AR and ER Interaction with a p21-Activated Kinase (PAK6)
Suzanne R. Lee,
Sharon M. Ramos,
Andrew Ko,
David Masiello,
Kenneth D. Swanson,
Michael L. Lu and
Steven P. Balk
Cancer Biology Program, Hematology-Oncology Division (S.R.L.,
S.M.R., A.K., D.M., K.D.S., S.P.B.), Department of Medicine, Beth
Israel Deaconess Medical Center and Harvard Medical School, Boston,
Massachusetts 02215; and Urology Division (M.L.L.), Department of
Surgery, Brigham and Womens Hospital, Boston, Massachusetts
02115
Address all correspondence and requests for reprints to: Steven P. Balk, Hematology-Oncology Division, Beth Israel Deaconess Medical Center, HIM Building-Room 1050, 330 Brookline Avenue, Boston, Massachusetts 02215. E-mail sbalk{at}caregroup.harvard.edu
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ABSTRACT
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A human protein termed p21-activated kinase 6 (PAK6), based on
homology to the PAK family of serine/threonine kinases, was cloned as
an AR interacting protein. PAK6 was a 75-kDa protein with a predicted
N-terminal Cdc42/Rac interactive binding domain and a C-terminal
kinase domain. PAK6 bound strongly to GTP-Cdc42 and weakly to GTP-Rac.
In contrast to most PAKs, kinase activity was not stimulated by Cdc42
or Rac, but could be stimulated by AR binding. PAK6 interacted with the
intact AR in a mammalian one-hybrid assay and bound in
vitro, without ligand, to the hinge region between the AR DNA-
and ligand-binding domains. PAK6 also bound to the ER
, and binding
was enhanced by 4-hydroxytamoxifen. AR and ER
transcriptional
activities were inhibited by PAK6 in transient transfections with
episomal and integrated reporter genes. AR inhibition was not reversed
by transfection with an activated Cdc42 mutant, Cdc42V12, which by
itself also inhibited AR transactivation. Epitope-tagged PAK6 was
primarily cytoplasmic in the absence or presence of AR and hormone.
PAK6 transcripts were expressed most highly in brain and testis, with
lower levels in multiple tissues including prostate and breast. PAK6
interaction provides a mechanism for cross-talk between steroid hormone
receptors and Cdc42-mediated signal transduction pathways and could
contribute to the effects of tamoxifen in breast cancer and in other
tissues.
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INTRODUCTION
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THE AR is a steroid hormone receptor member
of the larger nuclear receptor family that mediates the biological
functions of androgens (1, 2). In addition to its
physiological roles in many tissues, the AR also plays a central role
in prostate cancer development and progression (3, 4). The
steroid hormone receptors share a relatively conserved central DNA
binding domain (DBD) and a C-terminal ligand binding domain (LBD),
which also has a ligand-dependent transactivation function (referred to
as AF-2). Their N-termini are more diverse, but generally possess
an independent transactivation function (AF-1), with the AR N-terminal
domain being particularly large and having a strong AF-1. Hormone
binding causes conformational changes that result in receptor
dissociation from an HSP90 chaperone complex, homodimerization, and the
generation of a binding site for proteins containing
leucine-x-x-leucine-leucine (LXXLL) motifs, including the p160
family of steroid receptor coactivator (SRC) proteins
(5, 6, 7). The SRC proteins appear to be the major
coactivators mediating the ligand-dependent AF-2 transactivation
function of the LBD, through histone acetyl or methyltransferase
activity and association with cAMP response element binding
protein or p300. The SRC proteins can also interact directly
with the N terminus of steroid hormone receptors, and this interaction
may be particularly critical for the AR (8, 9, 10, 11).
In addition to the p160 steroid receptor coactivator proteins, there is
a growing list of proteins that interact with the N-terminal, DNA, or
ligand binding domains of steroid hormone receptors (5, 6). These include proteins linked to the general transcriptional
machinery, proteins that function as transcriptional coactivators or
corepressors by other mechanisms, and proteins the functions of which
remain to be determined. Some of these interactions are regulated by
ligand binding and mediated by LXXLL or related motifs, while others
are independent of ligand. There is also a ligand- dependent
interaction between N-terminal and C-terminal domains of the AR and
other steroid hormone receptors, which appears to be particularly
important for AR transactivation (12, 13, 14, 15, 16, 17, 18). Finally, there
is evidence for nontranscriptional functions of steroid hormone
receptors (19, 20, 21) and an association between the ER
and PI3K (22).
This report describes the isolation and characterization of an AR and
ER
interacting protein termed p21-activated kinase 6 (PAK6), based
upon its homology to previously identified PAKs. PAKs form an
evolutionarily conserved family of serine/threonine kinases that bind
to, and are regulated by, the active (GTP-bound) form of the Rho family
small (p21) GTPases, Cdc42 and Rac (23, 24, 25, 26). Cdc42 and Rac
binding are mediated by a conserved N-terminal Cdc42/Rac interactive
binding (CRIB) domain (27). PAKs are presumed to mediate
some of the downstream effects of activated Cdc42 and Rac, although the
targets of their kinase activity and precise functions remain to be
determined. The yeast PAK homolog (STE20) activates a MAPK
kinase kinase analogous to mammalian Raf (28), and
mammalian PAKs have been reported to similarly activate MAPK pathways
in response to activated Cdc42 and/or Rac (29, 30, 31, 32, 33, 34).
Additional possible roles for PAKs are in cytoskeleton organization
(35, 36, 37), cell cycle regulation (38),
heterotrimeric G protein signaling (39), and apoptosis
(40, 41, 42, 43). Therefore, the AR- and ER
-PAK6 interactions
provide potential direct links between these steroid hormone receptors
and signal transduction pathways regulating diverse cellular
functions.
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RESULTS
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Isolation of a PAK-Related Kinase Interacting with the AR in
Yeast
A fragment of the human AR containing the DNA and ligand binding
domains (AR505919), was fused to the GAL4 DNA binding domain and used
as the bait in a series of yeast two-hybrid screens, in the presence of
1 µM dihydrotestosterone (DHT). Positive clones were
subsequently screened without DHT, and several clones that showed
strong DHT-dependent growth were isolated and sequenced. Two clones
contained in-frame fusions to previously identified proteins, gelsolin,
an actin-binding protein (44), and ARA70. ARA70 (fused at
alanine 168) was identified previously as an AR interacting protein,
although its functional significance remains unclear (45, 46). The significance of the AR interaction with gelsolin was
also unclear, although an AR interaction with another actin-binding
protein, filamin, was recently reported (47).
