIsite (underlined) to amplify a region of the hER ${alpha}$ cDNA (pCR3.1-hER ${alpha}$ ).The PCR product was then digested with XmaI and XbaI and usedto replace the corresponding region of pCR3.1-hER ${alpha}$ . The expressionplasmid (pCR3.1-hER ${alpha}$ KVL) for the hER ${alpha}$ triple mutant (K362D/V376D/L539Aas KVL) was constructed by replacing a BglII-ApaI fragment ofpCR3.1-hER ${alpha}$ KV with the corresponding region of pCR3.1-hER ${alpha}$ L539Acontaining the L539A mutation. All the mutant plasmid constructsdescribed above were sequenced to ensure that only the intendedmutations were introduced.

The expression plasmids HEG0 and HE241G were described previously(78). The plasmid HEG0 encodes a wild-type human ER ${alpha}$ , and HE241Gencodes a human ER ${alpha}$ mutant (ER ${alpha}$ ${Delta}$ NLS) with a deletion of aa 250to 303, which encodes an NLS. The expression plasmid encodingthe GFP-tagged human ER ${alpha}$ , pEGFP-C1-hER ${alpha}$ (GFP-ER ${alpha}$ WT), was a giftfrom M. A. Mancini (Baylor College of Medicine) (63). The expressionplasmid encoding the GFP-tagged ER ${alpha}$ ${Delta}$ NLS, pEGFP-C1-hER ${alpha}$ ${Delta}$ NLS (GFP-ER ${alpha}$ ${Delta}$ NLS),was constructed by replacing the XmaI-BlpI fragment of pEGFP-C1-hER ${alpha}$ with the corresponding region of the plasmid HE241G containingthe ${Delta}$ NLS mutation.

The expression plasmid pCR3.1-hER ${alpha}$ CC, which encodes an hER ${alpha}$ mutantcontaining the double mutation (C202H/C205H as CC) within theDBD, was described previously (34). The expression plasmid pCR3.1-hER ${alpha}$ EGencodes an hER ${alpha}$ mutant containing the double mutation (E203A/G204Aas EG) within the DBD. It was constructed by replacing the XmaI-BglIIfragment of pCR3.1-hER ${alpha}$ with the corresponding region of pHAhER ${alpha}$ E203AG204A(a gift of P. Brown, Baylor College of Medicine).

Cell cultures and transfections. HEK293T and MCF-7 cells were obtained from the American TypeCulture Collection and maintained in Dulbecco's modified Eagle'smedium supplemented with 10% fetal bovine serum. Twenty-fourhours prior to transfection, cells were plated in phenol red-freeDulbecco's modified Eagle's medium with 5% charcoal-strippedfetal bovine serum. For plasmid DNA transfection, cells weretransfected using TransIT-LT1 (3 µl LT1/µg plasmidDNA), while for siRNA transfection, cells were transfected usingTransIT-TKO (30 µl TKO/0.1 nmol siRNA) according to themanufacturer's protocol. For immunoprecipitating SRC-3 and itsassociated proteins, HEK293T cells (6 x 10⁶ cells/10-cm dish)were transfected with a total of 10 µg of plasmid DNA(4 µg of the plasmid DNA encoding Flag-tagged SRC3 orthe empty vector pCMVTag2B [Stratagene, La Jolla, CA]; 2 µgof the plasmid DNA encoding hER ${alpha}$ , hERß, hPR-B, or theempty vector pCR3.1 [Invitrogen, Carlsbad, CA]; and 4 µgof the plasmid pBluescript [Stratagene]) for 48 h. Cells werethen treated with various nuclear receptor agonists or antagonists(10 nM) or ethanol (0.01%) for different periods of time. Forluciferase reporter assays, HEK293T cells (2 x 10⁵ cells/wellin six-well plates) were transfected with 1 µg of plasmidDNA (5 ng of the plasmid DNA encoding hER ${alpha}$ or the empty vectorpCR3.1, 500 ng of pERE-E1b-Luc reporter plasmid [45], and 495ng of pBluescript) for 24 h and treated with E2 (10 nM) or ethanol(0.01%) for an additional 20 h. For immunofluorescence, HEK293Tcells (6 x 10⁵ cells/well in six-well plates with coverslips)were transfected with 1 µg of plasmid DNA (400 ng of theplasmid DNA encoding Flag-tagged SRC-3, 200 ng of the plasmidDNA encoding hER ${alpha}$ , and 400 ng of the pBluescript DNA) for 48h. Cells were then treated with E2 (10 nM) or ethanol (0.01%)for 1 h.

