Truncated Activin Type I Receptor Alk4 Isoforms Are Dominant Negative Receptors Inhibiting Activin Signaling
Yunli Zhou,
Huiping Sun,
Daniel C. Danila,
Stacey R. Johnson,
Daniel P. Sigai,
Xun Zhang and
Anne Klibanski
Neuroendocrine Unit Massachusetts General Hospital and Harvard
Medical School Boston, Massachusetts 02114
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ABSTRACT
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Activin, a member of the transforming growth
factor ß (TGFß) superfamily of cytokines, inhibits cell
proliferation in a variety of cell types. The functions of activin are
mediated by type I and type II serine/threonine kinase receptors. The
main type I receptor mediating activin signaling in human cells is
ActRIB, also called Alk4. We have previously reported that several
truncated Alk4 receptor isoforms are exclusively expressed in human
pituitary tumors, and that the majority of such tumors did not exhibit
activin-induced growth arrest in culture. We therefore studied the
function of these truncated receptor isoforms. Transient expression of
these truncated receptors inhibited activin-activated transcription
from an activin-responsive reporter construct, 3TPLux. When each of
these truncated Alk4 receptors was stably transfected into K562 cells,
activin-induced expression of an endogenous gene, junB, was
blocked, indicating that inhibition of gene expression also occurred at
the chromosomal level. Furthermore, activin administration failed to
cause growth inhibition and an increase of the
G1 population in these cells.
Coimmunoprecipitation experiments showed that the truncated Alk4
receptors formed complexes with type II activin receptors, but were not
phosphorylated. These data indicate that the truncated activin type I
receptors, predominantly expressed in human pituitary adenomas,
function as dominant negative receptors to interfere with wild-type
receptor function and block the antiproliferative effect of activin.
This may contribute to uncontrolled pituitary cell growth and the
development of human pituitary tumors.
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INTRODUCTION
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Activin is a member of the transforming growth factor-ß (TGFß)
superfamily of cytokines regulating cell proliferation and
differentiation. Activin induces growth suppression in a variety of
normal cell types (1, 2, 3, 4) as well as a number of human cancer cell
lines, such as erythroleukemia K562 (5) and prostate LNCap (6).
Cytokines of the TGFß family signal primarily through their
ligand-specific heteromeric complexes comprising two related
transmembrane serine/threonine kinase receptors, named type I and type
II (7). The known type II activin receptors include ActRII and ActRIIB,
while the main type I activin receptor in mammalian cells is Alk4
(ActRIB) (7). In the presence of activin, type II and type I receptors
form complexes whereby the type II receptors activate Alk4 through
phosphorylation. The activated Alk4, in turn, transduces signals
downstream by phosphorylation of its effectors, such as Smads, to
regulate gene expression and affect cellular phenotype (7).
Receptors of the TGFß family of cytokines contain 11 kinase
subdomains within their intracellular regions (8, 9). The receptor
kinase activity has been shown to be critical in the transduction of
TGFß/activin signals (10, 11, 12, 13, 14). Study of the Alk4 gene predicts
several possible mRNA species, generated by alternative splicing,
encoding truncated Alk4 receptors that lack specific carboxyl kinase
subdomains (15). The majority of these truncated receptor isoforms have
been found to be exclusively expressed in human pituitary adenomas
(16). Molecular cloning and sequence analysis reveal that among these
alternative spliced variants, Alk41 is the full-length Alk4 receptor,
Alk42 has most of subdomain XI removed, and Alk43 lacks subdomains
X and XI. Both Alk42 and Alk43 also contain new carboxyl termini
with novel sequences not found in Alk41. Alk44, the most truncated
splice variant, lacks kinase subdomains IX-XI and part of subdomain
VIII (16). In this study, we investigated their function in mediating
activin signaling. We found that these truncated isoforms inhibit
activin signaling and block the antiproliferative effect of activin.
Our results suggest that the truncated receptors disrupt the activin
signaling pathway in pituitary adenomas and may contribute to pituitary
tumorigenesis by blocking the antiproliferative effects of activin.