Partial sequencing of a third isolate (clone 56) indicated that
it was a novel protein. A specific interaction between the AR and clone
56 in yeast was confirmed by cotransforming clone 56 (fused to the GAL4
transactivation domain) with additional plasmids encoding GAL4
DNA-binding domain (GAL4 DBD) fusion proteins and assessing
ß-galactosidase production from an integrated GAL4 responsive
reporter. In the absence of DHT, only low levels of ß-galactosidase
activity were detected in all cases (not shown). DHT increased
ß-galactosidase activity 27-fold in yeast expressing both clone 56
and the GAL4 DBD-AR (505919) fusion protein used in the yeast screen
(Table 1
). This level of induction was
greater than that seen with a transactivation domain fused to GR
interacting protein 1 (GRIP1) (5631121), which
contains three LXXLL motifs and an additional AR-interacting domain
(48). In contrast, no induction was observed when clone 56
was expressed with GAL4 DBD fusion proteins containing the AR
N-terminal transactivation domain, AR (2506), the DBD
and nuclear localization signal (NLS), AR (553635), or
with the irrelevant protein cortactin. These results demonstrated that
the protein encoded by clone 56 interacted with the AR in yeast and
that the interaction required a region C-terminal to the NLS.
Sequence Analysis of Full-Length PAK6
Complete sequencing of clone 56 revealed a consensus kinase domain
at the C terminus, but no homology to previously reported proteins at
the amino terminus (Fig. 1A
). However,
the first 35 nucleotides of the cDNA insert (nucleotides 874908,
boxed in Fig. 1A
) were identical to the 3'-end of an
expressed sequence tag (EST) from a testis cDNA library (GenBank
accession no. AA815255), indicating that this EST encoded the
5'-end of clone 56. The plasmid containing this EST was obtained and
sequenced to provide the 5'-end of the transcript. The assignment of
the initiation methionine was based upon an in-frame stop codon (tga)
39 bases upstream (Fig. 1A
, boxed). Since the overlap
between the EST and clone 56 was only 35 bases immediately before a
poly A tract in the EST, RT-PCR was used to confirm that this EST
represented the 5'-end of the clone 56 transcript. RT-PCR from prostate
cancer-derived cDNAs using 5'-primers derived from the EST and
3'-primers from clone 56 generated a product of the predicted size and
sequence (data not shown), confirming that EST AA815255 represented the
5'-end of clone 56.

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Figure 1. Complete Sequence of PAK6 and Homology to Other
PAKs
A, PAK6 nucleotide and predicted amino acid sequence. An in-frame stop
codon (tga) 5' of the predicted initiation ATG and the N-terminal CRIB
domain are boxed. Nucleotides at the 5'-end of yeast
clone 56 that were identical to the 3'-end of the EST from a testis
library (nucleotides 874908) are also boxed. Amino
acids highly conserved in all kinase domains are boxed.
S531, which is an asparagine in most other kinases, is
boxed and in bold. A possible
heterotrimeric G protein-binding site is boxed at the C
terminus. B, Homology in CRIB and autoinhibitory domains. The
alignments are based on PAK6 with periods (.) indicating
identical residues and dashes () marking gaps
introduced to maximize homology. PAK13 have an N-terminal extension
past the CRIB domain that is absent in PAK46. ß-Strands (ß1 and
ß2) and -helices (H13) are from the PAK1 crystal structure.
Residues underlined in the CRIB domain mediate
dimerization. The autoinhibitory lysine in PAK13 is indicated by an
asterisk (*).
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The full-length sequence predicted a protein of 681 amino acids with a
molecular mass of 75 kDa (Fig. 1A
). Analysis of the full-length
coding region revealed homology at the 5'- and 3'-ends to the PAK
family of serine/threonine kinases, of which four had been previously
reported in humans (24, 37, 49, 50, 51). The sequences of two
additional human PAK-related proteins, termed PAK5 and PAK6, were more
recently deposited in GenBank. The protein isolated here was identical
to PAK6, located on chromosome 15q15.
The N terminus of PAK6 had homology with the CRIB domains of the
previously characterized PAKs, containing six of eight of the CRIB
domain consensus residues (Fig. 1B
) (27). The greatest
homology was with PAK4 (37) and PAK5, also with CRIB
domains at the immediate N terminus. The recently reported crystal
structure of human PAK1 indicated that this protein formed a dimer
through an antiparallel ß-ribbon formed by a ß-strand that
overlapped the CRIB domain (ß1 in Fig. 1B
) (52). The
critical contact residues in this ß-strand are conserved in PAK16
(underlined in the CRIB consensus in Fig. 1B
), suggesting
that PAK6 is similarly a dimer. The PAK1 crystal structure also showed
that the autoinhibition of PAK1 kinase activity was due to a bundle of
three helices (H13 in Fig. 1B
) that packed against the kinase domain
and positioned a lysine residue (amino acid 141 in PAK1, indicated with
an asterisk) into the active site. These structural features
were highly conserved in PAK13, but were not evident in PAK46,
suggesting alternative mechanisms for regulating the kinase activity of
these latter PAKs.
The C terminus of PAK6 encoded a consensus kinase domain with
homology to PAK1, 2, and 3 (50% compared with PAK1), but again with
much greater homology to PAK4 (80% homology). Residues highly
conserved in kinase domains were also conserved in PAK6 (Fig. 1A
, boxed), with the exception of an asparagine that was
replaced by a serine (S531), a replacement also seen in PAK4
(boxed and in bold in Fig. 1A
). Other conserved structural
features of previously described PAK family members include N-terminal
proline-rich SH3-binding motifs, which can target PAKs to the membrane
through the Nck adapter protein (31, 53, 54, 55), and a
heterotrimeric G protein ß-subunit binding domain at the C terminus
(39, 56). The former N-terminal SH3-binding motifs were
not present in PAK4, 5, or 6, as the CRIB domains were at the extreme N
terminus. Three of the four residues defining a putative heterotrimeric
G protein binding motif were present in the C terminus of PAK6 (Fig. 1A
, boxed).
