Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M602567200 on May 11, 2006

J. Biol. Chem., Vol. 281, Issue 28, 19407-19416, July 14, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/28/19407    most recent
M602567200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van de Wijngaart, D. J.
Right arrow Articles by Dubbink, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van de Wijngaart, D. J.
Right arrow Articles by Dubbink, H. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Novel FXXFF and FXXMF Motifs in Androgen Receptor Cofactors Mediate High Affinity and Specific Interactions with the Ligand-binding Domain*

Dennis J. van de Wijngaart{ddagger}, Martin E. van Royen§, Remko Hersmus§, Ashley C. W. Pike1, Adriaan B. Houtsmuller§, Guido Jenster{ddagger}, Jan Trapman§, and Hendrikus J. Dubbink§2

From the Departments of {ddagger}Urology and §Pathology, Josephine Nefkens Institute, Erasmus MC, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands and Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom

Received for publication, March 20, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Upon hormone binding, a hydrophobic coactivator binding groove is induced in the androgen receptor (AR) ligand-binding domain (LBD). This groove serves as high affinity docking site for {alpha}-helical FXXLF motifs present in the AR N-terminal domain and in AR cofactors. Study of the amino acid requirements at position +4 of the AR FXXLF motif revealed that most amino acid substitutions strongly reduced or completely abrogated AR LBD interaction. Strong interactions were still observed following substitution of Leu+4 by Phe or Met residues. Leu+4 to Met or Phe substitutions in the FXXLF motifs of AR cofactors ARA54 and ARA70 were also compatible with strong AR LBD binding. Like the corresponding FXXLF motifs, interactions of FXXFF and FXXMF variants of AR and ARA54 motifs were AR specific, whereas variants of the less AR-selective ARA70 motif displayed increased AR specificity. A survey of currently known AR-binding proteins revealed the presence of an FXXFF motif in gelsolin and an FXXMF motif in PAK6. In vivo fluorescence resonance energy transfer and functional protein-protein interaction assays showed direct, efficient, and specific interactions of both motifs with AR LBD. Mutation of these motifs abrogated interaction of gelsolin and PAK6 proteins with AR. In conclusion, we have demonstrated strong interaction of FXXFF and FXXMF motifs to the AR coactivator binding groove, thereby mediating specific binding of a subgroup of cofactors to the AR LBD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The androgen receptor (AR)3 is a key player in development and maintenance of male reproductive tissues (1, 2). AR is a ligand-inducible transcription factor of the nuclear receptor (NR) superfamily. Members of this family share a common structural and functional organization, including an N-terminal domain (NTD) harboring activation function 1 (AF-1), a central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD) containing activation function 2 (AF-2) (3-5). Upon binding of its ligand, testosterone or 5{alpha}-dihydrotestosterone (DHT), AR LBD undergoes conformational changes leading to dissociation from heat shock proteins and translocation to the nucleus (6). At the DNA, AR binds to specific androgen response elements to initiate target gene expression. Cofactors facilitate AR transcription function by histone modifications, chromatin remodeling, and bridging of the receptor to other components of the transcription initiation process, including general transcription factors and RNA polymerase II (7-9).

Although cofactors may functionally interact with all three NR domains, the most extensive knowledge is available for LBD interaction. Crystal structures of NR LBDs have shown that ligand binding triggers repositioning of helix 12 (10-13). As a result, a hydrophobic groove is formed that serves as a docking site for amphipathic {alpha}-helical LXXLL motifs present in many cofactors. The specific affinity of LXXLL motifs for distinct NR LBDs depends on amino acid residues flanking the core Leu residues (10, 14-16). Until now, only a limited number of LXXLL motifs have been reported to interact with the AR LBD (17-20). Instead, AR LBD prefers binding of FXXLF motifs, one of which is located in the AR NTD (17, 21, 22). Although the function of the FXXLF motif-mediated interaction of AR NTD with AR LBD (N/C interaction) is not fully understood, it contributes to slowing of the androgen dissociation rate and selectively affects transcription of AR target genes (17, 22-25). Functional FXXLF motifs are also essential for interaction between AR LBD and cofactors ARA54, ARA70, and RAD9 (17, 26-28). However, for the majority of AR-binding proteins the mode of interaction remains to be elucidated (29).

Alanine scan mutagenesis of the AR FXXLF motif demonstrated that amino acid residues at positions +1, +4, and +5 are essential for interaction with the coactivator groove (21). Modeling and crystal structures of AR LBD in complex with FXXLF-like peptides, including AR and ARA70 FXXLF motifs, showed that amino acid residues at positions +1 and +5 are buried in the coactivator groove, rendering these residues entirely solvent inaccessible (17, 30-32). In contrast, the amino acid residue at position +4 rests in a shallow pocket on the periphery of the coactivator groove and is largely solvent exposed. Phage display screens for AR LBD-interacting peptides and directed mutagenesis studies of the AR FXXLF motif demonstrated that not only Phe but also Met, Tyr, and Trp residues at positions +1 and +5 could be compatible with binding to the AR coactivator binding groove, although Phe residues seem to be preferred (17, 18, 30).


Figure 1
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 1.
The effect of substitution of Leu+4 in the AR FXXLF motif on AR LBD interaction. A, amino acid sequence of the AR 18-30 peptide motif applied for mutagenesis of Leu+4. B, yeast two-hybrid analysis of Leu+4 substitution of the AR FXXLF motif for interaction with AR LBD. Y190 yeast cells were transformed with expression constructs encoding Gal4DBD-AR LBD and Gal4AD-peptide fusion proteins as described under "Experimental Procedures." The amino acid single-letter code of Leu+4 substitutions is indicated on the x-axis. Bars represent mean beta-galactosidase activity of three independent experiments (± S.D.) in the presence of 1 µM DHT. No interactions were observed in the absence of hormone (data not shown). AR LBD interaction with wild-type AR FXXLF motif is indicated with a hatched bar. The lower panel represents a Western blot visualizing the expression of Gal4AD-peptide fusion proteins by Gal4AD antibody staining.