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RESULTS
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Human Pituitary Adenomas Express High Levels of Truncated Alk4
Receptor Isoforms
We have previously reported that human pituitary adenomas
express at least one or more truncated Alk4 receptor isoforms (16). To
determine their relative expression levels in pituitary tumors, we
examined several human nonfunctioning pituitary adenomas by comparative
RT-PCR, using truncated Alk4 isoform-specific primers. We found that a
low level of the full-length wild-type (wt) Alk4 transcript was
detected in both normal pituitary and most pituitary adenomas (Fig. 1
). In contrast, the truncated Alk42
and Alk43 were exclusively detected in pituitary tumors. The
expression of Alk42 transcript was significantly greater than that of
wt Alk41, while the mRNA level of Alk43 was much higher than or
comparable to that of wt Alk41 in tumors (Fig. 1
). Although Alk44
was detected in some normal pituitary tissues, the expression was
substantially less compared with that found in the pituitary tumors
(Fig. 1
).

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Figure 1. Expression of Alk4 Receptor Isoforms in Human
Pituitary Adenomas
Comparative RT-PCR was performed using primers specifically for the
wild-type Alk4 and the truncated Alk4 isoforms. A product of
cyclophylin A was also amplified for each reaction as a control. PCR
products were visualized by incorporation of
[ -32P]dCTP (100 nCi/reaction) in the
amplification reactions, and autoradiography onto Kodak
XO-Mat film for 248 h. C, RT-PCR negative control; NFT,
non-functioning tumors; NP, normal pituitary; Cycl A, cyclophylin A.
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Truncated Alk4 Receptors Block Activin-Activated Transcription from
3TPLux Reporter
To study their function, we tagged the truncated Alk4 receptors
Alk42, Alk43, and Alk44 as well as the full-length Alk41 with
FLAG epitopes (Fig. 2A
). The expression
of these tagged receptors was checked by transient transfection
experiments, and the proteins with correct molecular weights from these
constructs were detected by Western blotting (data not shown). In
addition, as described later, we established several stable K562 clones
using vectors containing the FLAG-tagged cDNAs, which expressed
receptor proteins with correct sizes of approximately 58, 54.5, 55, and
43 kDa for FLAG-tagged Alk41, -2, -3, and -4, respectively (Fig. 2B
).

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Figure 2. Expression of FLAG-Tagged Alk4 Receptor
Isoforms
A, Schematic representation of FLAG epitope-tagged Alk4 receptor
isoforms. E, Extracellular domain; TM, transmembrane domain; GS,
glycine and serine-rich domain; Roman numerals, kinase
subdomains; solid box, novel amino acid sequences. B,
Expression of Alk4 receptor isoforms in stably transfected K562 cells.
Cell lysates from established K562 cells containing FLAG-tagged Alk4
isoforms were immunoprecipitated with anti-FLAG antibody-agarose
(M2-agarose). Different clone numbers are indicated. MW, Mol wt marker;
Parental, lysates from untransfected K562 cells; Vector, lysates from
empty vector-transfected cells.
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Activin induced approximately an 8-fold increase in luciferase activity
in L17 cells transfected with wt Alk41 (Fig. 3A
). In contrast, no significant
induction of luciferase activity by activin was observed in cells
transfected with expression vectors carrying truncated Alk4 isoforms
(Fig. 3A
). Activin-induced luciferase activity was determined in cells
transfected with FLAG-tagged, HA-tagged, or untagged Alk41. The
induction mediated by these tagged and untagged receptors was very
similar (data not shown).

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Figure 3. Effects of Truncated Alk4 Receptor Isoforms on
Activin-Activated Transcription from 3TPLux Reporter
These data were analyzed using paired Students t test
and represented as mean ± SD for results from at
least three independent experiments. A, L17 cells transfected with 0.4
µg reportor 3TPLux in the absence or presence of 0.2 µg FLAG-tagged
Alk4 isoforms in pCI-neo expression vector. B, L17 cells cotransfected
with 0.3 µg 3TPLux, Alk4HA expression vector, and various amounts of
FLAG-tagged Alk4 isoform expression vectors as indicated. *,
P < 0.05 compared with values from cells
transfected with only 3TPLux and Alk4HA. C, K562 cells transfected with
0.4 µg 3TPLux in the absence or presence of 1 µg Alk4 isoform
expression vectors. *: P < 0.05 compared with
values from cells transfected with only 3TPLux.
