©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Important Regions in the Cytoplasmic Juxtamembrane Domain of Type I Receptor That Separate Signaling Pathways of Transforming Growth Factor- (*)

(Received for publication, August 21, 1995; and in revised form, November 20, 1995)

Masao Saitoh (1) (2) Hideki Nishitoh (1) (3) Teruo Amagasa (2) Kohei Miyazono (4) (5) Minoru Takagi (1) Hidenori Ichijo (1) (4)(§)

From the  (1)Department of Oral Pathology and the (2)First and (3)Second Departments of Oral and Maxillofacial Surgery, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan, the (4)Department of Biochemistry, The Cancer Institute, Tokyo, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan, and the (5)Ludwig Institute for Cancer Research, Biomedical Center, S-751 24 Uppsala, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Proteins in the transforming growth factor-beta (TGF-beta) superfamily exert their effects by forming heteromeric complexes of their type I and type II serine/threonine kinase receptors. The type I and type II receptors form distinct subgroups in the serine/threonine kinase receptor family based on the sequences of the kinase domains and the presence of a highly conserved region called the GS domain (or type I box) located just N-terminal to the kinase domain in the type I receptors. Recent studies have revealed that upon TGF-beta binding several serine and threonine residues in the GS domain of TGF-beta type I receptor (TbetaR-I) are phosphorylated by TGF-beta type II receptor (TbetaR-II) and that the phosphorylation of GS domain is essential for TGF-beta signaling. Here we investigated the role of cytoplasmic juxtamembrane region located between the transmembrane domain and the GS domain of TbetaR-I by mutational analyses using mutant mink lung epithelial cells, which lack endogenous TbetaR-I. Upon transfection, wild-type TbetaR-I restored the TGF-beta signals for growth inhibition and production of plasminogen activator inhibitor-1 (PAI-1) and fibronectin. A deletion mutant, TbetaR-I/JD1(Delta150-181), which lacks the juxtamembrane region preceding the GS domain, bound TGF-beta in concert with TbetaR-II and transduced a signal leading to production of PAI-1 but not growth inhibition. Recombinant receptors with mutations that change serine 172 to alanine (S172A) or threonine 176 to valine (T176V) were similar to wild-type TbetaR-I in their abilities to bind TGF-beta, formed complexes with TbetaR-II, and transduced a signal for PAI-1 and fibronectin. Similar to TbetaR-I/JD1(Delta150-181), however, these missense mutant receptors were impaired to mediate a growth inhibitory signal. These observations indicate that serine 172 and threonine 176 of TbetaR-I are dispensable for extracellular matrix protein production but essential to the growth inhibition by TGF-beta.


INTRODUCTION

The cell growth and differentiation in a multicellular organism are critically regulated by members of transforming growth factor-beta (TGF-beta) (^1)superfamily including TGF-beta, activin/inhibin, bone morphogenetic protein (BMP), Müllerian inhibiting substance, and glial cell line-derived neurotrophic factor. TGF-beta is a prototype in this superfamily of structurally related molecules and regulates cell proliferation, extracellular matrix formation, migration, adhesion, and many other cellular functions important for development and homeostasis (reviewed in (1, 2, 3, 4) ).

Certain members of the TGF-beta superfamily exert their biological actions through heteromeric complexes of two types (type I and type II) of transmembrane receptors with a serine/threonine kinase domain in their cytoplasmic region(5, 6, 7, 8) . To date, more than 15 receptor serine/threonine kinases have been cloned in flies through humans (reviewed in (4) and (9) -12). Among them six different type I receptors have been identified in mammals(5, 8, 13, 14, 15, 16, 17, 18, 19, 20) , including one TGF-beta type I receptor (TbetaR-I), two activin type I receptors (ActR-I and ActR-IB), two BMP type I receptors (BMPR-IA and BMPR-IB), and one additional type I receptor called activin receptor-like kinase-1 (also termed TGF-beta superfamily receptor type I or R3) that has recently been shown to mediate certain signals in response to BMP-7 (osteogenic protein-1). (^2)The type I receptors have similar sizes (502-532 amino acid residues) and 60-90% amino acid sequence identities to each other in their kinase domains. In addition, type I receptors contain a conserved sequence known as the GS domain (also called type I box) in their cytoplasmic juxtamembrane region(10, 11) . Type I receptors are more similar to each other than they are to the known type II receptors, including TGF-beta type II receptor (TbetaR-II) and two activin type II receptors (ActR-II and ActR-IIB), and thus form a subgroup of mammalian type I receptors in the family of receptor serine/threonine kinases.