 
Although it is presumed that the requirements for the amino acid residue at +4 in the FXXLF motif are less stringent than those at +1 and +5, our actual knowledge in this respect is limited. Here we performed a systematic functional analysis of the AR FXXLF motif mutated at +4. Yeast two-hybrid and mammalian one-hybrid experiments demonstrated that Leu to Phe and Leu to Met substitutions in the AR FXXLF motif are compatible with high affinity and specific AR LBD interaction. Strong and specific interaction was also obtained if the same substitutions were introduced in the ARA54 and ARA70 FXXLF motifs. As assessed by in vivo fluorescence resonance energy transfer (FRET) analysis, functional protein-protein interaction assays, and mutagenesis, the AR partners gelsolin and PAK6 were found to contain an FXXFF and FXXMF motif, respectively, necessary and sufficient for AR LBD interaction.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Yeast and mammalian expression plasmids encoding Gal4AD-, Gal4DBD-, and YFP-peptide fusion proteins were generated by in-frame insertion of double-stranded synthetic oligonucleotides with 5'-BamHI and 3'-EcoRI cohesive ends into the corresponding sites of pACT2 (Takara Bio, Otsu, Shiga, Japan), pM-B/E (17), or in the BglII and EcoRI sites of pEYFP-C2 (Takara Bio) as described previously (17). Mutagenesis of position +4 in the AR FXXLF motif was performed in oligonucleotides encoding AR18-30. Mutant oligonucleotides were inserted into pACT2 as described above. All peptide expression constructs were verified by sequence analysis.

Yeast expression construct pGalDBD-AR LBD (AR661-919) has been described previously (33). Constructs encoding Gal4DBD fusions with LBDs of ER{alpha}, PR, and RXR{alpha} were generously provided by Michael Stallcup (34). Mammalian constructs expressing wild-type AR (pCMVARo) and F23L/F27L-AR (pCMVF23L/F27L-AR) have been described previously (17). pM-PAK612-681 was generated by subcloning a BglII-XbaI fragment from pSPORT6-PAK6 (IRAKp961I1968Q; RZPD, Berlin, Germany) into the BamHI and XbaI sites of pM (Takara Bio). pM-Gelsolin was obtained by subcloning an EcoRI-digested PCR fragment encoding amino acid residues 281-731 of gelsolin into pM. PCR was performed using primers 5'-GATCGAATTCTTCATCCTGGACCACG-3' and 5'-GATCGAATTCCTCAGGCAGCCAGCTC-3' (EcoRI sites in bold) on pOTB7-Gelsolin (IMAGp958I211459Q; RZPD). FXXAA variants of pM-PAK6 and pM-gelsolin were generated by QuikChange (Stratagene, La Jolla, CA) using primer pair 5'-CTATTCCGAAGCGCGGCCCTGTCCACTG-3' and 5'-CAGTGGACAGGGCCGCGCTTCGGAATAG-3' for PAK6 and 5'-CTGTTCAAGCAGGCCGCCAAGAACTGGCGG-3' and 5'-CCGCCAGTTCTTGGCGGCCTGCTTGAACAG-3' for gelsolin, respectively (substitutions in bold) according to the manufacturer's instructions. For generation of pCFP-ARLBD (AR612-919) a BamHI-digested PCR fragment from pAR0 (35) was cloned into the corresponding site of pECFP-C2 (Takara Bio). Primers used were 5'-AATTGGGGATCCGACCATCTTCTCGTCTTCGGAAATG-3' and 5'-AATTGGGGATCCGATCACTGGGTGTGGAAATAGATG-3' (BamHI sites in bold). pCYFP encoding the ECFP-EYFP chimera was kindly provided by Dr. Claude Gazin. The (ARE)2TATA-LUC reporter construct has been previously described as (PRE)2-E1b-LUC (36). The (UAS)4TATA-LUC reporter construct was kindly provided by Magda Meester. All constructs generated with PCR fragments and QuikChange mutagenesis were verified by sequence analysis.

Yeast Culture, Transformation, and beta-Galactosidase Assay—Y190 yeast culture, transformation, and liquid culture beta-galactosidase assays to quantify NR LBD-peptide interactions were performed as described previously (33, 37). Liquid culture beta-galactosidase assays were performed in the presence of 1 µM DHT (for AR; Steraloids, Wilton, NH), 1 µM progesterone (PR; Steraloids), 100 nM estradiol (ER{alpha}) (Steraloids), 10 nM retinoic acid (RXR{alpha}) (Sigma), or vehicle.


Figure 2
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 2.
FXXFF and FXXMF variants of AR, ARA54, and ARA70 FXXLF motifs interact with AR LBD in mammalian cells. A, amino acid sequences of AR, ARA54, and ARA70 FXXLF peptides. B-G, mammalian one-hybrid analysis of Leu to Phe- and Leu to Met-substituted FXXLF motifs of AR (B, C), ARA54 (D, E), and ARA70 (F, G) with full-length wild-type AR (B, D, F) or F23L/F27L-AR (C, E, G). Hep3B cells were cotransfected with expression constructs encoding the indicated Gal4DBD-peptide fusion protein and AR in the presence of the (UAS)4TATA-LUC reporter. Interactions were determined in the absence and presence of 100 nM DHT. Each bar represents mean luciferase activity of two independent experiments (± S.D.). Mean -fold inductions are shown above the bars.

 
Mammalian Cell Culture, Transient Transfections, and Luciferase Activity—Hep3B cells were cultured and transfected as described previously (37). For one-hybrid assays, cells were transfected with 50 ng of Gal4DBD-peptide or Gal4DBD-protein expression construct, 50 ng of AR expression construct, and 150 ng of (UAS)4TATA-LUC construct in the presence of 100 nM DHT or vehicle. Luciferase activity was determined as described previously (17, 37).

For FRET experiments, Hep3B cells were cultured overnight on glass coverslips in 9.5-cm2 wells in {alpha}-minimal essential medium supplemented with 5% fetal calf serum, L-glutamine, and antibiotics. 4 h prior to transfection the medium was substituted by 1 ml of {alpha}-minimal essential medium containing 5% charcoal-stripped fetal calf serum. Cells were transfected with 1 µg of pCYFP or 1 µg of pCFP-AR-LBD and 0.5 µg of YFP peptide expression construct, together with 3 µl of FuGENE 6 (Roche Diagnostics)/µg of DNA in 100 µlof {alpha}-minimal essential medium. 4 h after transfection the medium was substituted by 2 ml of {alpha}-minimal essential medium containing 100 nM DHT. FRET assays were done the next day.