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Since truncated Alk4 receptors did not transduce the activin signal in
the reporter assay, we examined their ability to interfere with
wild-type receptor function as dominant negative receptors. When
cotransfected into L17 cells with Alk41, each of the truncated Alk4
isoforms significantly suppressed activin-induced
transcription from 3TPLux in a dose-dependent manner (Fig. 3B
). In
K562 cells, a human erythroleukemia cell line containing endogenous
functional Alk4, luciferase activity was significantly decreased in
cells transfected with the truncated Alk4 receptors compared with
3TPLux-transfected control cells. Conversely, luciferase activity in
cells transfected with Alk41 was increased compared with the control
(Fig. 3C
). In addition, when an equal amount of wt Alk41 and combined
truncated receptor were cotransfected into L17 cells, the
activin-activated transcription from 3TPLux was significantly
suppressed (Fig. 3B
).
Truncated Alk4 Receptors Inhibit Activin-Induced Expression of
Endogenous junB Gene in K562 Cells
To investigate whether the truncated Alk4 receptors suppress
activin-regulated expression of endogenous genes under native
conditions, we established stable clones expressing truncated Alk4
isoforms from K562 cells. Expression of the truncated receptors in
established K562 clones was checked by direct Western blotting (data
not shown) and confirmed by immunoprecipitation (Fig. 2B
). Alk41FLAG
migrated as a broad band and contained proteins with various molecular
weights. In contrast, the truncated receptors migrated as sharp bands
(Fig. 2B
).
It has been reported that activin stimulates junB expression
rapidly in K562 cells (17); we therefore examined activin-induced
junB expression in these stably transfected cells by Western
blotting. 12-O-Tetradecanoylphorbol 13-acetate (TPA) was
used as a control in these experiments, since it has been shown to
up-regulate junB mRNA expression in K562 cells independently
of activin (17). As shown in Fig. 4
, a
significant amount of junB protein was induced in all
TPA-treated cells independently of truncated receptor expression (Fig. 4
, lanes 3, 6, 9, 12, 15, and 18). Also, activin induced expression of
junB in parental, empty vector-transfected and full-length
Alk41 transfected cells (Fig. 4
, lanes 2, 5, and 8). However, activin
failed to induce junB expression in cells with the truncated
receptors (Fig. 4
, lanes 11, 14, and 17).

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Figure 4. Truncated Alk4 Receptor Isoforms Inhibit
Activin-Induced junB Expression in K562 Cells
Lysates were prepared from activin or TPA-treated cells and resolved by
SDS-PAGE, transferred to Immobilon P membrane, and probed with an
anti-junB antibody. ß-Actin was also analyzed by
Western blotting as a control for equal protein loading. -, No
treatment; A, 2 nM activin A treatment; T, 200
nM TPA treatment; Parental, untransfected K562 cells; Vector, empty vector transfected cells.
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Truncated Alk4 Receptors Prevent Activin-Mediated Growth
Suppression in K562 Cells
We also studied the antiproliferative function of activin in the
established K562 cell clones expressing truncated Alk4 isoforms.
Activin treatment resulted in approximately a 40% decrease in
proliferation of parental cells as well as of empty vector-transfected
cells, and a 60% decrease in cells expressing Alk41. However, no
significant decreases in cell growth were observed in the
activin-treated clones expressing Alk42, Alk43, or Alk44 (Fig. 5A
). Further cell cycle analysis revealed
that activin treatment increased the G1
population by 3035% in control K562 cells and induced an even more
dramatic G1 population increase (5060%) in
cells expressing Alk41 (Fig. 5
, B and C). In contrast, activin failed
to induce any G1 population increase in cells
stably transfected with any truncated Alk4 receptors (Fig. 5
, B and
C).