Immunoprecipitation and Western blotting. Cell lysates were prepared by incubating cells in the lysisbuffer (20 mM Tris-HCl [pH 7.5], 1% Triton X-100, 150 mM NaCl,1 mM EDTA, 1 mM EGTA, 5% glycerol, supplemented with phosphataseinhibitors [1 mM NaF, 1 mM Na₃VO₄, 1 mM ß-glycerophosphate,and 1 mM sodium pyrophosphate], and protease inhibitor cocktail[Roche, Indianapolis, IN]) for 20 min at 4°C, followed bycentrifugation at 14,000 x g for 15 min at 4°C. The proteinconcentration of the lysates was determined using the BCA proteinassay kit according to the manufacturer's protocol (Pierce,Rockford, IL). For immunoprecipitating Flag-tagged SRC-3 andits associated proteins, cell lysates containing 500 µgof protein were incubated with EZview red anti-Flag M2 affinitygel (30 µl of packed beads) for 2 h at 4°C with constantrotation. For immunoprecipitating endogenous SRC-3, MCF-7 cellswere lysed and 500 µg of protein was incubated with ananti-SRC-3 antibody (1 µg) for 2 h at 4°C with constantrotation; protein G PLUS-Agarose beads (30 µl of packedbeads; Santa Cruz Biotechnology) were then added, and the incubationwas continued for an additional 2 h. Beads were then washedthree times using the lysis buffer. Between washes, beads werecollected by centrifugation at 8,000 x g for 30 s at 4°C.The precipitated proteins were eluted from beads by resuspendingthe beads in NuPage sample buffer (Invitrogen) and heating themat 75°C for 10 min. The resulting materials from immunoprecipitationor total cell lysates for detecting total protein levels wereseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) using 3 to 8% NuPage Novex gels (Invitrogen) andtransferred onto nitrocellulose membranes. For Western blotanalysis, membranes were first blocked using 5% nonfat dry milkin TBS-T buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1%Tween 20) for 1 h at room temperature (RT) and then blottedusing indicated antibodies which were diluted in TBS-T bufferfor 1 h at RT or overnight at 4°C followed by incubationwith the appropriate horseradish peroxidase-conjugated secondaryantibodies for 1 h at RT. Immunoreactive bands were visualizedusing ECL Plus reagents as recommended by the manufacturer (AmershamBiosciences, Piscataway, NJ). The experiments were repeatedat least two times, and representative results are shown inthe figures.

Luciferase reporter assay. After transfection and treatment, HEK293T cells were harvested,and extracts were assayed for luciferase activity using theLuciferase Assay Systems kit (Promega, Madison, WI), which wasmeasured using a Luminoskan Ascent Thermo Labsystems instrument(Thermo Electron Corporation, Vantaa, Finland). Relative luciferaseunits were normalized to total cellular protein and standardizedto a negative control (cells transfected with empty vector andtreated with ethanol) which is set as 1. Experiments were donein duplicate, and values represent averages ± standarderrors of the means of at least three individual experiments.

Immunofluorescence microscopy. HEK293T cells were prepared according to the protocol describedpreviously (63). Briefly, after transfection and treatment,cells on the coverslips were fixed in 4% formaldehyde (PolysciencesInc., Warrington, PA) in PEM buffer {80 mM potassium PIPES [piperazine-N,N'-bis(2-ethanesulfonicacid)] [pH 7.5], 5 mM EGTA, and 2 mM MgCl₂ } for 30 min on ice.Cells were then washed three times with PEM buffer and incubatedfor 10 min in 0.1 M ammonium chloride to quench autofluorescence.The cells were then washed two times in PEM buffer and incubatedin 0.5% Triton X-100 in PEM buffer for 5 min to permeabilizethe cells. After being washed three times with PEM buffer, cellswere counterstained for 1 min with 1 µg/ml 4,6-diamidino-2-phenylindole(DAPI) in TBS-T and mounted in Slow Fade reagent (MolecularProbes/Invitrogen). Cells on the coverslips were examined ona Carl Zeiss AxioPhot microscope, and GFP and DAPI signals wererecorded using a color charge-coupled device camera with fluoresceinand DAPI filters. Images were processed using Adobe PhotoshopCS (Adobe Systems Incorporated, San Jose, CA).

RESULTS

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Results

Estradiol induces a mobility change of SRC-3 on SDS-PAGE gels in an ER-dependent manner. It is well known that posttranslational modifications of proteins(e.g., phosphorylation) can change their electrophoretic mobilityon SDS-PAGE gels. Protein modification pathways can be activatedby various stimuli including hormones and growth factors. Toinvestigate the effect of estradiol (E2) on the migration patternof SRC-3 on an SDS-PAGE gel, E2 induction (10 nM, 1 h) was performedin HEK293T cells transiently transfected with Flag-tagged wild-typeSRC-3 (SRC-3) with or without ERs. As shown in Fig. 1B, E2 treatmentcaused an upward smearing migration pattern of SRC-3 in thepresence of ER ${alpha}$ or ERß (lanes 4 and 6). The mobilityshift was not observed in the absence of ER (Fig. 1B, lane 2).Treatment with E2 did not change the mobility pattern of anA6 SRC-3 mutated at all six phosphorylation sites (Fig. 1B,lanes 7 and 8). These results indicated that E2 could alterthe electrophoretic mobility of the wild-type SRC-3 but notits phosphorylation-site A6 mutant, implying that the E2-inducedmobility change of SRC-3 is the result of SRC-3 phosphorylation.Since this was observed only in the presence of an ER, the E2-inducedSRC-3 mobility shift depends on expression of either ER ${alpha}$ or ERß.