Western Blot Analysis—Yeast protein extraction and Western blot analysis for detection of Gal4AD fusion proteins were performed as described previously (21, 33). Proteins were visualized using a monoclonal antibody against Gal4AD (Takara Bio).

FRET Measurement by Acceptor Photobleaching—Live cell imaging was performed using a Zeiss LSM510 confocal laser scanning microscope equipped with a PlanNeofluar x40/1.3 NA oil objective (Carl Zeiss, Jena, Germany) at a lateral resolution of 100 nm. CFP and YFP images were collected sequentially at 458 and 514 nm excitation, respectively, using a 458/514-nm dichroic beam splitter, a 515-nm beam splitter, and specific emission filters. CFP was excited with the 458-nm laser line of an Argon laser at moderate laser power and detected using a 470-500-nm band pass emission filter. YFP excitation was at 514 nm at moderate laser power and detected using a 560-nm emission filter. After sequential collection of YFP and CFP images, YFP was bleached by scanning 25 times a nuclear region of ~100 µm2, covering a large part of the nucleus, using the 514-nm argon laser line at high laser power. After acceptor photobleaching a second YFP and CFP image pair was collected. The apparent FRET efficiency was calculated after background subtraction as shown in Equation 1

Formula(Eq.1)

where CFPbefore and YFPbefore are the average fluorescence intensities measured in the nuclei before bleaching and CFPafter and YFPafter the average fluorescence intensities after bleaching.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 3.
Leu to Phe- and Leu to Met-substituted FXXLF motifs specifically interact with AR LBD. Yeast two-hybrid experiments were carried out to assess hormone-dependent interactions of Leu to Phe- and Leu to Met-substituted FXXLF motifs of AR, ARA54, and ARA70 fused to Gal4AD with the indicated NR LBDs fused to Gal4DBD. Interaction was determined in the presence of 1 µM DHT for AR (A) and 1 µM progesterone for PR (B). Bars represent mean beta-galactosidase activity of two independent experiments (± S.D.).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Leu to Phe- and Leu to Met-substituted AR FXXLF Motifs Strongly Interact with AR LBD—Although the importance of the core hydrophobic amino acid residues at positions +1 and +5in FXXLF motifs has been described (17, 21, 26), little is known about the amino acid requirements at +4 for AR LBD binding. To study this, we tested every amino acid at this position in the context of the AR FXXLF motif using a yeast two-hybrid read-out system (Fig. 1A). In this assay, peptides were expressed as fusions to Gal4AD and AR LBD was fused to Gal4DBD. All assays were done in the presence of DHT (17). Western blot analysis of transformed yeast cells demonstrated that all Gal4AD-peptide fusion proteins were appropriately expressed (Fig. 1B). The yeast two-hybrid screening showed that most Leu+4 substitutions completely abolished AR LBD interaction (Fig. 1B). Reduced interaction was observed with peptides containing a Trp, Thr, Ile, Val, Cys, or Tyr residue at position +4 instead of a Leu. In contrast, AR LBD interactions were identical or even stronger than wildtype motif if Leu+4 were substituted by Phe or Met.

FXXFF and FXXMF Variants of AR, ARA54, and ARA70 FXXLF Motifs Interact with AR LBD in Mammalian Cells—Next, we evaluated interaction capacities of FXXFF and FXXMF variants of AR FXXLF with full-length wild-type AR in a mammalian one-hybrid assay (Fig. 2A and Ref. 17). Interaction was assayed in Hep3B cells cotransfected with Gal4DBD-peptide and full-length wild-type AR expression constructs and a (UAS)4TATA-LUC reporter. The results of this assay closely resembled the results obtained in yeast, as both FXXFF and FXXMF variants displayed hormone-dependent binding capacities comparable with the wild-type motif (Fig. 2B). We also investigated the interaction of the peptides with full-length F23L/F27L-mutated AR (F23L/F27L-AR), which abrogates AR N/C interaction (17). This resulted in increased interactions of the FXXFF and FXXMF variants (Fig. 2C), indicating that both compete with the FXXLF motif in the AR NTD for AR LBD binding.

Subsequently, interaction of Phe+4 and Met+4 variants of ARA54 and ARA70 FXXLF motifs with AR LBD were assessed (Fig. 2A). The variants of both ARA54 (Fig. 2D) and ARA70 (Fig. 2F) interacted strongly with wild-type AR. All variants showed increased interactions with F23L/F27L-AR, indicative of interaction with the coactivator binding groove (Fig. 2, E and G). Summarizing, Leu+4 can be substituted by Phe or Met residues in distinct FXXLF peptide motifs, thereby retaining AR LBD interaction.

Effects of Phe and Met Residues at Position +4onAR Specificity—Previously, we and others have demonstrated that FXXLF motifs, including those of AR and ARA54, display high specificity for AR (17, 26, 38, 39). However, some FXXLF motifs, including the ARA70 motif, also interacted with PR (39). We studied in yeast two-hybrid experiments the effect of Leu to Phe and Leu to Met substitutions at position +4 in the AR, ARA54, and ARA70 FXXLF motifs on AR specificity. Peptides were fused to Gal4AD, and LBDs of ER{alpha}, PR, and RXR{alpha} were fused to Gal4DBD. Upon ligand binding all NR LBDs adopted a functional conformation because strong interaction with a control LXXLL peptide, D11, was observed (Ref. 40 and data not shown). Contrary to a potent interaction with AR LBD (Fig. 3A), none of the FXXFF and FXXMF variant motifs interacted with LBDs of ER{alpha}, PR, or RXR{alpha} (Fig. 3B and data not shown). The specificity of the ARA70 FXXLF motif even increased upon Leu to Phe and Leu to Met substitutions as no PR LBD interaction was observed with the variant ARA70 motifs (Fig. 3B). The weak beta-galactosidase activities detected with PR LBD were due to the intrinsic activity of Gal4DBD-PR LBD because similar values were obtained when this construct was expressed in the absence of a peptide expression construct (data not shown). These results demonstrate that Leu to Phe and Leu to Met substitution variants of FXXLF motifs remain AR specific or become even more specific.