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Figure 5. Truncated Alk4 Receptor Isoforms Prevent K562 Cells
from Activin-Induced Growth Inhibition
A, Stably transfected K562 cells (2.5 x 104/ml) were
seeded in triplicate into 24-well plates and cultured in the presence
or absence of 2 nM activin A. Four days later, the numbers
of cells in each well were counted. Cell number counts for the
activin-treated cells are normalized to those for untreated cells, which are set to 100%. The data are
represented as mean ± SD for counts from at least
three independent experiments. *, P < 0.001
compared with values from the untreated cells. B, Representative
histograms of flow cytometry analysis for stably transfected K562 cells
with or without activin treatment. C, Percentage increases in
G1 population of activin-treated cells compared with that
of the untreated. Flow cytometry analysis was performed at least three
times using two established K562 cell clones for each Alk4 isoform. The
portion of cells in G1 phase was determined using
Becton Dickinson and Co. CellFIT software.
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Truncated Alk4 Receptors Form Complexes with Type II Activin
Receptors but Fail to be Phosphorylated in the Presence of Activin
A two-step coimmunoprecipitation procedure was used to determine
whether the truncated Alk4 receptor isoforms form complexes with type
II activin receptors. Lysates from
[35S]methionine-labeled cells cotransfected
with ActRIIB and Alk41 contained activin receptor complexes
regardless of activin treatment (Fig. 6A
, lanes 6 and 7). Also, all truncated Alk4 receptors formed complexes
with ActRIIB independently of activin treatment (Fig. 6A
, lanes 813).
Interestingly, two forms of the full-length Alk41 were found in the
complexes from cells treated with activin; one appeared on SDS-PAGE as
a sharp band, while the other appeared as a broader, slow-migrating
band (Fig. 6A
, lane 7, arrow A). However, no
apparent corresponding slow migrating bands were observed for the
truncated Alk4 isoforms (Fig. 6A
, lanes 813).

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Figure 6. Truncated Alk4 Receptors Form Complexes with, but
Fail to be Phosphorylated by, Type II Activin Receptors
COS1 cells were transiently transfected with FLAG-tagged Alk4 receptor
isoforms and myc-tagged ActRIIB as indicated, labeled
with either [35S]methionine (for panel A) or
[32P]phosphate (for panel B). Receptor complexes were
purified using the two-step procedure as described in Materials
and Methods. Lysates from cells transfected with only
ActRIIB-myc were included in both experiments as
controls (panel A, lanes 4 and 5; panel B, lanes 1 and 2). Panel A
shows that truncated Alk4 receptors formed complexes with type II
activin receptors. Lane 1, Cells transfected with empty vector only.
Lanes 2 and 3, Lysates were same as lanes 4 and 5 and
immunoprecipitated with anti-myc antibody. Panel B shows
no phosphorylation in truncated Alk4 isoforms. Arrow
A, Activin-induced modification of Alk41.
Arrow B, Phosphorylation of ActRIIB.
Arrow C, Activin-induced phosphorylation
of ActRIIB. RIIB, Type II activin receptor ActRIIB.
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We further investigated activin-induced phosphorylation of each Alk4
isoform. In COS1 cells ActRIIB-complexed Alk41 from activin-treated
cells was heavily phosphorylated (Fig. 6B
, lane 4, arrow
A). In contrast, none of the truncated ALK4 isoforms was
phosphorylated (Fig. 6B
, lanes 510), although the coprecipitated
ActRIIB was phosphorylated independently of activin treatment (Fig. 6B
, lanes 510, arrow B). The activin type II receptor ActRIIB
that complexed with full-length Alk41 was further modified by
phosphorylation upon activin stimulation (Fig. 6B
, lane 4,
arrow C). However, no significant activin-induced
modification was observed in ActRIIB complexed with any truncated
receptor (Fig. 6B
, lanes 510). These results indicate that truncated
ALK4 receptor isoforms cannot be phosphorylated by type II receptors
and, therefore, are not active.