Cell and Tissue Distribution of PAK6
The full-length PAK6 was fused to the C-terminus of green
fluorescent protein (GFP) and used to assess cellular distribution. The
GFP-PAK6 fusion protein transfected into HeLa cells localized primarily
to the plasma membrane and cytoplasm (Fig. 2A
). PAK6 remained
primarily cytoplasmic when cotransfected with the AR, in the absence
(Fig. 2B
) or presence of DHT (Fig. 2D
). HeLa cells were similarly
transfected with a GFP-AR expression vector to assess PAK6 effects on
AR distribution. The AR in HeLa cells was primarily cytoplasmic in the
absence of DHT (Fig. 2
, E and F) and nuclear in the presence of DHT
(Fig. 2
, G and H). This distribution was not altered by PAK6
cotransfection (Fig. 2
, F and H). Similar results were obtained
by indirect immunofluorescence with AR and an epitope-tagged
PAK6, indicating that cellular localization was not altered by the GFP
fusion (data not shown).

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Figure 2. PAK6 Cell and Tissue Distribution
AD, HeLa cells transiently transfected with GFP-PAK6 (pEGFP-PAK6)
alone (A and C) or cotransfected with the pSVARo expression
vector (B and D). EH, HeLa cells transiently transfected with GFP-AR
(pEGFP-AR) alone (E and G) or cotransfected with pcDNA-PAK6 (F and H).
Cells were in steroid hormone-depleted medium (A, B, E, and F) or were
treated for 30 min with 10 nM DHT (C, D, G, and H). I,
Multiple tissue expression array probed with unique
32P-labeled fragment of PAK6. Column 1 (AH), Whole brain, cerebral cortex, frontal
lobe, parietal lobe, occipital lobe, temporal lobe, paracentral gyrus
of the cerebral cortex, pons. Column 2 (AH), Blank, right cerebellum,
corpus callosum, amygdala, caudate nucleus, hippocampus, medulla
oblongata, putamen. Column 3 (AE), Substantia nigra, nucleus
accumbens, thalamus, pituitary gland, spinal cord; FH, blank. Column
4 (AH), Heart, aorta, left atrium, right atrium, left ventricle,
right ventricle, interventricular septum, apex of heart. Column 5
(AH), Esophagus, stomach, duodenum, jejunum, ileum, ileocecum,
appendix, ascending colon. Column 6 (AC), Transverse colon,
descending colon, rectum; DH, blank. Column 7 (AH), kidney,
skeletal muscle, spleen, thymus, peripheral blood leukocyte, lymph
node, bone marrow, trachea. Column 8 (AH), Lung, placenta, bladder,
uterus, prostate, testis, ovary, blank. Column 9 (AF), Liver,
pancreas, adrenal gland, thyroid gland, salivary gland, mammary gland;
G and H, blank. Column 10 (AH), Promyelocytic leukemia (HL-60), HeLa
S3, chronic myelogenous leukemia (K-562), lymphoblastic leukemia
(MOLT-4), Burkitts lymphoma (Raji), Burkitts lymphoma (Daudi),
colorectal adenocarcinoma (SW480), lung carcinoma (A549). Column 11
(AH), Fetal brain, fetal heart, fetal kidney, fetal liver, fetal
spleen, fetal thymus, fetal lung, blank. Negative controls: Column 12
(AH), Yeast total RNA, yeast tRNA, E. coli rRNA,
E. coli DNA, poly r(A), human C0t-1 DNA, 100
ng human DNA, 500 ng human DNA.
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Hybridization of a unique internal fragment from PAK6 (nucleotides
451-1256) to a human multiple tissue expression array revealed the
strongest expression in testis and in many areas of the brain,
particularly cortical structures (Fig. 2I
). Lower level expression was
seen in prostate, thyroid, adrenals, placenta, kidney, esophagus,
mammary gland, and heart. There was little or no detectable expression
in ovary, uterus, intestine, liver, lung, spleen, thymus, peripheral
blood leukocytes, lymph node, or bone marrow.
PAK6 Binding to the AR in Mammalian Cells and in
Vitro
PAK6 binding to the AR in mammalian cells was assessed by fusing
the VP16 transactivation domain to the N-terminal of the PAK6 fragment
isolated from the yeast screen, VP16-PAK6(256681). DHT induced AR
transcription 60-fold from a luciferase reporter gene regulated by a
multimerized androgen response element (ARE4)
(Fig. 3
). Cotransfection with vectors
encoding only the VP16 activation domain (AASVVP16) or a
VP16-SRC1(595780) fusion, containing the first LXXLL motif of SRC-1,
were used as negative controls as this region of SRC-1 was shown
previously to interact very weakly or not at all with the AR (10, 11, 16). Both vectors markedly repressed AR activity (Fig. 3
and
data not shown), likely reflecting competition for transcription
factors by the noninteracting VP16 transactivation domain. In contrast,
cotransfection with the VP16-PAK6(256681) vector increased the
induction to 175-fold. These results indicated that PAK6 interacted
with the intact ligand-bound AR on DNA in mammalian cells.

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Figure 3. Mammalian One-Hybrid Analysis of PAK6-AR
Binding
CV1 cells were cotransfected with ARE4-Luc reporter (100
ng), pRL-SV40 (Renilla control), and with the pSVARo (200
ng), VP16-PAK6(256681) (20 ng), or VP16-SRC(595780) (20 ng), as
indicated. DHT was added at 24 h, and cells were harvested at
48 h and assayed for luciferase and Renilla activity.
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AR binding to PAK6 was next assessed in vitro by
precipitation with a series of glutathione-S-transferase
(GST) fusion proteins. The fragment of PAK6 isolated in the yeast
screen was expressed as a GST fusion protein, GST-PAK6(256681), and
full-length 35S-labeled AR was generated by
coupled in vitro transcription/translation. AR binding to
GST-PAK6(256681) was compared with binding to a GST-GRIP1 (6241122)
fusion protein, containing multiple LXXLL motifs and shown to interact
with agonist-bound LBDs of nuclear receptors including the AR
(48, 57). AR binding to the GST-GRIP1(6241122) fusion
protein was detectable, but was very weak (Fig. 4A
). Binding was not enhanced by DHT,
which may reflect nonnative folding of the in
vitro-generated AR. In contrast, full-length AR was precipitated
efficiently by the GST-PAK6(256681) fusion protein. The binding was
ligand independent as it did not require added DHT and was not blocked
by bicalutamide (BIC), a competitive antagonist of DHT binding and AR
function (58).