Figure 4
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 4.
An FXXFF motif in gelsolin and an FXXMF motif in PAK6 interact with AR LBD. A and B, Hep3B cells were cotransfected with expression constructs encoding Gal4DBD-peptide (for peptide sequences see panels C and D, left) and wild-type (A) or F23L/F27L-substituted (B) full-length AR and a (UAS)4TATA-LUC reporter plasmid. Interaction was determined in the absence and presence of 100 nM DHT. Bars represent mean luciferase activity of two independent experiments (± S.D.). Mean -fold inductions are indicated above the bars. C and D, left, schematic representation of gelsolin (C) and PAK6 (D) proteins. Positions are indicated of the FXXFF motif in gelsolin and the FXXMF motif in PAK6 and the corresponding peptide sequences tested for interaction with AR. The dotted lines represent gelsolin (C) and PAK6 (D) fragments originally identified in yeast two-hybrid screenings (43, 48, 49). C and D, right, helical wheel presentation of gelsolin FXXFF and PAK6 FXXMF motifs. Polar residues are indicated in white boxes and hydrophobic residues in gray boxes.

 
Naturally Occurring AR-interacting FXXFF and FXXMF Motifs—To assess a role of FXXFF and FXXMF motifs in cofactor-AR LBD interaction, we screened all AR-interacting proteins present in the AR gene mutations data base (www.mcgill.ca/androgendb; Ref. 29) and in the human protein reference data base (www.hprd.org) for the presence of these motifs. This yielded two proteins with an FXXFF (gelsolin and cdc37) and two with an FXXMF motif (PAK6 and supervillin). Mammalian one-hybrid experiments showed that the cdc37 FXXFF and supervillin FXXMF motifs weakly interacted with F23L/F27L-AR, but not with wild-type AR (data not shown). In contrast, the gelsolin FXXFF and PAK6 FXXMF motifs displayed strong hormone-dependent interactions with both F23L/F27L-AR and wild-type AR (Fig. 4, A and B). AR N/C interaction did not affect gelsolin FXXFF binding to AR LBD but reduced binding of the AR FXXLF and PAK6 FXXMF motifs, indicating that the gelsolin FXXFF motif had a higher affinity for AR LBD than the AR FXXLF and PAK6 FXXMF motifs. Both motifs are predicted to adopt an amphipathic {alpha}-helical structure (Fig. 4, C and D). FXXFF and FXXMF motifs present in AR cofactors gelsolin and PAK6 may thus be essential for interaction with AR.

To extend our knowledge on the interactions between AR LBD and gelsolin FXXFF and PAK6 FXXMF peptide motifs, in vivo FRET experiments were carried out (Fig. 5A). Hep3B cells were transiently cotransfected with constructs expressing CFP-tagged AR LBD and YFP-tagged peptide motifs. Close association of ligand-bound CFP-tagged AR LBD and YFP-tagged peptide results in energy transfer (FRET) by excitated CFP donor to YFP acceptor (Fig. 5A, left) (41). FRET efficiency was estimated by acceptor photobleaching (Fig. 5A, middle and right) (42, 43). FRET intensity was calculated based on the difference in CFP emission intensities before and after YFP photo destruction as described under "Experimental Procedures."

FRET signals between AR LBD and FXXLF motifs of AR, ARA54, and ARA70 were readily detected in the presence of ligand (Fig. 5B). FRET signals between AR LBD and either gelsolin FXXFF or PAK6 FXXMF motifs were similar (Fig. 5C). These findings demonstrate direct in vivo interactions of AR LBD with gelsolin FXXFF and PAK6 FXXMF peptides.


Figure 5
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 5.
Direct in vivo interaction of AR LBD with FXXLF, FXXFF, and FXXMF motifs of AR NTD and AR cofactors. A, schematic representation of acceptor photobleaching FRET. Photo destruction of YFP of an interacting pair of CFP-tagged AR LBD and YFP-tagged peptide will result in enhanced CFP emission. B and C, direct interaction of AR LBD with the FXXLF motifs of AR, ARA54, and ARA70 (B) and with gelsolin FXXFF and PAK6 FXXMF motifs (C) as determined by in vivo FRET. Hep3B cells were transiently cotransfected with constructs expressing CFP-tagged AR LBD and YFP-tagged peptides. Western blot analysis demonstrated that all fusion proteins were expressed at the correct size (not shown). Confocal microscopy showed that in both the absence and presence of DHT CFP-AR LBD was localized in the nucleus, whereas the YFP-tagged peptides distributed over both nucleus and cytoplasm (data not shown). FRET was estimated based on emission intensities of CFP and YFP before and after YFP photo destruction as described under "Experimental Procedures." FRET efficiency is expressed relative to the values of co-expressed CFP-AR LBD and YFP without peptide (B) and normalized to values obtained for the CFP-YFP chimera. FRET efficiencies of peptides were determined in three independent experiments in a total of 30 cells in the presence of 100 nM DHT. Error bars represent 2 x S.E.

 
Alanine Scanning and AR LBD Specificity of Gelsolin FXXFF and PAK6 FXXMF Motifs—To further characterize the gelsolin FXXFF and PAK6 FXXMF motifs we performed an alanine scan by substituting consecutive doublet residues in both motifs into alanine residues (Fig. 6A). Mammalian one-hybrid results show that alanine substitutions encompassing the core hydrophobic residues at positions +1, +4, and +5 of both gelsolin and PAK6 completely abrogated AR interactions (Fig. 6, B and C). Residues at positions +6 and +7 of the PAK6 motif, but not of the gelsolin FXXFF motif, also appeared important for AR LBD interaction. All other alanine substitutions hardly interfered with AR binding. As found for AR, ARA54, and ARA70 peptide motifs, gelsolin FXXFF and PAK6 FXXMF strongly bound to AR LBD but hardly bound or did not bind to the LBDs of ER{alpha}, PR, and RXR{alpha} (Fig. 7).