Estradiol-induced SRC-3 phosphorylation requires ER. In cells that express ER ${alpha}$ , E2 induces SRC-3 phosphorylation atall six phosphorylation sites identified (75). To determinewhether E2-induced SRC-3 phosphorylation is dependent on thepresence of ER, HEK293T cells transfected with SRC-3 togetherwith or without either ER ${alpha}$ or ERß were treated withE2. SRC-3 phosphorylation state-specific antibodies were usedto determine the level of phosphorylation of immunoprecipitatedFlag-tagged SRC-3. As shown in Fig. 2A and B, E2 induced SRC-3phosphorylation at all six sites (T24, S505, S543, S857, S860,and S867) in cells transfected with an expression vector forwild-type ER ${alpha}$ (Fig. 2A, lanes 3 and 4, rows 1 to 6) or ERß(Fig. 2B, rows 1 to 6), but not in cells transfected with anempty expression vector (Fig. 2A, lanes 1 and 2, rows 1 to 6).As a negative control, E2-induced SRC-3 phosphorylation wasnot observed when the SRC-3 phosphorylation mutant A6 was used(Fig. 2A, lanes 5 and 6, rows 1 to 6), even though the A6 formof SRC-3, like wild-type SRC-3, could still bind to ER ${alpha}$ in responseto E2 (Fig. 2A, lane 6, row 7). These results indicated thatE2-induced SRC-3 phosphorylation requires either form of ER.

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FIG. 2. Estradiol-induced SRC-3 phosphorylation requires the presence of ER. (A) HEK293T cells were transfected with Flag-tagged SRC-3 (SRC-3) or its phosphorylation mutant (A6), in the absence or presence of ER ${alpha}$ . Forty-eight hours later, 10 nM E2 (+) or 0.01% ethanol vehicle (–) was added for 1 h. Cells were then lysed, and Flag-SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. Immunoblots were probed with antibodies against each of the six SRC-3 phosphorylation sites (rows 1 to 6). The amount of SRC-3-bound ER ${alpha}$ was detected by probing with an anti-ER ${alpha}$ antibody (row 7). The total amounts of ER ${alpha}$ and SRC-3 in the lysates were determined by immunoblotting with an anti-ER ${alpha}$ and an anti-SRC-3 antibody (rows 8 and 9, respectively). (B) Cells were transfected with plasmids for wild-type SRC-3 and ERß, and the level of SRC-3 phosphorylated at each of the six sites was assessed as described above. (C) siRNA against ER ${alpha}$ (siER ${alpha}$ ) or against luciferase (siLuc) was transfected into MCF-7 cells. Cells were treated as described above. Cells were then lysed, and endogenous SRC-3 was immunoprecipitated using an anti-SRC-3 antibody and separated by SDS-PAGE. The levels of SRC-3 phosphorylated at T24, S505, and S543 were assessed. (D) Cells were transfected as in panel A and treated with 10 nM E2 (+) or 0.01% ethanol (–) for various periods of time (min). The level of SRC-3 phosphorylated at T24, S505, and S543 and the amount of SRC-3-bound ER ${alpha}$ were assessed. Shown in the bottom two rows of all the panels are total cell lysates separated by SDS-PAGE and probed with the anti-ER ${alpha}$ , anti-ERß, or anti-SRC-3 antibodies to assess the total cellular levels of ER ${alpha}$ , ERß, and SRC-3, respectively.

In order to confirm the ER dependence with endogenous proteins,siRNA against ER ${alpha}$ (siER ${alpha}$ ) was transfected into MCF-7 cells toreduce the expression of endogenous ER ${alpha}$ . Forty-eight hours posttransfection,cells were treated with E2 (10 nM, 1 h), and phosphorylationof immunoprecipitated endogenous SRC-3 was determined usingSRC-3 phosphorylation state-specific antibodies as describedabove. As shown in Fig. 2C, in the presence of siER ${alpha}$ , expressionof endogenous ER ${alpha}$ was greatly reduced (lanes 3 and 4, row 4),and E2-induced phosphorylation of endogenous SRC-3 was abolished(lanes 3 and 4, rows 1 to 3). In contrast, SRC-3 phosphorylationwas still induced when a control siRNA against luciferase (siLuc)was used (lanes 1 and 2, rows 1 to 3). This confirmed the datafrom transient-transfection experiments indicating that E2-inducedphosphorylation of SRC-3 requires ER.

The kinetics of E2-induced SRC-3 phosphorylation also were assessedin HEK293T cells cotransfected with Flag-tagged SRC-3 and ER ${alpha}$ .As shown in Fig. 2D, SRC-3 phosphorylation could be observedby 15 min after E2 treatment, represented by phosphorylationat sites T24, S505, and S543 (lanes 3 to 7, rows 1 to 3).