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Figure 4. PAK6 Binds to the AR
A, GST, GST-GRIP1(6241122), or GST-PAK6(256681) were used to pull
down 35S-labeled AR, plus or minus DHT (10 nM)
or BIC (10 or 50 µM), as indicated. B, Lysates from LNCaP
cells grown in medium with 10% FCS were precipitated with GST,
GST-GRIP1(6241122), or GST-PAK6(256681), and bound AR was detected
by immunoblotting. C, LNCaP cells grown in medium with 10% CDS FCS or
in this medium with 10 nM DHT or 5 µM BIC
added overnight were lysed in glycerol lysis buffer, precipitated with
GST-PAK6(256681) or GST beads (25 µg each), and AR immunoblotted.
D, 35S-Labeled full-length PAK6 was precipitated with the
indicated GST-AR fusion proteins. E, 35S-labeled
full-length PAK1 or PAK6 was precipitated with GST, GST-AR(505919),
or GTP-loaded GST-Cdc42V12. The input lanes contain 10% of the
material incubated with the beads.
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GST-PAK6(256681) pull-down experiments were next carried out
using the endogenous AR from LNCaP cells, the only generally
available AR-expressing human prostate cancer cell line. LNCaP cells
express a mutant AR (T877A) that still responds to DHT but has altered
responses to other ligands and AR antagonists (59).
LNCaP lysates were precipitated by GST-PAK6(256681),
GST-GRIP1(6241122), or control GST beads, and AR was detected by
immunoblotting. The results similarly demonstrated specific binding of
the intact AR to PAK6 (Fig. 4B
). In these experiments comparable
binding to the GST-GRIP1(6241122) control was observed, possibly
reflecting native folding and androgen binding by the AR in
vivo. The hormone dependence of binding by the LNCaP AR was
further assessed by culturing the cells overnight in medium with
charcoal dextran-stripped (CDS) FCS (steroid hormone-depleted medium),
or with added DHT (10 nM) or BIC (5
µM), which is also an antagonist of the LNCaP
AR. GST-PAK6(256681) bound specifically to the AR from DHT, BIC, and
untreated (CDS) LNCaP cells (Fig. 4C
), further demonstrating that
binding was not ligand dependent.
Binding of full-length PAK6 to AR was investigated using
35S-PAK6, which was labeled by coupled in
vitro transcription/translation. Strong binding was observed to a
GST-AR(505919) fusion protein, containing the DBD, hinge, and LBD
(Fig. 4D
). Binding was also detected to deletion mutants
GST-AR(634804), with the LXXLL binding AF-2 removed, and to
GST-AR(634668). The GST-AR(634668) protein corresponds to a
fragment from the AR nuclear localization signal (amino acids 617633)
to the beginning of helix 3, which marks the beginning of the
LBD. These latter results indicated that PAK6 bound to a site distinct
from the LXXLL motif binding AF-2 and suggested that binding was
through the hinge region between the DBD and LBD.
Finally, the specificity of the PAK6-AR interaction was assessed by
examining GST-AR binding to PAK1. In contrast to the results with PAK6,
there was no specific binding of in
vitro-transcribed/translated PAK1 to the GST-AR
(505919) fusion protein (Fig. 4E
). However, PAK1 was
found to bind to a GTP-loaded GST-Cdc42 fusion protein (see below).
This result indicated that the AR interaction was not a general
property of PAKs.
PAK6 Binding to ER
The ER
was next examined to determine whether PAK6 binding was
specific for the AR. 35S-Labeled ER
, generated
by in vitro transcription/translation in rabbit reticulocyte
lysates, bound specifically to the positive control GST-GRIP(6241122)
fusion protein (Fig. 5A
). This GRIP1
binding in the absence of added estrogen likely reflected estrogen in
the rabbit reticulocyte lysate and could be augmented with added E2. As
expected, ER
binding to GST-GRIP1(6241122) was markedly reduced by
the partial agonist 4-hydroxytamoxifen (OHT). A comparable level of
ER
binding to GST-PAK6(256681) was observed in the absence or
presence of E2. However, in marked contrast to GST-GRIP1 results, ER
binding to PAK6 was enhanced (3.4-fold) by OHT (Fig. 5A
). This latter
finding was consistent with the AR results and indicated that PAK6
bound to a site distinct from the agonist-generated LXXLL coactivator
binding site, as this site is occluded in the OHT-bound ER
(60, 61, 62).
It was next determined whether full-length PAK6 bound
specifically to the ER
. These experiments used a GST-ER
(281595)
fusion protein, encompassing the region C terminal to the NLS and the
complete ER
LBD, and shown previously to bind LXXLL containing
coactivator proteins in an agonist-dependent manner (63).
Full-length in vitro-transcribed/translated PAK6
bound specifically to the GST-ER
(281595) fusion protein in the
absence of added E2, and binding was not augmented by added E2
(Fig. 5B). Moreover, consistent with the results above, binding
was enhanced (2.7-fold) by OHT. PAK6 binding by the OHT-ligated
GST-ER
was comparable to the CRIB domain-mediated binding of
GST-GTP-Cdc42V12 (see below).
PAK6 Interaction with p21 GTPases
It was next determined whether PAK6 had a functional CRIB domain
capable of binding to GTP-Cdc42 and/or -Rac. These experiments used a
full-length PAK6 cDNA with a C-terminal myc/his epitope tag,
constructed in the pcDNA3 mammalian expression vector. GST pull-down
experiments were carried out using GTPase-deficient (activated)
Cdc42V12 and RacV12 mutants expressed as GST fusion proteins.
Equal amounts of GST fusion proteins bound to glutathione agarose beads
were first loaded with GTP. Comparable GTP loading was confirmed on
parallel samples using 32P-GTP (not shown). The
beads were then used to precipitate the myc/his-tagged PAK6 from
transfected CV1 cell lysates. GST-GTP-Cdc42V12 pulled down a
substantial amount of PAK6, identified as a 75-kDa protein by
immunoblotting with an anti-myc monoclonal antibody (mAb) (Fig. 6A
). In contrast, only very weak binding
to GST-GTP-RacV12 was detected, although this binding appeared to be
specific as no binding was detected to either an inactive control
(GST-RacN17) or to other control GST proteins.