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DISCUSSION
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Disruption of the TGFß signaling pathway has been linked to the
development of many human malignancies (18). Although little is known
about the involvement of activin pathways in human tumorigenesis,
evidence suggests that the activin signaling pathway may also play a
role in tumor development. For example, Smad2 and Smad4, two of the
downstream mediators shared by activin and TGFß pathways, are
frequently mutated in cancers of the colon and pancreas (18). In
addition, it has been suggested that certain breast cancer cell lines
are insensitive to activin-induced growth arrest due to loss of activin
receptor expression (19). Recently, we reported that about 70% of
human nonfunctioning tumors are resistant to activin-induced growth
arrest (20) and that approximately two thirds of nonfunctioning tumors
and virtually all of the somatotroph adenomas and prolactinomas express
one or more truncated Alk4 receptor isoforms (16). We found that the
expression levels of these truncated Alk4 isoforms are typically
expressed at much higher levels than that of wt Alk41 in human
pituitary adenomas. We therefore hypothesized that these truncated
isoforms may function as dominant negative receptors that block activin
signaling and contribute to the development of pituitary tumors.
Consistent with our hypothesis, transient transfection assays
demonstrate that the truncated receptors suppress activin-induced
transcription from the 3TpLux reporter in L17 and K562 cells. When
stably transfected, they also inhibit activin-induced endogenous
junB gene expression in K562 cells. In addition, the
truncated receptors completely block the antiproliferative function of
activin. Furthermore, these activin receptor isoforms form heteromeric
complexes with ActRIIB, but fail to be phosphorylated by the type II
receptor. Therefore, these truncated Alk4 receptors act as dominant
negative inhibitors to interfere with wild-type receptor-mediated
activin signaling.
In our in vitro reporter assay in L17 cells, inhibition of
the full-length Alk4-mediated transcription from 3TPLux was observed
when the truncated receptors were overexpressed. Although the ratio of
each truncated receptor to the full-length Alk4 is from 2 to 4 in the
cotransfection experiments, we believe that the results are still
physiologically relevant. As demonstrated in Fig. 1
, mRNA levels of
Alk42 and Alk44 are significantly higher than that of Alk41 in
pituitary tumors, indicating that either Alk42 or Alk44 alone may
be enough to inhibit activin signaling in these tumor cells. One of the
tumors examined showed very high mRNA level of Alk43, suggesting that
Alk43 alone is also able to significantly inhibit activin signaling
in some pituitary tumors. In addition, when we cotransfected Alk41
with an equal amount of combined truncated receptors into L17 cells,
the activin-induced transcription from 3TPLux is significantly
suppressed (Fig. 3B
). This result indicates that the truncated Alk4
isoforms can act synergistically to interfere with activin signaling.
Since human pituitary tumors usually contain more than one truncated
Alk4 isoform and the combined expression of these receptors is
substantially greater than that of wt Alk4, the activin signaling may
be effectively inhibited even in tumors with relatively low expression
of an individual truncated receptor type.
The K562 cell line was chosen to investigate whether truncated Alk4
receptors inhibited transcriptional activation and growth suppression
by activin, because it contains a functional activin pathway and has
been used previously in activin signaling studies (5, 11, 21). In
addition, expression of the protooncogene junB has been
shown to be rapidly induced by activin in K562 cells (17). We observed
that, in K562 clones stably transfected with wt Alk41, activin
induced more junB expression and caused more cells arrested
in the G1 phase of cell cycle than in parental
and vector-transfected K562 cells. This is probably due to an increase
in the total number of type I activin receptors on the cell membrane,
which intensifies activin signaling. These results are consistent with
a previous report showing that overexpression of ActRIB potentiates
activin-induced transcriptional activation in K562 cells (21). In
contrast, expression of any truncated Alk4 receptors significantly
inhibited both activin-induced junB expression and growth
suppression in K562 cells. These data indicate that the truncated Alk4
receptors are potent dominant negative inhibitors capable of
interfering with activin signaling under native conditions.