TGF-beta initiates the signaling of its multiple responses through formation of a heteromeric complex of TbetaR-I and TbetaR-II. TGF-beta binds directly to TbetaR-II that is a constitutively active kinase, which then recruits TbetaR-I into the complex. TbetaR-II in the complex then phosphorylates the GS domain of TbetaR-I, resulting in propagation of further downstream signals(21, 22) . The catalytic activities of the kinases of TbetaR-I and TbetaR-II are indispensable for signaling (22, 23, 24, 25) . Mutational analyses altering serine and threonine residues in the TbetaR-I GS domain have revealed that phosphorylation of certain serines and threonines by TbetaR-II is essential for TGF-beta signaling, although its signaling activity does not appear to depend on the phosphorylation of any particular serine or threonine residue in the TTSGSGSG sequence of the GS domain(22, 26, 27) . In addition, recent identification of a constitutively active form of TbetaR-I that does not require TbetaR-II and TGF-beta for signaling suggested that TbetaR-I acts as a downstream signaling molecule of TbetaR-II(27) .

Despite the functional importance of the GS domain for initiating intracellular signals, little is known about how the signals are propagated after phosphorylation of the GS domain. Based on the knowledge of receptor tyrosine kinases, one possible mechanism could be that the phosphorylated serine and/or threonine residues in the GS domain may act as the binding sites for the intracellular substrate to be activated by the TbetaR-I kinase. This hypothesis is attractive to explain the signaling mechanism for certain common effects induced by the members of TGF-beta superfamily because the GS domain of the known type I receptors is highly conserved(10, 11, 12) . On the other hand, amino acid sequences of the GS domain of the type I receptors might be too similar to each other to confer specificities to the signals that mediate a wide variety of responses induced by the TGF-beta superfamily. In fact, a TbetaR-I chimeric receptor substituting the GS domain of ActR-I for that of TbetaR-I still transduces the TGF-beta-induced antiproliferative signal, which is not mediated through intact ActR-I (27, 28) . Thus, certain region(s) other than the GS domain in the type I receptors may also be important for diverse signaling activities of the proteins in the TGF-beta superfamily.

In the present study we focused on the role of the TbetaR-I juxtamembrane region preceding the GS domain, and serine 172 and threonine 176 within this region were found to be essential for signaling a TGF-beta antiproliferative response but not plasminogen activator inhibitor-1 (PAI-1) and fibronectin induction. Identification of such cytoplasmic regions important only for a limited response may suggest that at least two different signals are specified through different cytoplasmic parts of TbetaR-I.


EXPERIMENTAL PROCEDURES

List and Sequences of the Oligonucleotides Used to Generate Expression Constructs

The sequences of the oligonucleotide primers are presented in the 5` to 3` direction. Numbering is based on the nucleotide sequence of TbetaR-I(8) , and restriction enzyme sites incorporated into the primers are underlined. The junction of a deletion primer RISdel5 is indicated by a hyphen. The sequences are: RIS1-sma, GTCCCGGGCTGCCACAACCGCACT (nucleotides 441-445); RISdel2-sma, GCCCCGGGTTATGATATGACA (nucleotides 544-555); RIS0-hind, GGAAGCTTGACCATGGAGGCG (nucleotides 1-13); RIAS-not, AGGCGGCCGCTTACATTTTGATGCC (nucleotides 1512-1498); RIASdel1, GTGGCAGATATAGACCATCAAC (nucleotides 446-425); RISdel5, CTATATCTGCCAC-TATGATATGACA (nucleotides 433-445, 544-555); S-1, CCTGCATTAGATCGCCCTTTTAT (nucleotides 492-514); S-2, CGCCCTTTTATTGCAGAGGGTACT (nucleotides 504-527); S-3, GAGGGTACTGTGTTGAAAGAC (nucleotides 519-539); AS-1, ATAAAAGGGCGATCTAATGCAGG (nucleotides 514-492); AS-2, AGTACCCTCTGCAATAAAAGGGCG (nucleotides 527-504); and AS-3, GTCTTTCAACACAGTACCCTC (nucleotides 539-519).