Selectivity of hormones and nuclear receptors for induction of SRC-3 phosphorylation. It has been shown that E2 and the synthetic androgen R1881 caninduce SRC-3 phosphorylation in the presence of ER and androgenreceptor, respectively (Fig. 2) (75). In order to investigatewhether other hormones and their cognate nuclear receptors caninduce SRC-3 phosphorylation, HEK293T cells transfected withan SRC-3 expression vector in the absence or presence of progesteronereceptor B (PR-B) plasmid were treated with progesterone (P;10 nM, 1 h). As shown in Fig. 3A, P did not induce SRC-3 phosphorylationin the presence of PR-B (lanes 5 and 6), although in parallelexperiments E2 induced ER-dependent SRC-3 phosphorylation (lanes3 and 4). Representative data for SRC-3 phosphorylation at S505and S867 were shown, and other sites had a similar pattern (datanot shown). As in the case of PR-B, dexamethasone did not induceSRC-3 phosphorylation in cells expressing the glucocorticoidreceptor (data not shown). These data indicated that differentsteroids and their receptors have differential effects on SRC-3phosphorylation.

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FIG. 3. Effects of different agonists and antagonists and their receptors on SRC-3 phosphorylation. HEK293T cells were transfected with Flag-tagged wild-type SRC-3 (SRC-3) or its phosphorylation-site mutant (A6) together with either ER ${alpha}$ (A and B), ERß (C), or progesterone receptor B (PR-B) (A). Forty-eight hours posttransfection, vehicle (–), 10 nM E2, 10 nM progesterone (P), 10 nM 4HT, 10 nM ICI 182,780 (ICI), or 10 nM Ral was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The levels of SRC-3 phosphorylated at S505 and S867 were assessed by Western blotting using SRC-3 phosphorylation state-specific antibodies. Total cellular levels of ER ${alpha}$ , ERß, SRC-3, and PR-B were also determined by immunoblotting total cell lysates separated by SDS-PAGE with the appropriate antibodies.

SERMs do not induce SRC-3 phosphorylation. In order to investigate whether selective estrogen receptormodulators (SERMs) such as 4HT and Ral and pure antagonistssuch as ICI 182,780 (ICI) can induce SRC-3 phosphorylation,drug treatment (10 nM, 1 h) was performed in HEK293T cells cotransfectedwith plasmids for Flag-tagged SRC-3 and ER ${alpha}$ (Fig. 3B) or ERß(Fig. 3C). Tamoxifen and ICI (Fig. 3B) and 4HT and Ral (Fig.3C), unlike E2, did not noticeably induce SRC-3 phosphorylationat sites S505 and S867 in the presence of ER ${alpha}$ and ERß,respectively (lanes 3 and 4, rows 1 and 2). ICI treatment significantlyreduced the level of total ER ${alpha}$ (Fig. 3B, lane 4, row 3) as reportedpreviously (13, 18, 54). Since SERMs do not promote interactionbetween SRC coactivators and ER in HEK293T (data not shown)and other cell lines (23, 48), the data are consistent witha requirement for a direct binding between ER and SRC-3 forinduction of coactivator phosphorylation.

AF-1 and LBD/AF-2 of ER ${alpha}$ are required for maximal induction of SRC-3 phosphorylation. The results above demonstrated that ER is required for E2-inducedSRC-3 phosphorylation and implied that an interaction, eitherdirect or indirect, between ER and SRC-3 is required. In orderto investigate the contribution of the AF-1 and AF-2 domainsof ER ${alpha}$ for E2-induced SRC-3 phosphorylation, wild-type full-lengthER ${alpha}$ and an ER ${alpha}$ truncation mutant, ER ${alpha}$ 1-282 (N282) lacking AF-2/LBDor ER ${alpha}$ 179-595 (179C) lacking AF-1, together with Flag-taggedSRC-3, were transfected into HEK293T cells. Cells were thentreated, and SRC-3 phosphorylation was examined. As shown inFig. 4A, N282 did not support E2-induced SRC-3 phosphorylation(lanes 3 and 4), which is not surprising since N282 lacks anLBD and is not responsive to E2. The 179C mutant supported onlyminimal E2-induced SRC-3 phosphorylation at S505 but not atother sites as represented by T24, S543, and S867 (Fig. 4A,lanes 5 and 6). Similarly, an ERß truncation mutant,143C (aa 143 to 530), lacking AF-1, supported only minimal E2-inducedSRC-3 phosphorylation at S505 and S543 but not at other sitesas represented by T24 and S867 (Fig. 4B). These data revealthat the AF-1 or LBD/AF-2 domains are required for maximal inductionof E2-induced coactivator phosphorylation.

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FIG. 4. Both AF-1 and LBD/AF-2 domains of ER ${alpha}$ are required for maximal induction of SRC-3 phosphorylation in response to E2. HEK293T cells were cotransfected with either Flag-SRC-3 (SRC-3) or Flag-SRC-3 phosphorylation mutant (A6) together with either the full-length wild-type ER ${alpha}$ (ER ${alpha}$ ) (A and C), or ER ${alpha}$ 1-282 (N282) or ER ${alpha}$ 179-595 (179C) (A), or the full-length wild-type ERß (ERß) or ERß143-530 (143C) (B), or ER ${alpha}$ AF-1 phosphorylation mutant 3A (S104/106/118A) (C). Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. Immunoblots were probed with SRC-3 phosphorylation state-specific antibodies for T24, S505, S543, and S867. Total cellular levels of wild-type ER and its mutants and SRC-3 were also determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies.