The key event initiating the cascade of activin signaling is activation
of the type I receptor through phosphorylation by constitutively active
type II receptor kinase. To investigate the mechanism of dominant
negative inhibition by the truncated Alk4 receptors, we examined
whether they formed complexes with, and were phosphorylated by, the
type II receptors. Our results demonstrate that truncated Alk4
receptors can form complexes with ActRIIB comparable to the full-length
Alk41. This indicates that alterations in the kinase domain do not
affect the ability of Alk4 to bind to type II receptors. This is in
agreement with previous reports showing that a deletion or amino acid
substitution mutation in the cytoplasmic region of either the type I or
type II TGFß/activin receptors typically does not affect complex
formation (10, 11, 12, 14, 22). Alk4 exists in two forms: one is the less
phosphorylated precursor and the other is a heavily phosphorylated
mature form (22). Although both forms can be found in type I/II
receptor complexes from activin-treated and untreated cells, previous
reports (22, 23) and our studies demonstrate that activin treatment
leads to a dramatic increase in the mature forms. This phenomenon is
attributed to the phosphorylation of Alk4 in the GS domain by the type
II activin receptors (22). Despite containing an intact GS domain, the
truncated Alk4 isoforms are not able to be phosphorylated in type I/II
receptor complexes. The common structural characteristics shared by the
truncated Alk4 isoforms are lack of kinase subdomain XI. Also, it has
been reported that a point mutation that changes Pro 525 to Leu in
kinase subdomain XI of the TGFß type II receptor abolishes its
ability to phosphorylate type I TGFß receptors, although the mutant
TßR-II forms complexes with TßR-I (14). These data suggest that the
C-terminal structures involving subdomain XI are critical for
signaling transduction. Mutation in this subdomain
could induce conformational changes, which makes the receptors unable
to recognize their binding partners correctly and causes the failure in
phosphorylation of the type I receptor. This possibility is also
supported by the fact that an ActRIB KR mutant carrying a point
mutation that changes Lys 234 to Arg in its kinase subdomain II is able
to form complexes with type II activin receptors and is phosphorylated
in a way similar to the wild-type ActRIB, but is unable to transduce
activin signals. Apparently, the truncated Alk4 isoforms and the ActRIB
KR mutant fail to transduce activin signaling via different mechanisms.
The KR mutant retains an intact subdomain XI and, therefore, can be
phosphorylated by the type II receptors. However, it is unable to
transduce signals to downstream Smads due to the lack of kinase
activity. In contrast, the truncated Alk4 isoforms do not have
subdomain XI and cannot be activated by phosphorylation; therefore, the
activin signal transduction stops before the activation of the type I
receptors.
It is known that ActRIIB is constitutively autophosphorylated
independently of type I receptor kinase activity (22, 24).
Interestingly, our data show that activin increases phosphorylation of
ActRIIB significantly in complexes containing Alk41 compared with
that seen with basal phosphorylation. A phosphorylation of ActRIIB
slightly above basal level has also been observed in complexes
containing truncated Alk4 isoforms from cells treated with activin.
This is probably due to the presence of endogenous wild-type type I
receptors. Nevertheless, the activin-induced phosphorylation of ActRIIB
complexed with wt Alk41 is much greater compared with that complexed
with truncated isoforms, indicating that activin-induced
phosphorylation of ActRIIB is wt Alk4 dependent. This raises the
possibility that, in addition to the basal level of phosphorylation,
ActRIIB is further phosphorylated upon the formation of a functional
type I/II receptor complex in the presence of activin, either by
itself, Alk41, or other kinases. Truncated Alk4 receptors may inhibit
ActRIIB modification in type I/II receptor complexes, since they fail
to induce ActRIIB phosphorylation in the presence of activin. Taken
together, our data indicate that these naturally occurring truncated
Alk4 receptor isoforms act as dominant negative inhibitors to interfere
with activin signaling by blocking kinase function and activin-induced
modification of type II receptors. Since these truncated receptors
block the antiproliferative effect of activin, the high levels of
expression of such truncated receptors in human pituitary tumors may
contribute to uncontrolled pituitary cell growth.
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MATERIALS AND METHODS
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RT-PCR
Tumor tissue from five clinically nonfunctioning pituitary
macroadenomas operated by transphenoidal surgery, and four postmortem
pituitary specimens from control subjects (Harvard Brain Bank, McLean
Hospital, Belmont, MA) were snap frozen in liquid nitrogen before total
RNA was extracted and reverse transcribed as previously described (20).