cDNA Constructions

Stable expression vectors of wild-type TbetaR-I and its mutant derivatives were prepared by subcloning the polymerase chain reaction (PCR)-generated cDNA fragments into pMEP4 vector, a Zn-inducible mammalian expression vector(25) . To construct wild-type TbetaR-I-pMEP4, primer RIS0-hind and primer RIAS-not were used to amplify the coding region of TbetaR-I cDNA. Reaction conditions were 1 min at 94 °C, 1 min at 48 °C, and 2 min at 72 °C for 30 cycles. The PCR products were digested with HindIII and NotI and subcloned into the pMEP4 vector. To construct the deletion mutant TbetaR-I/JD1(Delta150-181), the primers RIS0-hind and RIASdel1 were used to amplify the 5` part of TbetaR-I cDNA fragment, and the primers RISdel5 and RIAS-not were used for the 3` fragment. The two primary PCR products were gel-purified, mixed, and subjected to reamplification with primers RIS0-hind and RIAS-not. The secondary PCR products were digested with HindIII and NotI and subcloned into the pMEP4 vector. Likewise, for the constructions of single missense mutants TbetaR-I/JM1(S165A), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V), primer RIS0-hind and the mutant antisense primer (AS-1, AS-2, and AS-3, respectively) were used to amplify the 5` fragments, and the mutant sense primer (S-1, S-2, and S-3, respectively) and primer RIAS-not were used to amplify the 3` fragments. PCR products were mixed in respective combinations and reamplified with primers RIS0-hind and RIAS-not. For TbetaR-I/JM123(S165A/S172A/T176V), PCR was performed using TbetaR-I/JM1 as a template for the 5` fragment with primers RIS0-hind and AS-2 and using TbetaR-I/JM3 as a template for the 3` fragment with primers S-2 and RIAS-not. The two PCR fragments were mixed and reamplified with primers RIS0-hind and RIAS-not. The SmaI-XbaI fragments of the mutant PCR products were swapped for the corresponding region of wild-type TbetaR-I plasmid.

Expression vectors for bacterial expression of wild-type TbetaR-I glutathione S-transferase (GST) fusion protein (GST-WT), its deletion mutant GST-JD1(Delta150-181), and missense mutants GST-JM1(S165A), GST-JM2(S172A), GST-JM3(T176V), and GST-JM123(S165A/S172A/T176V) were obtained by insertion of PCR-generated fragments of the corresponding cytoplasmic regions of TbetaR-I into pGEX-4T-1 (Pharmacia) using their stable expression plasmids as templates with RIS1-sma or RISdel2-sma as sense primers and RIAS-not as an antisense primer. PCR conditions were 1 min at 94 °C, 1 min at 54 °C, and 1 min at 72 °C for 25 cycles. The resulting PCR products for the GST fusion protein constructs were digested with SmaI and NotI and ligated in-frame into pGEX-4T-1. The structures of PCR-amplified region of the recombinants were all confirmed by sequencing using a Sequenase DNA sequencing kit (U. S. Biochemical Corp.).