AR LBD Binding of Cofactors Gelsolin and PAK6 Is FXXFF and FXXMF Mediated—Next, we investigated the importance of the FXXFF and FXXMF motifs for interaction of gelsolin and PAK6 with AR. PAK6 (amino acids 12-681) and gelsolin (amino acids 281-731) were fused to Gal4DBD and allowed to interact with AR in the mammalian read-out system. As expected, both proteins interacted with wild-type AR (Fig. 8A), and binding was increased if the competing FXXLF motif in AR NTD was inactivated (Fig. 8B). However, if the FXXFF motif in gelsolin and the FXXMF motif in PAK6 were mutated into FXXAA, interactions with both wild-type AR and F23L/F27L-AR were abolished. Gelsolin and PAK6 protein expression levels were not affected by the mutations (data not shown). These data clearly demonstrate that the FXXFF and FXXMF motifs in gelsolin and PAK6, respectively, are necessary and sufficient for AR interaction.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Upon agonist binding, the architecture of the AR LBD surface is rearranged to allow high affinity binding of FXXLF motifs present in AR NTD and in AR cofactors. Binding of these short amphipathic {alpha}-helical structures turned out to depend strongly on optimal docking of the Phe residues at +1 and +5in the coactivator binding groove of AR LBD (17, 21). Although the coactivator groove is sufficiently flexible to accommodate other large hydrophobic amino acid residues, Phe residues at +1 and +5 are preferred (17, 18, 30). Based on functional assays we have here provided insight into the requirements of the amino acid at position +4 of peptide motifs for optimal AR LBD binding. We demonstrated that Leu+4 can be substituted by Phe and Met residues in the AR, ARA54, and ARA70 FXXLF motifs, retaining strong and selective AR binding. Novel AR-interacting FXXFF and FXXMF motifs were identified in AR cofactors gelsolin and PAK6, respectively.


Figure 6
View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 6.
Alanine scan of gelsolin FXXFF and PAK6 FXXMF motifs interacting with AR LBD. A, amino acid sequences of peptides used for the alanine scan of gelsolin FXXFF and PAK6 FXXMF motifs. B and C, Gal4DBD-fused gelsolin (B) and PAK6 (C) peptides were studied for interaction with F23L/F27L-AR using (UAS)4TATA-LUC as reporter in transiently cotransfected Hep3B cells. Interaction was assayed in the absence and presence of 100 nM DHT. Bars represent mean luciferase activity of two independent experiments (± S.D.). Mean -fold inductions are shown above the bars.

 


Figure 7
View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 7.
Gelsolin FXXFF and PAK FXXMF motifs specifically interact with AR LBD in a yeast two-hybrid assay. Y190 yeast cells were transformed with gelsolin FXXFF (A) and PAK6 FXXMF (B) motifs fused to Gal4AD and LBDs of AR, ER{alpha}, PR, and RXR{alpha} fused to Gal4DBD. Interaction was determined in the presence of 1 µM DHT, 100 nM 17beta-estradiol, 1 µM progesterone, and 10 µM all-trans-retinoic acid, respectively. Bars represent mean beta-galactosidase units of two independent experiments (± S.D.). A positive control, LXXLL peptide D11 (39), interacted with all NR LBDs, ensuring proper LBD expression (data not shown).

 
Systematic mutation screening of position +4 of the AR FXXLF motif resulted in the identification of three categories of peptides (Fig. 1). (i) The largest group of peptides does not interact with AR LBD. This group includes small hydrophobic, charged, or polar residues at +4. (ii) Several peptides showed reduced interaction with AR LBD (Cys, Ile, Thr, Val, Trp, and Tyr). Most of these variants have a hydrophobic residue at +4 and (iii) strongly interacting variants containing bulky hydrophobic side chains (Leu, Phe, and Met). Strong binding by Leu, Phe, and Met residues indicates that hydrophobic contacts underlie the ability to interact with AR LBD. The inability or limited potency of most +4 variants to bind AR LBD can be due to destabilization of the peptide by distortion of the helical structure, active interference with LBD interaction caused by the charge or size of the side chains, or incapability of forming sufficient hydrophobic contacts with the AR LBD. Our findings underscore the importance of the amino acid residue at +4 for optimal binding of peptide motifs to AR LBD, even though this amino acid residue is not deeply buried in the binding pocket (30, 31).

Phage display screens of random peptide libraries with full-length AR or AR LBD as bait yielded different AR-interacting motifs containing Phe residues at positions +1 and +5 (30, 38). Besides the classical FXXLF sequences, FXXVF, FXXYF, and FXXFF motifs were identified in these screens. The FXXVF-containing peptide weakly interacted with AR, in agreement with our screening results, and strong interactions were observed with FXXYF and FXXFF sequences (38). In our +4 mutation screen of AR FXXLF, the FXXYF variant showed decreased interaction with AR LBD, suggesting that in this specific FXXYF motif the Tyr residue has a less optimal position for AR LBD binding. Similar data were found for ARA54 and ARA70 FXXLF-based FXXYF variants (data not shown). Therefore, the requirement for the amino acid at +4 might depend on the further context of the motif. Chang et al. (38) demonstrated that most FXXYF and FXXFF peptide motifs picked up in phage display screens interacted with AR LBD not only in the presence but also in the absence of ligand. Repetition of these experiments in our interaction assay indicated that ligand was essential for AR LBD interaction (data not shown). This apparent discrepancy might be due to differences in read-out systems.


Figure 8
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 8.
AR LBD binding of cofactors gelsolin and PAK6 is mediated by FXXFF and FXXMF motifs, respectively. A and B, Hep3B cells were cotransfected with expression constructs for Gal4DBD-PAK6 (amino acids 12-681) or Gal4DBD-gelsolin (amino acids 281-731) containing either wild-type or FXXAA-mutated motifs and wild-type (A) or F23L/F27L-substituted (B) full-length AR and a (UAS)4TATA-LUC reporter plasmid. Interaction was assayed in the absence and presence of 100 nM DHT. Bars represent mean luciferase activity of two independent experiments (± S.D.). Mean -fold inductions are indicated above the bars.