To further test the role of AF-1 in SRC-3 phosphorylation, anER ${alpha}$ phosphorylation mutant (S104A/S106A/S118A as 3A) was usedto determine whether phosphorylation of AF-1 is required. HEK293Tcells were transfected with either wild-type ER ${alpha}$ or its mutant3A together with Flag-tagged SRC-3 and treated as describedabove. Interestingly, this ER ${alpha}$ AF-1 phosphorylation mutant couldstill induce SRC-3 phosphorylation, at a level comparable tothe wild-type ER ${alpha}$ (Fig. 4C), suggesting that phosphorylationof ER ${alpha}$ is not a prerequisite for promoting SRC-3 phosphorylation.

The coactivator-binding groove of ER ${alpha}$ is required for E2-induced SRC-3 phosphorylation. It has been shown that the amino acids K362, V376, and L539of the ER ${alpha}$ LBD are among the residues critical for forming acoactivator-binding groove in response to E2 (38). In orderto investigate whether this surface is essential for E2-inducedSRC-3 phosphorylation, several mutations which disrupt the directinteraction of ER ${alpha}$ with coactivator were examined for their abilityto impact E2 induction of SRC-3 phosphorylation in HEK293T cells.The mutants (the single mutants K362D [K], V376D [V], and L539A[L]; the double mutant K362D/V376D [KV]; and the triple mutantK362D/V376D/L539A [KVL]) were transcriptionally impaired asdemonstrated in a luciferase reporter assay (Fig. 5A), consistentwith previous reports on the effect of the single mutations(34). As shown in Fig. 5B, the ER ${alpha}$ single mutants (K, V, or L)were severely compromised in their ability to support E2-inducedSRC-3 phosphorylation (lanes 5 to 10, row 1), while the triplemutant (KVL) was unable to induce any phosphorylation of SRC-3(lanes 3 and 4, row 1). As predicted, the E2-induced interactionbetween these ER ${alpha}$ mutants and SRC-3 was greatly reduced or totallyabolished (Fig. 5B, lanes 3 to 10, row 2) in comparison to thepositive control (lanes 1 and 2, row 2). These data indicatedthat an intact coactivator-binding groove within the LBD isessential for E2-induced SRC-3 phosphorylation and imply thata direct interaction between SRC-3 and ER ${alpha}$ is necessary.

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FIG. 5. The coactivator-binding groove of ER ${alpha}$ is required for estradiol-induced and ER-dependent SRC-3 phosphorylation. (A) HEK293T cells were cotransfected with pERE-E1b-Luc and either the empty vector (Vector), the ER ${alpha}$ wild type (WT), the ER ${alpha}$ single mutants (K362D as K, V376D as V, or L539A as L), the ER ${alpha}$ double mutant (K362D/V376D as KV), or the ER ${alpha}$ triple mutant (K362D/V376D/L539A as KVL). Twenty-four hours posttransfection, vehicle or E2 (10 nM) was added for an additional 20 h before cells were harvested for luciferase assays. The error bars represent the standard errors of the means of three individual experiments. (B) HEK293T cells were transfected with either Flag-SRC-3 or Flag-SRC-3 phosphorylation mutant (A6) together with full-length wild-type, single mutant, or triple mutant forms of ER ${alpha}$ . Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The level of SRC-3 phosphorylated at S505 was assessed by Western blotting using an SRC-3 phosphorylation state-specific antibody. The level of SRC-3-bound ER ${alpha}$ was determined by immunoblotting with an anti-ER ${alpha}$ antibody. Total cellular levels of wild-type ER ${alpha}$ and its mutants and SRC-3 were also determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies.

The LXXLL motifs of SRC-3 are required for E2-induced SRC-3 phosphorylation. SRC family members contain several LXXLL motifs located in theirreceptor-interacting domains that mediate their direct bindingto the LBD of nuclear receptors such as ER ${alpha}$ . The SRC-3-AAA mutanthas two amino acid substitutions within each of its three NRboxes, such that the LXXLL motifs are mutated to LXXAA (36);this abolishes the ligand-dependent binding between SRC-3 andthe ER ${alpha}$ LBD (10, 12, 21, 40). To investigate whether the SRC-3-AAAmutant can be phosphorylated in response to E2, plasmids forthe SRC-3 mutant and ER ${alpha}$ were transfected into HEK293T cells.As shown in Fig. 6, unlike the wild-type SRC-3 (lanes 2 and3), the AAA mutant could not be phosphorylated in response toE2 treatment (lanes 4 and 5), substantiating the notion thata direct ligand-induced interaction between coactivator andER ${alpha}$ is necessary for E2-dependent SRC-3 phosphorylation. Theinteraction observed between the AAA mutant and ER ${alpha}$ in the absenceof E2 (Fig. 6, lane 4, row 7) likely reflects a hormone- andLXXLL-independent interaction of the ER ${alpha}$ AF-1 domain with SRC-3(72), which is inhibited by E2 as a result of ligand-inducedinteractions of the N and C termini of the receptor (41).