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Figure 6. PAK6 Binding by p21 GTPase and Kinase Activation
A, GST fusion proteins were used to pull down myc-tagged PAK6 from a
transfected CV1 cell lysate. Equivalent amounts of GST fusion proteins
loaded with GTP were used as indicated, and precipitated PAK6 was
detected by immunoblotting with an anti-myc mAb. GST-CD1d is an
irrelevant control fusion protein. Lysate alone (2% of the material
used for each pull down) is shown in the first lane, and the position
of the full-length PAK6 (75 kDa) is indicated. B, PAK6 programmed or
unprogrammed rabbit reticulocyte lysates (PAK6 +/-, respectively) were
pulled down with the indicated AR or GTP-loaded GST fusion proteins.
The precipitates were then split in half and analyzed for kinase
activity with MBP substrate (top panel), or analyzed for
bound 35S-PAK6 (bottom panel).
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GTP-Cdc42 or -Rac binding activates the kinase activity of other PAKs
by blocking an autoinhibitory domain carboxy to the CRIB domain (Fig. 1B
) (52, 64, 65, 66). However, this domain appears to be
absent in PAK46 (see Fig. 1B
), and PAK4 kinase activity is not
stimulated by Cdc42 (37). Therefore, kinase assays were
carried out to assess PAK6 activation by GTP-Cdc42. For these
experiments, full-length 35S-labeled PAK6 was
expressed in vitro and precipitated by a series of GST
fusion proteins bound to glutathione beads. The kinase activity of the
precipitated proteins was compared with proteins precipitated by the
same beads from a control unprogrammed rabbit reticulocyte lysate,
using myelin basic protein (MBP) as a substrate. No kinase activity was
detected in the GST-GTP-Cdc42V12 precipitate (Fig. 6B
, top
panel), although PAK6 binding to these beads was confirmed by
recovery of the labeled PAK6 protein (Fig. 6B
, bottom
panel). There was also no detectable kinase activity or PAK6
binding to GST-Rac fusion proteins.
Kinase activity was precipitated by the GST-AR(505919) fusion protein
from the unprogrammed and PAK6 programmed lysates (Fig. 6B
). However,
the kinase activity from the PAK6 programmed lysate was consistently
greater (
3-fold). This indicated that it reflected PAK6 kinase
activity, or possibly PAK6 activation of another AR- associated kinase,
stimulated by AR binding. It is not yet clear whether the lower levels
of kinase activity precipitated from the unprogrammed lysate were due
to an endogenous PAK or other kinases. Taken together, these results
demonstrated that the PAK6 CRIB domain was functional with respect to
p21 binding, with a marked preference for binding GTP-Cdc42
vs. GTP-Rac. The results further indicated that PAK6 kinase
activity was not regulated by Cdc42 binding, with the data suggesting
instead a role for AR in regulating PAK6 kinase activity.
PAK6 Inhibition of AR and ER
Transcriptional Activity
Cotransfection experiments were carried out to determine whether
PAK6 could modulate the transcriptional activity of the AR. Cells were
cotransfected with an ARE4-luciferase reporter
plasmid, AR expression vector (pSVARo)
(67), pcDNA-PAK6 or control (pcDNA-LacZ) expression
vectors, and an internal control Renilla vector (pRL-SV40). AR
transcriptional activity was stimulated 33-fold by DHT in the absence
of PAK6 and was not inhibited by the control pcDNA-LacZ vector (Fig. 7A
). In contrast, AR transcriptional
activity was markedly inhibited by PAK6, with induction reduced
approximately 5-fold by 200 ng of the PAK6 vector. This inhibition was
not a nonspecific effect on transcription or on the pSV promoter
regulating the AR, as expression of the control Renilla reporter
regulated by a pSV promoter was unaffected by PAK6 (Fig. 7B
).
Immunoblotting for AR protein further showed that the inhibition was
not due to decreased AR protein expression (Fig. 7C
).

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|
Figure 7. PAK6 Inhibition of AR Transcriptional Activity
A, CV1 cells were transiently transfected with an
ARE4-luciferase reporter (100 ng), pRL-SV40 (0.2 ng),
pSVARo (200 ng), and pcDNA-LacZ or pcDNA-PAK6 expression
vectors. Luciferase and Renilla activity were assessed after 24 h
± DHT. B, Renilla activity from the experiment in panel A. C, AR
expression in lysates from the cells used in panel A.
|
|
The effect of PAK6 on transcription from another AR-responsive
promoter, the mouse mammary tumor virus long terminal repeat (MMTV-LTR)
containing two steroid response elements recognized by the AR, was next
examined. DHT stimulated AR transcriptional activity on the
MMTV-LTR-luciferase reporter by 5.3-fold (Fig. 8A
). Similar to the above result with the
ARE4 reporter, this DHT-stimulated activity was
inhibited by cotransfection with PAK6. It was also determined whether
PAK6 affected AR transcriptional activity in the more physiological
setting of an integrated ARE. For this experiment, CV1 cells were
stably transfected with the MMTV-LTR-luciferase reporter plasmid, in
conjunction with a neomycin resistance plasmid. G418-resistant clones
were then screened for ligand-dependent stimulation of luciferase
activity by transfected AR, and a clone with a low level of background
luciferase activity and a relatively high level of DHT-inducible AR
activity was selected. DHT augmented the transcriptional activity of
the transfected AR by approximately 6-fold in these cells (Fig. 8B
).
This was reduced to 2.4-fold induction by cotransfection with PAK6,
indicating that PAK6 could inhibit AR activity on an integrated as well
as an episomal reporter.

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[in a new window]
|
Figure 8. PAK6 Inhibition of AR on Episomal and Integrated
MMTV-LTR
A, CV1 cells were transiently transfected with an MMTV-LTR-luciferase
reporter. Cotransfected plasmids and DHT treatments were as in Fig. 7 .
B, CV1(MMTV-Luc) cells were cotransfected with AR, Renilla, and PAK6
plasmids and treated with DHT as in panel A.
|
|
Similar cotransfection experiments were carried out with an ER
expression vector and an estrogen responsive element (ERE)-regulated
luciferase reporter plasmid to determine whether PAK6 modulated ER
transcriptional activity. Luciferase activity was stimulated about
4.5-fold by E2 (Fig. 9A
). Cotransfection
with PAK6 reduced this stimulation by approximately 50%, while a
control LacZ vector had no effect. This result demonstrated a similar
inhibitory effect of PAK6 on AR and ER
transcriptional activity.