 


Figure 9
View larger version (92K):
[in this window]
[in a new window]
  		FIGURE 9.
Variable binding modes of the +4 residue in FXXLF, FXXFF, and FXXYF peptide motifs to the coactivator groove. Surface representation of the coactivator groove region of the AR LBD (PDB accession code 1XOW). Residues that form the +4 binding site are in magenta. The binding modes of the FXXLF (green; 1XOW), FXXFF (orange, 1T73), and FXXYF (yellow, 1T7M) peptides are shown after global superposition of the various crystal structures. For clarity, only peptide side chains at positions +1, +4, and +5 are shown. AR side chains that line the +4 binding site are highlighted along with the two charge clamp residues (blue).

 
Recently, crystal structures of AR LBDs in complex with the AR FXXLF and ARA70 FXXLF motifs and FXXLF, FXXFF, and FXXYF peptides selected by phage display have been resolved (30-32). Comparing LBDs with and without bound peptide showed that the side chains of amino acid residues in AR LBD that line the coactivator groove rearrange upon binding of the peptide motif. The largest conformational changes were observed for Lys-720, Met-734, Met-894, and Glu-897, leading to optimal binding sites for residues +1, +4, and +5of interacting peptide motifs (30-32). The Phes at positions +1 and +5 are buried in a deep solvent-inaccessible groove in AR LBD. This mode of interaction is largely conserved, suggesting that these residues drive the interaction of the peptide motif (30). In contrast, the binding mode of the residue at position +4 seems less critical. This residue binds to a shallow hydrophobic depression formed by Leu-712, Val-713, Val-716, and Met-894 in AR LBD (Fig. 9) (30-32). Based on the crystal structures, the side chains of the different amino acids at +4 studied so far (Leu, Phe, and Tyr) form hydrophobic contacts with Val-713, Val-716, and Met-894 in the groove with an additional contact formed between the FXXYF peptide and Lys-717 of AR LBD. As shown in Fig. 9, the FXXFF peptide has shifted in the coactivator groove toward the Lys-720 residue as compared with the FXXLF and FXXYF peptide motifs (30). This shift together with a less optimal helical geometry of the peptide backbone makes the Phe at +4 have a different orientation than a Leu or Tyr at this position (30). We have shown in this study that +4 of the peptide motif can also be a Met. Because of the variability of the position of the +4 residue in the complex with AR LBD and because Met has a highly flexible side chain, its precise positioning in the coactivator groove cannot be accurately predicted.

In contrast to LXXLL motifs, FXXLF motifs show a strong preference for AR (17, 26). Some FXXLF motifs, including the ARA70 FXXLF motif, also interact with PR (38, 39). The FXXFF and FXXMF motifs tested in the present study also specifically interacted with AR. Leu to Phe and Leu to Met substitutions increased specificity of the ARA70 FXXLF motif. We hypothesize that Met and Phe residues at position +4 select against binding to the coactivator binding groove of PR. In agreement with this hypothesis, AR-interacting FXXLF and FXXFF peptides selected by phage display show a similar selectivity for AR: only 1 of 5 FXXFF peptides interacted with PR LBD as compared with 4 of 6 FXXLF peptides (38). Of the residues in the AR coactivator binding groove that contact the +4 side chains in FXXLF and FXXFF peptides (see Fig. 9), only Val-713 differs from the corresponding Leu-727 residue in PR. As recently shown by He et al. (31), V713L substitution in AR LBD reduced binding of the AR FXXLF motif. Vice versa, L727V substitution in PR LBD increased binding of the AR FXXLF motif. We presume that the size and orientation of Leu-727 in PR LBD precludes binding of peptide motifs with bulky Phe and Met residues at position +4.

Although the mode of interaction of the majority of cofactors with AR LBD is unknown, for several, including ARA54, ARA70, and RAD9, an essential FXXLF motif has been established (17, 26-28). Here we demonstrated that two other AR-interacting proteins, gelsolin and PAK6, interact with AR LBD via an FXXFF and an FXXMF motif, respectively. Gelsolin is an actin-capping and -severing protein and is presumed to act as an AR cofactor by facilitating nuclear translocation (44). Interestingly, other members of the gelsolin family, including flightless-1 and supervillin, have also been identified as cofactors of AR and other NRs, suggesting an important role in NR function (45, 46). The gelsolin FXXFF motif is highly conserved not only among different species but also among different members of the gelsolin family. Our preliminary data revealed that the conserved FXXFF motif present in adseverin also strongly binds AR LBD, suggesting that adseverin may act as an AR cofactor as well (data not shown). PAK6 is a member of the PAK family of serine/threonine kinases, which is, based on homology, divided into two subfamilies (Group I: PAK1, PAK2, and PAK3; Group II: PAK4, PAK5, and PAK6) (47). Although the FXXMF motif in PAK6 is conserved in other species, it is not conserved in any of the other members of the PAK family, and so far PAK6 is the only member known to modulate NR function. PAK6 might repress AR function by phosphorylation of the DBD (48). Hormone-dependent interactions with AR LBD were observed in yeast two-hybrid and co-immunoprecipitation experiments, whereas glutathione S-transferase pulldown experiments indicated that these LBD interactions were hormone independent and also involved the hinge region (49, 50). Our results unambiguously demonstrated that the novel FXXMF motif is sufficient and necessary for hormone-dependent interaction of PAK6 with AR LBD.

The identification of the FXXFF and FXXMF motifs in AR cofactors raises the possibility that other so far unrecognized proteins interact with AR LBD via similar motifs. Based on these findings, it would be of interest to perform a proteomewide in silico screen for FXX(L/F/M)F peptide motifs combined with functional protein-protein interaction assays to identify new candidate AR partners.

Prostate cancer growth is dependent on the androgen-AR axis (51, 52). Nonetheless, endocrine treatment of metastatic prostate cancer by androgen withdrawal or blocking AR activity by antagonists is not curative, even though AR is still active in progressive disease in most cases (53). AR N/C interaction and cofactor interactions are important steps in AR activation. Disruption of these interactions might be a complementary or alternative approach to more efficiently inhibit AR function. Increased knowledge of the mode of AR LBD-peptide interaction will be instrumental in the design of small molecules that fit in the AR coactivator binding groove and block protein interactions.