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FIG. 6. The receptor-interacting domain of SRC-3 is required for estradiol-induced SRC-3 phosphorylation. HEK293T cells were transfected with either wild-type Flag-SRC-3 (SRC-3) or SRC-3 mutant (AAA) having all three LXXLL motifs in the receptor-interacting domain mutated to LXXAA, together with wild-type ER ${alpha}$ . Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The level of SRC-3 phosphorylated at each of the six phosphorylation sites was assessed by immunoblotting with each SRC-3 phosphorylation state-specific antibody (rows 1 to 6). The level of SRC-3-bound ER ${alpha}$ was determined by immunoblotting with an anti-ER ${alpha}$ antibody (row 7). Total cellular levels of wild-type SRC-3 and its mutant and ER ${alpha}$ were also determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies (rows 8 and 9).

Nuclear localization of ER ${alpha}$ is not required for E2-induced SRC-3 phosphorylation. To determine whether phosphorylation of SRC-3 can take placein the cytoplasm, an ER ${alpha}$ mutant (ER ${alpha}$ ${Delta}$ NLS) lacking amino acids 250to 303, which encompass the ER ${alpha}$ NLS, was used. This mutant wastranscriptionally impaired in a luciferase reporter assay andis localized predominantly in the cytoplasm in the absence ofligand (78). In order to determine whether SRC-3 overexpressionor ligand would affect the mutant vs subcellular localization,cells were transfected with vectors for Flag-tagged SRC-3 andeither wild-type or mutant ER ${alpha}$ and then treated with either E2or vehicle. In the presence of ligand, GFP-ER ${alpha}$ ${Delta}$ NLS was localizedin the cytoplasm, as opposed to the wild-type ER ${alpha}$ (GFP-hER ${alpha}$ WT),which was localized primarily in the nucleus (Fig. 7A), regardlessof the presence or absence (data not shown) of ligand. Next,the ability of the mutant to promote coactivator phosphorylationwas tested. As for wild-type ER ${alpha}$ , the mutant was able to induceSRC-3 phosphorylation at all six sites (Fig. 7B), indicatingthat nuclear localization of ER ${alpha}$ may not be required for inducingSRC-3 phosphorylation in response to E2. Furthermore, the mutantcould still bind to SRC-3 in the presence of E2 (Fig. 7B, lanes5 and 6, row 7). It is possible that there is a small amountof the ${Delta}$ NLS mutant receptor localized in the nucleus (78) whichmay contribute to SRC-3 phosphorylation. However, it shouldbe noted that, under the conditions used for these assays, thereis insufficient nuclear ER ${alpha}$ ${Delta}$ NLS to induce the transcription ofan ERE-containing reporter gene (data not shown) consistentwith a previous report (78).

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FIG. 7. Nuclear localization of ER ${alpha}$ is not required for estradiol-induced SRC-3 phosphorylation. (A) HEK293T cells were cotransfected with GFP-tagged wild-type ER ${alpha}$ (GFP-WT) or ER ${alpha}$ NLS deletion mutant lacking aa 250 to 303 (GFP- ${Delta}$ NLS), together with Flag-SRC-3. Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cell fixing and preparation were performed, and GFP fluorescence signals were determined microscopically (column 1). DNA was counterstained with DAPI (column 2), and the signals for GFP and DAPI were merged (column 3). (B) HEK293T cells were transfected with either Flag-SRC-3 wild type (SRC-3) or its phosphorylation mutant (A6), together with either wild-type ER ${alpha}$ (WT), or ER ${alpha}$ mutant ( ${Delta}$ NLS). Forty-eight hours posttransfection, E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The level of SRC-3 phosphorylated at each of the six phosphorylation sites was assessed by immunoblotting with each SRC-3 phosphorylation state-specific antibody (rows 1 to 6). The level of SRC-3-bound ER ${alpha}$ was determined by immunoblotting with an anti-ER ${alpha}$ antibody (row 7). Total cellular levels of ER ${alpha}$ and SRC-3 were determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies (rows 8 and 9).

ER ${alpha}$ DNA binding activity is not required for E2-induced SRC-3 phosphorylation. Previous data indicated that E2-induced SRC-3 phosphorylationrequires the formation of a complex containing SRC-3 and ER.Assembly of ER ${alpha}$ , coactivators, and general transcription factorsat target gene promoters is associated with recruitment of proteinkinases and receptor phosphorylation (8, 50). Whether E2-dependentSRC-3 phosphorylation requires ER ${alpha}$ to be bound to DNA was investigated.The receptor binds directly to estrogen response elements mainlythrough the P box in the first of the two functionally distinctcysteine-cysteine zinc fingers located within the DBD. Two ER ${alpha}$ DBD mutants were tested, ER ${alpha}$ CC and ER ${alpha}$ EG, each containing a doublemutation in different regions of the P box (C202H/C205H as CCand E203A/G204A as EG, respectively). Neither of the two ER ${alpha}$ DBD mutants could activate ERE-dependent transcription in aluciferase reporter assay (Fig. 8A). However, both of the CCand EG ER ${alpha}$ DBD mutants induced SRC-3 phosphorylation, representedby sites S505, S543, and S867 (Fig. 8B, lanes 5 and 6 and lanes7 and 8, respectively). The CC mutant appeared to be less effectiveat supporting SRC-3 phosphorylation than wild-type ER ${alpha}$ was (lanes3 and 4) or the EG mutant, even though the CC mutant binds toSRC-3 as well as does the wild-type receptor. In contrast, aweaker interaction between the EG mutant and SRC-3 was noted(Fig. 8B, row 4). It was observed that E2 treatment resultedin decreased SRC-3 levels in cells transfected with either ER ${alpha}$ DBD mutant (Fig. 8B, lanes 5 to 8, row 6); the reason for thisis unknown, but it does suggest that ER ${alpha}$ can exert control overSRC-3 levels in some contexts.