PCR for the wild-type and each Alk4 receptor isoform was carried out as
previously (16). PCR products were visualized by incorporation of
[
-32P]dCTP (100 nCi/reaction) in the
amplification reactions, and autoradiography onto XO-Mat film
(Kodak, Rochester, NY) for 248 h. The amount of
amplification products was compared between samples in correlation with
the equal amount of cyclophylin A products, as described previously
(20, 25).
Expression Vectors and Reporter Constructs
The activin-responsive reporter construct, 3TPLux, was kindly
provided by Dr. J. Massague (26). A plasmid containing a full-length
Alk4 tagged with the HA influenza virus hemagglutinin epitope in
pcDNA3, pcDNA3Alk4-HA, was kindly provided by Dr. P. ten Dijke.
Full-length ActRIIB cDNA in pcDNA3 (pcDNA3ActRIIB) was kindly provided
by Dr. O. Ritvos (27). Sequences encoding five copies of myc
epitope were added to the 3'-end of the ActRIIB coding region by PCR
and inserted into pcDNA3 to generate a myc-tagged ActRIIB
construct, named pcDNA3RIIB-myc. Activin type I Alk4
receptor isoforms were cloned previously from human pituitary tumors by
RT-PCR (16) and tagged with FLAG epitope sequences at the end of their
coding region. The FLAG-tagged Alk4 isoform cDNAs were further cloned
into a mammalian expression vector, pCI-neo (Promega Corp., Madison, WI) and verified by sequencing. The plasmids
generated were named pCI-Alk41FLAG, pCI-Alk42FLAG, pCI-Alk43FLAG,
and pCI-Alk44FLAG, respectively. The FLAG-tagged Alk4 isoforms were
also subcloned into the expression vector pLPCX (CLONTECH Laboratories, Inc. Palo Alto, CA), which confers purimycin
resistance after transfection into mammalian cells, and used to
establish stable K562 clones expressing each individual FLAG-tagged
Alk4 receptor isoform.
Cell Culture and Luciferase Assay
The mink lung epithelial cell line derivative L17 (kindly
provided by Dr. J. Massague) was maintained in histidine-free MEM as
described previously (22). L17 cells express very low levels of Alk4
(28), and the activin signaling pathway does not function unless
exogenous Alk4 is introduced into the cell (3). The human
erythroleukemia cell line K562 (American Type Culture Collection, Manassas, VA) was cultured in RPMI medium 1640
supplemented with 10% FBS. L17 or K562 cells were transfected in
12-well plates with 3TPLux and Alk4 isoform expression constructs using
Lipofectamine (Life Technologies, Inc., Gaithersburg, MD).
A plasmid (pCMXßGal) expressing ß-galactosidase under the control
of a cytomegalovirus immediate early promoter was included in each
transfection to monitor transfection efficiency. Twenty-four hours
after transfection, cells were incubated in the presence or absence of
2 nM activin A (National Hormone and Pituitary Program,
Torrance, CA) in 0.5 ml MEM containing 0.2% FBS for 1820 h. The
transfected cells were lysed and luciferase activity was measured as
described previously (23).
Western Blotting for junB Detection
K562 cells (3 x 106) were maintained
in serum-free medium overnight and treated with 2 nM
activin A or 200 nM TPA for 2.5 h. The cells were
lysed with 500 µl of RIPA lysis buffer [50 mM Tris-Cl
(pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1
mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1
mM Na3VO4, 1
mM NaF, and 1 mM phenylmethylsulfonyl fluoride
(PMSF)]. Thirty micrograms of total protein were resolved by SDS-10%
PAGE and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). The blot was probed with a polyclonal
antibody against junB (sc-73; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and detected using ECL Plus
chemiluminescent reagent (Amersham Pharmacia Biotech,
Arlington Heights, IL).