   FOOTNOTES
 
* This work was supported by Grant DDHK2001-2402 from the Dutch Cancer Society (KWF). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Structural Genomics Consortium, University of Oxford, Botnar Research Centre, Oxford OX3 7LD, UK. Supported by a Wellcome Trust Career Development fellowship (Grant 064803). Back

2 To whom correspondence should be addressed. Tel.: 31-10-4088467; Fax: 31-10-4089487; E-mail: h.dubbink{at}erasmusmc.nl.

3 The abbreviations used are: AR, androgen receptor; CFP, cyan fluorescent protein; DBD, DNA-binding domain; DHT, 5{alpha}-dihydrotestosterone; ER{alpha}, estrogen receptor {alpha}; FRET, fluorescence resonance energy transfer; LBD, ligand-binding domain; LUC, luciferase; N/C interaction, interaction between NTD and LBD; NR, nuclear receptor; NTD, N-terminal domain; PR, progesterone receptor; RXR{alpha}, retinoid x receptor {alpha}; YFP, yellow fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Michael Stallcup, Magda Meester, and Claude Gazin for providing plasmids.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lee, D. K., and Chang, C. (2003) J. Clin. Endocrinol. Metab. 88, 4043-4054[Abstract/Free Full Text]
  2. Quigley, C. A., De Bellis, A., Marschke, K. B., el-Awady, M. K., Wilson, E. M., and French, F. S. (1995) Endocr. Rev. 16, 271-321[CrossRef][Medline] [Order article via Infotrieve]
  3. Tsai, M. J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve]
  4. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. (1995) Cell 83, 835-839[CrossRef][Medline] [Order article via Infotrieve]
  5. Warnmark, A., Treuter, E., Wright, A. P., and Gustafsson, J. A. (2003) Mol. Endocrinol. 17, 1901-1909[Abstract/Free Full Text]
  6. Chang, C., Saltzman, A., Yeh, S., Young, W., Keller, E., Lee, H. J., Wang, C., and Mizokami, A. (1995) Crit. Rev. Eukaryotic Gene Expression 5, 97-125[Medline] [Order article via Infotrieve]
  7. Shang, Y., Myers, M., and Brown, M. (2002) Mol. Cell 9, 601-610[CrossRef][Medline] [Order article via Infotrieve]
  8. Heinlein, C. A., and Chang, C. (2002) Endocr. Rev. 23, 175-200[Abstract/Free Full Text]
  9. Kinyamu, H. K., and Archer, T. K. (2004) Biochim. Biophys. Acta 1677, 30-45[Medline] [Order article via Infotrieve]
  10. Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997) Nature 387, 733-736[CrossRef][Medline] [Order article via Infotrieve]
  11. Greschik, H., and Moras, D. (2003) Curr. Top. Med. Chem. 3, 1573-1599[CrossRef][Medline] [Order article via Infotrieve]
  12. Pike, A. C., Brzozowski, A. M., and Hubbard, R. E. (2000) J. Steroid Biochem. Mol. Biol. 74, 261-268[CrossRef][Medline] [Order article via Infotrieve]
  13. Feng, W., Ribeiro, R. C., Wagner, R. L., Nguyen, H., Apriletti, J. W., Fletterick, R. J., Baxter, J. D., Kushner, P. J., and West, B. L. (1998) Science 280, 1747-1749[Abstract/Free Full Text]
  14. Darimont, B. D., Wagner, R. L., Apriletti, J. W., Stallcup, M. R., Kushner, P. J., Baxter, J. D., Fletterick, R. J., and Yamamoto, K. R. (1998) Genes Dev. 12, 3343-3356[Abstract/Free Full Text]
  15. McInerney, E. M., Rose, D. W., Flynn, S. E., Westin, S., Mullen, T. M., Krones, A., Inostroza, J., Torchia, J., Nolte, R. T., Assa-Munt, N., Milburn, M. V., Glass, C. K., and Rosenfeld, M. G. (1998) Genes Dev. 12, 3357-3368[Abstract/Free Full Text]
  16. Needham, M., Raines, S., McPheat, J., Stacey, C., Ellston, J., Hoare, S., and Parker, M. (2000) J. Steroid Biochem. Mol. Biol. 72, 35-46[CrossRef][Medline] [Order article via Infotrieve]
  17. Dubbink, H. J., Hersmus, R., Verma, C. S., van der Korput, H. A., Berrevoets, C. A., van Tol, J., Ziel-van der Made, A. C., Brinkmann, A. O., Pike, A. C., and Trapman, J. (2004) Mol. Endocrinol. 18, 2132-2150[Abstract/Free Full Text]
  18. Hsu, C. L., Chen, Y. L., Yeh, S., Ting, H. J., Hu, Y. C., Lin, H., Wang, X., and Chang, C. (2003) J. Biol. Chem. 278, 23691-23698[Abstract/Free Full Text]
  19. Chang, C. Y., and McDonnell, D. P. (2002) Mol. Endocrinol. 16, 647-660[Abstract/Free Full Text]
  20. Hall, J. M., Chang, C. Y., and McDonnell, D. P. (2000) Mol. Endocrinol. 14, 2010-2023[Abstract/Free Full Text]
  21. Steketee, K., Berrevoets, C. A., Dubbink, H. J., Doesburg, P., Hersmus, R., Brinkmann, A. O., and Trapman, J. (2002) Eur. J. Biochem. 269, 5780-5791[Medline] [Order article via Infotrieve]
  22. He, B., Kemppainen, J. A., and Wilson, E. M. (2000) J. Biol. Chem. 275, 22986-22994[Abstract/Free Full Text]
  23. He, B., Bowen, N. T., Minges, J. T., and Wilson, E. M. (2001) J. Biol. Chem. 276, 42293-42301[Abstract/Free Full Text]
  24. He, B., Lee, L. W., Minges, J. T., and Wilson, E. M. (2002) J. Biol. Chem. 277, 25631-25639[Abstract/Free Full Text]