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FIG. 8. Estradiol-induced and ER-dependent SRC-3 phosphorylation does not require ER DNA binding. (A) HEK293T cells were cotransfected with pERE-E1b-Luc, and either the empty vector (Vector), or vectors for wild-type ER ${alpha}$ (WT), or the EG (E203A/G204A) or CC (C202H/C205H) double ER ${alpha}$ DNA-binding domain mutant. Twenty-four hours posttransfection, vehicle or E2 (10 nM) was added for an additional 20 h. The luciferase activity was assayed as described in Materials and Methods. (B) HEK293T cells were cotransfected with either Flag-SRC-3 wild type (SRC-3) or its phosphorylation mutant (A6), together with either the wild-type ER ${alpha}$ (ER ${alpha}$ ), or the ER ${alpha}$ double mutant EG or CC. Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The level of SRC-3 phosphorylated at S505, S543, and S867 was assessed by Western blotting using the SRC-3 phosphorylation state-specific antibody against each site (rows 1 to 3). The level of SRC-3-bound ER ${alpha}$ was determined by immunoblotting with an anti-ER ${alpha}$ antibody (row 4). Total cellular levels of wild-type ER ${alpha}$ and its mutants and SRC-3 were also determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies (rows 5 and 6).

DISCUSSION

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Discussion

The phosphorylation status of SRC family members has been shownto be important for their biological functions including theircoactivator activities and tumorigenic potentials (17, 20, 55,74, 75). Hormones and growth factors induce SRC phosphorylation,but the mechanisms are largely unknown. Estrogen can induceposttranslational modifications through either "genomic" or"nongenomic" pathways (3). The "genomic" effects of estradiolare mainly mediated by ERs over the time course of hours todays and relate to an activated receptor's ability to bind topromoters of estrogen-responsive target genes involved in cellularsignaling (3). The "nongenomic" effects of estrogen are mediatedthrough activation of cellular signaling pathways independentof transcription and can be either ER dependent or independent,occurring over a time course of seconds to minutes (3). Herewe report that E2-induced SRC-3 phosphorylation occurs withinminutes and requires a direct interaction between SRC-3 andER ${alpha}$ but does not require nuclear localization or the DNA bindingactivity of ER ${alpha}$ . However, both the AF-1 and LBD/AF-2 domainsof ER ${alpha}$ are essential for maximal E2 induction of SRC-3 phosphorylation.These results lead to a model in which the posttranslationalmodification of SRC-3 is the result of a transcription-independentand "nongenomic" action of ER that occurs in an extranuclearcomplex containing SRC-3 and ER ${alpha}$ .

We found that both the ER ${alpha}$ AF-1 and AF-2 domains are necessaryfor robust induction of SRC-3 phosphorylation by E2 and thata direct interaction between the ER ${alpha}$ LBD and coactivator is required.The inability of the SERMs, 4HT and raloxifene, to induce SRC-3phosphorylation is consistent with both of these points. Eventhough SERMs allow AF-1 to exert its ligand-independent function,they block AF-2 activity and inhibit interaction between coactivatorsand the LBD of ERs (5, 61). Moreover, steroid-induced phosphorylationof SRC-3 is nuclear receptor specific. Previously, we showedthat androgen can induce SRC-3 phosphorylation (75), but inthis report we demonstrate that neither PR-B nor GR is ableto induce phosphorylation of any of the six known SRC-3 phosphorylationsites in response to their cognate ligands. This dichotomy suggeststhat the extensive phosphorylation of SRC-3 induced by estrogenand androgen may relate to the well-known growth-promoting actionsof their cognate receptors. However, failure to induce extensivephosphorylation was surprising in view of the ability of SRC-3to coactivate and participate in nuclear PR-B- and GR-dependentgene expression, albeit in conjunction with SRC-1 and SRC-2,respectively (33). Conceivably, PR or GR and their cognate ligandsmay induce phosphorylation of SRC-3 at sites distinct from thoseknown to be phosphorylated in response to ER or AR ligands.Nonetheless, the differences between ER/AR and GR/PR and theircognate ligands, relative to their abilities to induce SRC-3phosphorylation, indicate that the relative importance of SRC-3phosphorylation at the six sites tested can be receptor specific.

