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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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, Alk4–1 is the full-length Alk4 receptor, Alk4–2 has most of subdomain XI removed, and Alk4–3 lacks subdomains X and XI. Both Alk4–2 and Alk4–3 also contain new carboxyl termini with novel sequences not found in Alk4–1. Alk4–4, 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). In contrast, the truncated Alk4–2 and Alk4–3 were exclusively detected in pituitary tumors. The expression of Alk4–2 transcript was significantly greater than that of wt Alk4–1, while the mRNA level of Alk4–3 was much higher than or comparable to that of wt Alk4–1 in tumors (Fig. 1Go). Although Alk4–4 was detected in some normal pituitary tissues, the expression was substantially less compared with that found in the pituitary tumors (Fig. 1Go).



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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 [{alpha}-32P]dCTP (100 nCi/reaction) in the amplification reactions, and autoradiography onto Kodak XO-Mat film for 2–48 h. C, RT-PCR negative control; NFT, non-functioning tumors; NP, normal pituitary; Cycl A, cyclophylin A.

 
Truncated Alk4 Receptors Block Activin-Activated Transcription from 3TPLux Reporter
To study their function, we tagged the truncated Alk4 receptors Alk4–2, Alk4–3, and Alk4–4 as well as the full-length Alk4–1 with FLAG epitopes (Fig. 2AGo). 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 Alk4–1, -2, -3, and -4, respectively (Fig. 2BGo).



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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.

 
Activin induced approximately an 8-fold increase in luciferase activity in L17 cells transfected with wt Alk4–1 (Fig. 3AGo). In contrast, no significant induction of luciferase activity by activin was observed in cells transfected with expression vectors carrying truncated Alk4 isoforms (Fig. 3AGo). Activin-induced luciferase activity was determined in cells transfected with FLAG-tagged, HA-tagged, or untagged Alk4–1. 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 Student’s 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.

 
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 Alk4–1, each of the truncated Alk4 isoforms significantly suppressed activin-induced transcription from 3TPLux in a dose-dependent manner (Fig. 3BGo). 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 Alk4–1 was increased compared with the control (Fig. 3CGo). In addition, when an equal amount of wt Alk4–1 and combined truncated receptor were cotransfected into L17 cells, the activin-activated transcription from 3TPLux was significantly suppressed (Fig. 3BGo).

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. 2BGo). Alk4–1FLAG migrated as a broad band and contained proteins with various molecular weights. In contrast, the truncated receptors migrated as sharp bands (Fig. 2BGo).

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. 4Go, a significant amount of junB protein was induced in all TPA-treated cells independently of truncated receptor expression (Fig. 4Go, lanes 3, 6, 9, 12, 15, and 18). Also, activin induced expression of junB in parental, empty vector-transfected and full-length Alk4–1 transfected cells (Fig. 4Go, lanes 2, 5, and 8). However, activin failed to induce junB expression in cells with the truncated receptors (Fig. 4Go, 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.

 
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 Alk4–1. However, no significant decreases in cell growth were observed in the activin-treated clones expressing Alk4–2, Alk4–3, or Alk4–4 (Fig. 5AGo). Further cell cycle analysis revealed that activin treatment increased the G1 population by 30–35% in control K562 cells and induced an even more dramatic G1 population increase (50–60%) in cells expressing Alk4–1 (Fig. 5Go, B and C). In contrast, activin failed to induce any G1 population increase in cells stably transfected with any truncated Alk4 receptors (Fig. 5Go, 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.

 
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 Alk4–1 contained activin receptor complexes regardless of activin treatment (Fig. 6AGo, lanes 6 and 7). Also, all truncated Alk4 receptors formed complexes with ActRIIB independently of activin treatment (Fig. 6AGo, lanes 8–13). Interestingly, two forms of the full-length Alk4–1 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. 6AGo, lane 7, arrow A). However, no apparent corresponding slow migrating bands were observed for the truncated Alk4 isoforms (Fig. 6AGo, lanes 8–13).



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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 Alk4–1. Arrow B, Phosphorylation of ActRIIB. Arrow C, Activin-induced phosphorylation of ActRIIB. RIIB, Type II activin receptor ActRIIB.

 
We further investigated activin-induced phosphorylation of each Alk4 isoform. In COS1 cells ActRIIB-complexed Alk4–1 from activin-treated cells was heavily phosphorylated (Fig. 6BGo, lane 4, arrow A). In contrast, none of the truncated ALK4 isoforms was phosphorylated (Fig. 6BGo, lanes 5–10), although the coprecipitated ActRIIB was phosphorylated independently of activin treatment (Fig. 6BGo, lanes 5–10, arrow B). The activin type II receptor ActRIIB that complexed with full-length Alk4–1 was further modified by phosphorylation upon activin stimulation (Fig. 6BGo, lane 4, arrow C). However, no significant activin-induced modification was observed in ActRIIB complexed with any truncated receptor (Fig. 6BGo, lanes 5–10). These results indicate that truncated ALK4 receptor isoforms cannot be phosphorylated by type II receptors and, therefore, are not active.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Alk4–1 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. 1Go, mRNA levels of Alk4–2 and Alk4–4 are significantly higher than that of Alk4–1 in pituitary tumors, indicating that either Alk4–2 or Alk4–4 alone may be enough to inhibit activin signaling in these tumor cells. One of the tumors examined showed very high mRNA level of Alk4–3, suggesting that Alk4–3 alone is also able to significantly inhibit activin signaling in some pituitary tumors. In addition, when we cotransfected Alk4–1 with an equal amount of combined truncated receptors into L17 cells, the activin-induced transcription from 3TPLux is significantly suppressed (Fig. 3BGo). 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 Alk4–1, 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 Alk4–1. 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 Alk4–1 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 Alk4–1 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, Alk4–1, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 [{alpha}-32P]dCTP (100 nCi/reaction) in the amplification reactions, and autoradiography onto XO-Mat film (Kodak, Rochester, NY) for 2–48 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-Alk4–1FLAG, pCI-Alk4–2FLAG, pCI-Alk4–3FLAG, and pCI-Alk4–4FLAG, 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 18–20 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 20–22 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
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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