  25. Callewaert, L., Verrijdt, G., Christiaens, V., Haelens, A., and Claessens, F. (2003) J. Biol. Chem. 278, 8212-8218[Abstract/Free Full Text]
  26. He, B., Minges, J. T., Lee, L. W., and Wilson, E. M. (2002) J. Biol. Chem. 277, 10226-10235[Abstract/Free Full Text]
  27. Hu, Y. C., Yeh, S., Yeh, S. D., Sampson, E. R., Huang, J., Li, P., Hsu, C. L., Ting, H. J., Lin, H. K., Wang, L., Kim, E., Ni, J., and Chang, C. (2004) J. Biol. Chem. 279, 33438-33446[Abstract/Free Full Text]
  28. Wang, L., Hsu, C. L., Ni, J., Wang, P. H., Yeh, S., Keng, P., and Chang, C. (2004) Mol. Cell. Biol. 24, 2202-2213[Abstract/Free Full Text]
  29. Gottlieb, B., Beitel, L. K., Wu, J. H., and Trifiro, M. (2004) Hum. Mutat. 23, 527-533[CrossRef][Medline] [Order article via Infotrieve]
  30. Hur, E., Pfaff, S. J., Payne, E. S., Gron, H., Buehrer, B. M., and Fletterick, R. J. (2004) PLoS Biol. 2, E274[CrossRef][Medline] [Order article via Infotrieve]
  31. He, B., Gampe, R. T., Jr., Kole, A. J., Hnat, A. T., Stanley, T. B., An, G., Stewart, E. L., Kalman, R. I., Minges, J. T., and Wilson, E. M. (2004) Mol. Cell 16, 425-438[CrossRef][Medline] [Order article via Infotrieve]
  32. Estebanez-Perpina, E., Moore, J. M., Mar, E., Delgado-Rodrigues, E., Nguyen, P., Baxter, J. D., Buehrer, B. M., Webb, P., Fletterick, R. J., and Guy, R. K. (2005) J. Biol. Chem. 280, 8060-8068[Abstract/Free Full Text]
  33. Doesburg, P., Kuil, C. W., Berrevoets, C. A., Steketee, K., Faber, P. W., Mulder, E., Brinkmann, A. O., and Trapman, J. (1997) Biochemistry 36, 1052-1064[CrossRef][Medline] [Order article via Infotrieve]
  34. Ding, X. F., Anderson, C. M., Ma, H., Hong, H., Uht, R. M., Kushner, P. J., and Stallcup, M. R. (1998) Mol. Endocrinol. 12, 302-313[Abstract/Free Full Text]
  35. Brinkmann, A. O., Faber, P. W., van Rooij, H. C., Kuiper, G. G., Ris, C., Klaassen, P., van der Korput, J. A., Voorhorst, M. M., van Laar, J. H., Mulder, E., and Trapman, J. (1989) J. Steroid Biochem. 34, 307-310[CrossRef][Medline] [Order article via Infotrieve]
  36. Jenster, G., Spencer, T. E., Burcin, M. M., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7879-7884[Abstract/Free Full Text]
  37. Steketee, K., Timmerman, L., Ziel-van der Made, A. C., Doesburg, P., Brinkmann, A. O., and Trapman, J. (2002) Int. J. Cancer 100, 309-317[CrossRef][Medline] [Order article via Infotrieve]
  38. Chang, C. Y., Abdo, J., Hartney, T., and McDonnell, D. P. (2005) Mol. Endocrinol. 19, 2478-2490[Abstract/Free Full Text]
  39. Dubbink, H. J., Hersmus, R., Pike, A. C., Molier, M., Brinkmann, A. O., Jenster, G., and Trapman, J. (2006) Mol. Endocinol., in press
  40. Chang, C., Norris, J. D., Gron, H., Paige, L. A., Hamilton, P. T., Kenan, D. J., Fowlkes, D., and McDonnell, D. P. (1999) Mol. Cell. Biol. 19, 8226-8239[Abstract/Free Full Text]
  41. Patterson, G. H., Piston, D. W., and Barisas, B. G. (2000) Anal. Biochem. 284, 438-440[CrossRef][Medline] [Order article via Infotrieve]
  42. Bastiaens, P. I., Majoul, I. V., Verveer, P. J., Soling, H. D., and Jovin, T. M. (1996) EMBO J. 15, 4246-4253[Medline] [Order article via Infotrieve]
  43. Bastiaens, P. I., and Jovin, T. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8407-8412[Abstract/Free Full Text]
  44. Nishimura, K., Ting, H. J., Harada, Y., Tokizane, T., Nonomura, N., Kang, H. Y., Chang, H. C., Yeh, S., Miyamoto, H., Shin, M., Aozasa, K., Okuyama, A., and Chang, C. (2003) Cancer Res. 63, 4888-4894[Abstract/Free Full Text]
  45. Lee, Y. H., Campbell, H. D., and Stallcup, M. R. (2004) Mol. Cell. Biol. 24, 2103-2117[Abstract/Free Full Text]
  46. Ting, H. J., Yeh, S., Nishimura, K., and Chang, C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 661-666[Abstract/Free Full Text]
  47. Jaffer, Z. M., and Chernoff, J. (2002) Int. J. Biochem. Cell Biol. 34, 713-717[CrossRef][Medline] [Order article via Infotrieve]
  48. Schrantz, N., da Silva Correia, J., Fowler, B., Ge, Q., Sun, Z., and Bokoch, G. M. (2004) J. Biol. Chem. 279, 1922-1931[Abstract/Free Full Text]
  49. Lee, S. R., Ramos, S. M., Ko, A., Masiello, D., Swanson, K. D., Lu, M. L., and Balk, S. P. (2002) Mol. Endocrinol. 16, 85-99[Abstract/Free Full Text]
  50. Yang, F., Li, X., Sharma, M., Zarnegar, M., Lim, B., and Sun, Z. (2001) J. Biol. Chem. 276, 15345-15353[Abstract/Free Full Text]
  51. Jenster, G. (1999) Semin. Oncol. 26, 407-421[Medline] [Order article via Infotrieve]
  52. Trapman, J. (2001) Eur. J. Cancer 37, Suppl. 7, S119-S125[Medline] [Order article via Infotrieve]
  53. Balk, S. P. (2002) Urology 60, 132-138[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us