As PAK6 also binds to GTP-Cdc42, it was possible that AR and ER
inhibition by PAK6 was due to sequestration of GTP-Cdc42 rather than a
direct interaction. This was addressed by cotransfection with activated
(GTPase-deficient) Cdc42V12. DHT stimulated AR transactivation by
26-fold, and this was reduced to approximately 7-fold by PAK6
(Fig. 9B
). This repression by PAK6 was not reversed by cotransfected
Cdc42V12. Moreover, transfection of Cdc42V12 by itself inhibited AR
transcriptional activity, consistent with a recent report that Rho
GTPases can negatively regulate steroid hormone receptors
(68). Therefore, these results demonstrated that AR
inhibition by PAK6 was not due to sequestration of Cdc42.
 |
DISCUSSION
|
---|
This study identified PAK6 as a strongly AR-interacting protein in
a yeast two-hybrid screen. The physiological significance of the
interaction was supported by in vitro studies demonstrating
specific binding of the full-length AR and PAK6 proteins and by
mammalian one-hybrid experiments demonstrating an interaction between
the ligated native AR and a VP16-PAK6 fusion protein. PAK6 also bound
to ER
, and this binding was enhanced by OHT, indicating that the
interaction was sensitive to the functionally critical conformational
changes in the AF-2 region that mediate coactivator binding to the
ER
and to other nuclear receptors in response to ligand (61, 62, 69, 70).
The enhanced ER
binding with OHT further indicated that the PAK6
interaction was not through the LXXLL binding AF-2 region, as this site
is occluded in the OHT- bound ER
(62). This was
consistent with the ligand independence of AR binding in
vitro and results using GST-AR deletion mutants that indicated a
binding site in the hinge region between the DBD and LBD. A number of
other proteins appear to bind to this region of the AR and/or other
steroid hormone receptors, including ANPK (a nuclear serine/threonine
kinase) (71), L7/SPA (72), and UBC9 (a SUMO
conjugating enzyme) (73). Although these proteins
can function as coactivators, a deletion in the hinge region enhances
AR transcriptional activity (74). Moreover, mutations at
the C terminus of the AR hinge region have been identified in human
prostate cancers and in an SV40 T/t antigen-induced mouse prostate
cancer (75), with the latter mutation enhancing AR
transcriptional activity, consistent with a corepressor binding to this
region.
AR transcriptional activity from two different episomal reporter genes,
as well as an integrated reporter, was inhibited by PAK6. Inhibition
was not due to Cdc42 sequestration by the PAK6 CRIB domain, as it was
not reversed by cotransfected Cdc42V12. Moreover, Cdc42V12 by itself
inhibited AR transcriptional activation. This latter result was
consistent with a previous report showing that Rho GDI
, a negative
regulator of Rho GTPases, could augment AR, ER
, ERß, and GR
transcriptional activity, and that the ER was inhibited by Rho, Rac,
and Cdc42 (68). Taken together, these results suggest that
the AR transcriptional inhibition by PAK6 could be due to recruitment
of Cdc42 to the AR complex. However, PAK6 is unlikely to mediate all of
the effects of Rho GTPases on steroid hormone receptors, as it
interacted only weakly with GTP-Rac.
Alternatively, PAK6 inhibition of the AR may be mediated by
phosphorylation of the AR or other AR-associated proteins. Studies
addressing possible substrates for PAK6 kinase activity are underway
but have not found AR phosphorylation by PAK6 in vitro or
increased AR phosphorylation in vivo in response to
transfected PAK6. Other possible mechanisms for AR inhibition by PAK6
are that PAK6 competes with coactivators for binding, stabilizes the AR
in a conformation unfavorable for coactivator binding, or blocks the
interaction between the AR N-terminal domain and LBD
(12, 13, 14, 15, 16, 17, 18). These latter mechanisms would be consistent
with the enhanced PAK6 binding to the OHT- ligated ER
, as OHT blocks
coactivator binding. Finally, it should of course be emphasized that AR
inhibition may reflect nonphysiological high levels of transfected
PAK6, and that PAK6 may instead selectively modulate AR activity on
particular promoters or in response to activation of other signal
transduction pathways.
Alternative functions for the PAK6-AR interaction may be to recruit
PAK6 and/or activate its kinase activity. The kinase activity of most
previously characterized PAKs is blocked by an autoinhibitory domain
that follows the CRIB domain (52, 64, 65, 66). Cdc42 or Rac
binding relieves this inhibition and results in PAK autophosphorylation
and activation of kinase activity. Although PAK6 clearly has a
functional CRIB domain, which selectively binds to Cdc42, there is
limited homology between PAK6 and human PAK13 in the CRIB-regulated
autoinhibitory domain (see Fig. 1B
), and PAK6 kinase activity is not
activated by GTP-Cdc42 binding. The N terminus of PAK6 is homologous to
PAK4, which also selectively binds to Cdc42 and is not activated by
Cdc42 binding (37). Studies are underway to determine
whether, and under what conditions, the AR can activate PAK4 or PAK6
kinase activity in vivo, as suggested by the in
vitro kinase activity associated with PAK6 bound to a GST-AR
fusion protein. This kinase activity could contribute to the rapid
nontranscriptional activation of MAPKs and other pathways demonstrated
previously for ER
(19, 20) and AR
(21).
The cellular distribution of transfected PAK6, plus or minus
cotransfected AR, was primarily in the cytoplasm and on plasma
membrane. However, lower levels of nuclear PAK6, alone or in
association with AR, could not be ruled out. Preliminary biochemical
fractionation studies similarly indicate that PAK6 is mostly
cytoplasmic but suggest that a small fraction might be nuclear. The
highest levels of PAK6 expression were in brain and testis, although
PAK6 could also be expressed at relatively high levels by
specific cell types in other tissues. While this manuscript was
under revision, another group similarly identified PAK6 as an
AR-interacting protein that was highly expressed in testis and could
repress AR transcriptional activity, although their data indicated
marked AR stimulated nuclear translocation of transfected PAK6
(76). It is clear that specific antibodies will be needed
to better assess the cellular and tissue distribution of the endogenous
protein.
Database searches have not revealed definite homologs of PAK6 in
other species. However, the mushroom bodies tiny (mbt) gene
from Drosophila encodes a PAK that appears related to human
PAK4, 5, and 6 (77). Mutations in mbt interfere
with brain development, which in conjunction with the high level
expression of PAK6 in human adult and fetal brain, suggest a role for
PAK6 in brain development. In this regard, a PAK3 mutation has been
identified in a family with mental retardation (51).