Consistent with the model that E2-induced coactivator phosphorylationcan be mediated through the transcription-independent and "nongenomic"signaling of ER, we showed that ER ${alpha}$ nuclear localization is notrequired for promoting SRC-3 phosphorylation. It has been shownthat both ER and SRC-3 shuttle between the cytoplasm and thenucleus (14, 39, 53), and the collective data indicate thatSRC-3 phosphorylation can occur in both cytoplasm and nucleusand that it generally promotes nuclear localization of SRC-3(53, 68, 71, 74). For example, TNF- ${alpha}$ induces phosphorylationand nuclear translocation of SRC-3, and nuclear SRC-3 is hyperphosphorylatedcompared to the cytoplasmic fraction (74), indicating that SRC-3may be partially phosphorylated in the cytoplasm and furtherphosphorylated after translocation to the nucleus and recruitmentto the promoter. However, the precise extent to which each ofthe six known sites of SRC-3 is phosphorylated in each componentis unknown.

Our model, in which E2-induced SRC-3 phosphorylation occursin a complex with either ER ${alpha}$ or ERß, suggests thatthe high proximity of receptor and coactivator may enable thesame kinase to efficiently phosphorylate SRC-3 and ER in responseto E2. Indeed, the kinases ERK1/2, p38 MAPK, protein kinaseA, and IKK ${alpha}$ can phosphorylate both ER and SRC family members(7, 24, 29, 35, 50, 56, 75). However, the mechanism of kinaserecruitment to the complex is not clear. It is possible thatE2-bound ER ${alpha}$ can first recruit kinases via its AF-1 or AF-2 domain,allowing subsequent phosphorylation of SRC-3, or vice versa.Alternatively, a kinase could be recruited by either ER or SRC-3to an E2-induced preexisting complex of SRC-3 and ER ${alpha}$ , followedby phosphorylation of both molecules. Although our current datacannot distinguish between these models, or whether the ER isattached to the membrane (31, 52, 62), our results indicatethat phosphorylation of three of the major phosphorylation sitesof the ER ${alpha}$ AF-1 domain is not a prerequisite for SRC-3 phosphorylation.

In addition to its phosphorylation by an E2/ER-dependent pathway,SRC-3 phosphorylation also can be induced by growth factorsand cytokines (17, 75). Previously, we reported that TNF- ${alpha}$ inducesphosphorylation of SRC-3 in an ER-independent fashion and thatthe patterns of phosphorylated residues induced by TNF- ${alpha}$ andE2 overlapped but were not identical (75). Moreover, these distinctphosphorylation patterns lead to distinct effects on SRC-3 associationwith nuclear receptors, other transcription factors (e.g., NF- ${kappa}$ B),and coregulators (e.g., CBP and CARM1), leading to distinctpotentials for maximal transcriptional output (75). It is highlyprobable that the stimuli encountered by SRC-3, and hence itsphosphorylation pattern and coactivator activity, will be celland tissue specific. For example, in HER2-overexpressing breastcancers, SRC-3 phosphorylation induced by HER2 signaling mayfacilitate coactivator function under conditions of low E2 ortamoxifen exposure and thereby contribute to endocrine resistance(4, 49, 60). Indeed, tamoxifen induces SRC-3 phosphorylationas shown by its mobility shift and phosphatase treatment andserves as an agonist of ER/SRC-3-dependent transcription inHER2-overexpressing breast cancer cells (60). However, the statusof SRC-3 phosphorylation at each of the six sites in the presenceof high levels of HER2 has not been investigated yet. In contrast,estrogen may attenuate cellular responses to inflammatory stimuli,such as the cytokine TNF- ${alpha}$ , because of E2-induced phosphorylationof SRC-3 that renders the coactivator less effective at cytokinetarget genes.

Taken together, our results expand the established model ofaction of coactivators relative to their activation of geneexpression. This "classical" model indicates that E2-bound ERis first recruited to the promoter of its target gene, whichin turn recruits coactivators and other transcription factors.Our data provide the first indication of an E2-induced assemblyof an extranuclear SRC-3- and ER ${alpha}$ -containing complex prior topromoter binding, which is essential for E2-induced SRC-3 phosphorylationand thus for its biological functions. The complex could serveas a convergence point for cross talk between cell signalingand ER pathways, resulting in fine tuning of coactivator activationto support receptor nuclear functions. Since this step servesas a middle step in the multistep process of target gene activationby nuclear receptor and coactivator from their upstream "nongenomic"to downstream "genomic" responses leading to activation of transcription,it also is an intervention point for drug development for diseasessensitive to SRC-3 action.

ACKNOWLEDGMENTS

We are grateful to N. L. Weigel, P. H. Brown, R. X. Song, andH.-W. Chen for reagents and to M. A. Mancini for reagents andmicroscopy assistance.

This work was supported by grants from the National Institutesof Health to B.W.O. (HD08818 and NIDDK NURSA) and to C.L.S.(DK53002).

FOOTNOTES

* Corresponding author. Mailing address: Molecular and Cellular Biology, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-6205. Fax: (713) 798-5599. E-mail: berto{at}bcm.tmc.edu.

REFERENCES

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