Immunoprecipitation of FLAG-Tagged Receptors
K562 cells (5 x 106) were lysed in
500 µl Triton lysis buffer [20 mM Tris-Cl (pH 7.5), 150
mM NaCl, 0.5% Triton X-100, 2 mM EDTA, 50
mM NaF, 100 µM
Na4VO4, 5 µg/ml
aprotinin, 2 µg/ml leupeptin and 1 mM PMSF]. The cell
suspension was vortexed briefly and kept on ice for 20 min. The cell
lysate was cleared by centrifugation. Total protein of 500 µg was
incubated with 50 µl anti-FLAG antibody gel matrix slurry (50%,
vol/vol) (M2-agarose; Sigma, St. Louis, MO) at 4 C for
1.5 h on a rotary shaker. The gel matrix was washed four times
with lysis buffer, and the gel matrix-bound receptors were eluted with
125 µl of 0.2 mg/ml FLAG peptide (Sigma) in lysis
buffer. A portion of the elute equivalent to 20 µg of total cell
lysate was analyzed using 10% SDS-PAGE and transferred to Immobilon-P
membranes. The membranes were probed with the FLAG epitope-specific M2
antibody and detected using ECL-Plus reagent.
Purification of Receptor Complexes
COS1 cells (ATCC) in 60-mm tissue culture dishes
were transiently transfected with 5 µg pcDNA3RIIB-myc
alone or plus 10 µg each of the FLAG-tagged Alk4 isoforms in pCI-neo
using calcium phosphate-DNA precipitation method (29). For detection of
total protein, the transfected cells were labeled with 0.1 mCi/ml
[35S]methionine (NEN Life Science Products, Boston, MA) as described previously (22). To detect
changes in the phosphorylation state of activin receptors, the
transfected cells were metabolically labeled with 1 mCi/ml
[32P]orthophosphate (Amersham Pharmacia Biotech) in DMEM lacking sodium phosphate for 1 h and 10
min. Activin A was then added into the labeling medium at a
concentration of 2 nM, and cells were incubated
for another 20 min. Lysates were immediately prepared from the labeled
cells as described above using 500 µl Triton lysis buffer. To purify
activin type I and type II receptor complexes, the FLAG-tagged Alk4
receptor isoforms, including both the free and the type II
receptor-bound forms, were first isolated using M2-agarose as described
above. Each elute was then diluted to 500 µl with lysis buffer and
incubated with 1.5 µg monoclonal anti-myc antibody 9E10
(Santa Cruz Biotechnology, Inc.) for 1 h to isolate
the type I receptors complexed with myc-tagged ActRIIB.
Twenty microliters of Protein G-agarose beads (Life Technology) were
then added and incubation continued for 1 h. After being washed
four times with lysis buffer, the precipitated receptors were separated
by 10% SDS-PAGE, transferred to Immobilon-P membrane, and exposed to
film.
Cell Cycle Analysis
K562 cells (1 x 106) were cultured
for 2022 h in the presence or absence of 2 nM activin A.
Cells were washed once with cold PBS and fixed in 2 ml ice-cold 80%
ethanol at 4 C overnight. The fixed cells were stained with 1 ml
staining solution in PBS (10% µg/ml propidium iodide, 0.1% Triton
X-100, 0.1 mM EDTA, 10 µg/ml RNase A) for 30 min at room
temperature and stored at 4 C in the dark. The cell samples were sorted
using a FACScan System (Becton Dickinson and Co., Mountain
View, CA) . Approximately 2 x 104
cells were analyzed per sample, and the fractions of cells in different
phases of cell cycle were represented as percentages of total cells
analyzed.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. J. Massague for providing the L17 cell line
and plasmid 3TPLux, Dr. P. ten Dijke for the plasmid pcDNA3Alk4HA, Dr.
O. Ritvos for the plasmid pcDNA3ActRIIB, and Dr. A. F. Parlow for
the recombinant human activin A.
 |
FOOTNOTES
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Address requests for reprints to: Anne Klibanski, M.D., Massachusetts General Hospital, Neuroendocrine Unit, 55 Fruit Street, Bulfinch 457, Boston, Massachusetts 02114. E-mail: aklibanski{at}partners.org
This work was supported by NIH Grant RO1-DK-40947 and the Jarislowsky
Foundation.
Received for publication May 18, 2000.
Revision received August 22, 2000.
Accepted for publication September 7, 2000.
 |
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