However, although the biological functions of PAK6 remain
unclear, they likely differ in detail from most previously described
PAKs based upon low-sequence homology, the absence of SH3
binding motifs that direct binding of the Nck adapter protein
(31, 53, 54, 55) or the Pix/Cool nucleotide exchange proteins
(78, 79), and the failure of GTP-Cdc42 to activate PAK6
kinase activity. PAK6 binding may be a mechanism through which diverse
signal transduction pathways, in particular those mediated through
Cdc42, can modulate the activity of the AR and ER
(and possibly
other nuclear receptors). Alternatively, or in addition, PAK6 binding
may mediate nontranscriptional functions of the AR or ER
. Finally,
while the OHT-enhanced PAK6-ER
interaction may or may not be
physiological, it could contribute to the therapeutic effects of
OHT in breast cancer and in other tissues.
 |
MATERIALS AND METHODS
|
---|
PAK6 Cloning
A fragment of the human AR from glycine 505 to the C
terminus (AR505919) was generated by PCR and cloned into the pAS2
yeast GAL4 DNA binding domain vector (CLONTECH Laboratories, Inc., Palo Alto, CA). A series of human GAL4 activation
domain libraries were screened in the presence of 1 µM
DHT, and a fragment of PAK6 was isolated from a prostate library in the
pACT2 vector (clone 56). A plasmid containing the N terminus of PAK6,
identified as an EST from a testis library (GenBank accession no.
AA815255), was obtained from the I.M.A.G.E. (Integrated
Molecular Analysis of Genomes and their Expression) consortium.
Additional AR yeast vectors, as indicated, were constructed by
PCR and confirmed by DNA sequencing. pGAD24-GRIP(5631121) was from
Michael Stallcup (University of Southern California, Los Angeles)
(57). Liquid ß-galactosidase assays were carried out on
extracts from transformed yeast containing an integrated GAL4
promoter regulating ß-galactosidase (strain HF7c) (CLONTECH Laboratories, Inc.) with
O-nitrophenyl-ß-D-galactopyranoside as the
substrate, as described by the manufacturer.
PAK6 Expression Vectors
Full-length PAK6 was assembled from three fragments in the
pcDNA3.1(-)/Myc-His C mammalian expression vector, containing
C-terminal myc and histidine epitope tags (Invitrogen,
Carlsbad, CA). The 5'-end through an internal XbaI site was
obtained from the I.M.A.G.E. consortium plasmid described above, a
middle fragment from the XbaI site to a downstream
BamHI site was generated by PCR amplification from prostate
cancer cDNA, and a fragment from the BamHI site to the
3'-end was from the yeast clone 56. The pcDNA 3.1 epitope tag (myc-his)
was placed in frame at the C terminus by cutting at a BlpI
site in the PAK6 stop codon and a downstream KpnI site in
the pcDNA polylinker, followed by blunting with mung bean nuclease and
ligation.
The GFP-PAK6 vector (pEGFP-PAK6) was constructed in the pEGFP-C1 vector
(CLONTECH Laboratories, Inc.). An oligonucleotide encoding
the first 11 amino acids of PAK6 with a C-terminal BglII
site was ligated to the BglII site encoding amino acids 11
and 12 in PAK6. This was then excised and an N-terminal
HindIII site introduced by the oligonucleotide was used to
ligate PAK6 in frame at the C terminus of GFP as a
HindIII-KpnI fragment. A GFP-AR expression vector
(pEGFP-AR) was similarly constructed in pEGFP-C1, but using an
EagI site located eight amino acids from the N terminus of
the AR. To generate GST-PAK6 (256681), an
XhoI site was introduced into the pGEX-2TK polylinker, and
the entire PAK6 fragment from clone 56 was inserted in frame using a
compatible SalI site in pACT2 at the junction between the
GAL4 activation domain and PAK6.
PAK6 Expression
pEGFP-PAK6, with or without pSVARo, was
transfected into HeLa cells by electroporation. Cells were then plated
onto coverslips, cultured for 24 h in DMEM with 10%
charcoal-dextran stripped FCS (HyClone Laboratories, Inc., Logan, UT), treated for varying times with DHT, and
fixed in 1% paraformaldehyde in PBS. HeLa cells were similarly
transfected with pEGFP-AR, with or without the pcDNA-PAK6 expression
vector. A human multitissue blot containing polyadenylated RNA from
multiple different human tissues and cell lines (Human Multiple Tissue
Expression Array, CLONTECH Laboratories, Inc.) was probed
with a PCR-generated 32P-labeled fragment of
PAK6 corresponding to amino acids 115383 (having no homology to
previously described PAKs or other proteins). Hybridization and washing
conditions were as recommended by the manufacturer.
GST Fusion Proteins and Pull Downs
GST-AR fusion proteins were constructed in pGEX-2TK by PCR
amplification and confirmed by sequencing (80).
GST-GRIP1(6241122) and GST-ER(281595) (63) were from
Myles Brown (Dana Farber Cancer Institute, Boston, MA). GST-RacV12,
-RacN17, and -Cdc42V12 mutant constructs were from Chris Carpenter
(Beth Israel Deaconess Medical Center, Boston, MA) (81).
For GTP loading, the GST-RacV12 and GST-Cdc42V12 fusion proteins bound
to glutathione agarose beads (5 µg) were initially incubated in 20
mM Tris, pH 7.5, 100 mM NaCl, 1 mM
EDTA, and 1 mM dithiothreitol (DTT), with a 10-fold molar
excess of GTP
S, for 15 min at 30 C. The beads were then placed on
ice, and MgCl2 was added to a concentration of 5
mM. After 5 min on ice, the beads were pelleted and lysates
were added. GTP loading was comparable for the Rac and Cdc42 fusion
proteins, based upon [
-32P]GTP binding in
parallel experiments.
For GST pull-down experiments, CV1 cells were transfected in 10-cm
plates with 10 µg of PAK6 in pcDNA3.1(-)/Myc-His C, using
Lipofectamine according to the manufacturers directions (Life Technologies, Inc., Gaithersburg, MD). Transfected CV1 cells or
LNCaP prostate carcinoma cells (with endogenous AR) were lysed in 50
mM Tris, pH 7.6, 150 mM NaCl, 5 mM
MgCl2, 0.5% Triton X-100, 5 mM DTT,
and protease inhibitors for 15 min at 4 C, followed by centrifugation
to remove nuclei. Lysates were then incubated with GST fusion proteins
(5 µg except where indicated) bound to glutathione agarose beads for
24 h at 4 C, followed by washes in lysis buffer and elution in
SDS-PAGE sample buffer. In the indicated experiments lysis and washes
were in 10 mM Tris, pH 7.5, 150 mM NaCl, 1
mM MgCl2, 1 mM EGTA, 1
mM DTT, 10% glycerol, 0.1% Triton X-100, and protease
inhibitors (glycerol lysis buffer). PAK6 was detected by immunoblotting
with mouse anti-myc mAb 9E10, and AR was detected with a mixture of
rabbit anti-AR antibodies against N and C-terminal peptides (all from
Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
35S-labeled proteins were generated by in
vitro transcription/translation (TNT T7 Quick Coupled
Transcription/Translation System, Promega Corp., Madison,
WI). AR, ER
(from Myles Brown, Dana Farber Cancer Institute), PAK1,
and PAK6 plasmids (in pGEM3 or pcDNA, 2 µg) in 50 µl of
reticulocyte lysate with 20 µCi of
35S-methionine were incubated at 30 C for 1
h, according to the manufacturers directions. GST fusion proteins (5
µg on glutathione agarose beads) were mixed with 10 µl of the
programmed lysate in 0.5 ml of PBS, pH 7.4, 1 mM
DTT, and protease inhibitors. After 24 h at 4 C, the beads were
washed in the same buffer, except with 0.05% NP-40. Proteins were
eluted in SDS-PAGE sample buffer, and labeled proteins were detected
with a PhosphorImager (Molecular Dynamics, Inc.,
Sunnyvale, CA). DHT was obtained from Sigma, OHT was from
Alexis Biochemicals, and bicalutamide was kindly provided by
Astra Zeneca Pharmaceuticals (Wilmington, DE).
Kinase Assays
Beads with precipitated proteins were washed once in kinase
buffer (40 mM HEPES, pH 7.4, 20 mM
MgCl2) and then resuspended in 30 µl of kinase
buffer with MBP (5 µg) (Sigma), 10 µCi
[
-32P]ATP (3000 Ci/mmol), and 20
µM cold ATP. Reactions were carried out at room
temperature for 15 min and stopped with SDS-PAGE loading buffer
containing 10 mM EDTA. Labeled MBP was analyzed on 15%
SDS-PAGE.
AR and ER
Transcriptional Activity
Transient transfections to assess AR transcriptional activity
were carried out using CV1 cells in 24-well plates (58, 82). Cells were transfected using LipofectAMINE or LipofectAMINE
2000 (Life Technologies, Inc.) with AR expression vector
(pSVARo) (67), a Renilla
expression vector to control for transfection efficiency (pRL-CMV or
pRL-SV40, Promega Corp.), and PAK6 or other experimental
or control plasmids as indicated. The VP16-SRC(595780) encodes the
first nuclear receptor binding domain of SRC-1 fused to the C
terminus of the VP16 activation domain in the AASVVP16 vector
(83). A VP16-PAK6(256681) expression vector was
generated in AASVVP16 by PCR to generate an in-frame EcoRI
site at amino acid 256 in PAK6 and a HindIII site at the
3'-end. The fragment was then cloned at the 3'-end of VP16 as an
EcoRI-HindIII fragment. The reporter plasmids
were an androgen-responsive luciferase reporter construct driven by
synthetic AREs and a minimal promoter
(ARE4-luciferase) or MMTVpA3Luc, driven by the
androgen-responsive MMTV-LTR (83). The
ARE4-luciferase reporter was constructed by
inserting four tandem ARE repeats
(5'-TGTACAGGATGTTCTGAATTCCATGTACAGGATGTTCT-3') in front of an E1b
minimal TATA box sequence, followed by a firefly luciferase gene. After
the 24-h transfection, cells were cultured for another 24 h in
DMEM with 5% CDS FCS, with or without added 10
nM DHT. Lysates were assayed for luciferase
activity and Renilla activity using a dual luciferase kit
(Promega Corp.), and luciferase activity was normalized
for Renilla to give relative light units.
ER
transcriptional activity was assessed similarly using a
pcDNA3.1-ER
expression vector and a luciferase reporter gene,
ERE2TK-Luc, regulated by two copies of the ERE
from the vitellogenin gene (kindly provided by Myles Brown, Dana Farber
Cancer Institute). The ER
experiments were done in phenol red-free
medium. All points were in triplicate or quadruplicate and
mean ± SEM are shown.
Additional experiments used CV1 cells containing an integrated
luciferase reporter gene regulated by the androgen responsive MMTV-LTR.
These CV1(MMTV-Luc) cells were generated by transfecting CV1 cells with
an MMTV-LTR-luciferase reporter plasmid (MMTVpA3Luc) (83)
and neomycin resistance plasmid, and selecting for G418-resistant
cells. A series of clones were then screened to identify ones with low
background luciferase activity and high level androgen-dependent AR
induction.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. C. Carpenter and A. Hollenberg for reagents and
very helpful discussions, and Drs. A. Brinkmann, M. Brown, M. Stallcup,
W. Chin, and R. Pestell for reagents.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grants R01-CA-65647 (S.P.B.),
T32-AI-07542 (S.M.R.), HL-07623, R29-GM-54713 (M.L.L.), a Department of
Defense Breast Cancer Research grant (to S.P.B.), and by the Hershey
Family Prostate Cancer Research Fund.
Abbreviations: ARE, Androgen response element; BIC,
bicalutamide; CDS, charcoal dextran-stripped; CRIB, Cdc 42/Rac
interactive binding; DBD, DNA-binding domain; DHT, dihydrotestosterone;
DTT, dithiothreitol; ERE, estrogen response element; EST, expressed
sequence tag; GFP, green fluorescent protein; GRIP, GR-interacting
protein; GST, glutathione-S-transferase; LBD,
ligand-binding domain; mAb, monoclonal antibody; MBP, myelin basic
protein; MMTV-LTR, mouse mammary tumor virus-long terminal repeat; NLS,
nuclear localization signal; OHT, 4-hydroxytamoxifen; PAK,
p21-activated kinase; SRC, steroid receptor coactivator.
Received for publication February 7, 2001.
Accepted for publication September 14, 2001.
 |
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