Cell Culture and Transfection

The Mv1Lu mink lung epithelial cells (CCL-64; American Type Culture Collection) and the R mutant Mv1Lu cells (clone 4-2; R4-2) (29, 30) were maintained in Dulbecco's modified Eagle's medium (DMEM; Nissui) supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin. To generate stable transfectants expressing the various mutant forms of TbetaR-I, R4-2 cells were transfected by the calcium phosphate precipitation method using a eukaryotic transfection kit (Promega). Selection of transfected cells was performed in the presence of 120 units/ml of hygromycin B (Wako Chemicals). Resistant cell colonies were examined for the expression of TbetaR-I and its mutants by the receptor affinity labeling assays using I-TGF-beta1 after induction of the recombinant proteins by ZnCl(2). More than two independent clones for each of the transfectants were subjected to the following experiments.

Receptor Binding Assay of TbetaR-I Mutants

Recombinant human TGF-beta1 (Kirin Brewery Company) was iodinated using the chloramine T method as described(31) . Affinity cross-linking experiments were performed as described previously with minor modifications(25) . Briefly, cells were pretreated in DMEM containing 0.2% FBS with or without 100 µM ZnCl(2) for 6 h, and the binding of I-TGF-beta1 was allowed in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin for 3 h at 4 °C. After washing the cells with PBS three times, the ligand-receptor complexes were cross-linked with 0.27 mM of disuccinimidyl suberate (Pierce). Cells were washed once with 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 10% glycerol and solubilized by incubation in TNE buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) containing 1.5% of aprotinin for 20 min at 4 °C. For immunoprecipitation of the cross-linked complexes, cell lysates were then incubated with an antiserum against TbetaR-II (8) for 60 min at 4 °C. Immune complexes were bound to protein A-Sepharose (Kabi-Pharmacia) for 45 min at 4 °C, washed once with TNE buffer, and eluted by boiling in the SDS sample buffer (100 mM Tris, pH 8.8, 0.01% bromphenol blue, 36% glycerol, 4% SDS) in the presence of 10 mM dithiothreitol (DTT). The samples were analyzed by SDS-8.5% polyacrylamide gel electrophoresis and a Fuji BAS 2000 Bio-Imaging Analyzer (Fuji Photo Film).

Cell Proliferation Assay

Cells were plated into 24-well plates at 5 times 10^4 cells/well in DMEM containing 10% FBS, grown overnight, and placed in DMEM containing 0.2% FBS in the presence or the absence of 100 µM ZnCl(2) for 5 h. The cells were then added with TGF-beta1, incubated for additional 16 h, and pulsed with 1 µCi/ml [^3H]thymidine (6.7 Ci/mmol, Amersham Corp.) for 2 h. They were fixed on ice with 12.5% trichloroacetic acid and lysed with 1 N NaOH, and the [^3H]thymidine incorporation into the DNA was determined by a liquid scintillation counter.

PAI-1 Assay

PAI-1 assays were performed as described previously with minor modifications(28, 32) . Briefly, subconfluent cells in 6-well plates were incubated for 5 h with DMEM containing 0.2% FBS and 100 µM ZnCl(2). Cells were washed once with PBS and incubated for 4 h in methionine- and cysteine-free DMEM (ICN Biomedicals Inc.) containing 100 µM ZnCl(2) with or without 50 ng/ml of TGF-beta1. During the final 2 h of incubation, 30 µCi of [S]methionine and [S]cysteine mixture (Pro-mix cell labeling mix; Amersham Corp.) were added to the cells. The cells were then removed by washing once in PBS, four times in 10 mM Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, two times in 2 mM Tris-HCl, pH 8.0, and once in PBS. Proteins were extracted from plastics by SDS sample buffer containing 10 mM DTT and were analyzed by SDS-10% polyacrylamide gel electrophoresis and Bio-Imaging Analyzer.

Fibronectin Assay

Measurement of fibronectin was performed as described with minor modifications(25) . Cells grown overnight in 6-well plates were incubated for 5 h with DMEM containing 0.2% FBS and 100 µM ZnCl(2). The cells were then added with or without 50 ng/ml of TGF-beta1, incubated for 20 h, and labeled with 50 µCi/ml [S]methionine and [S]cysteine mixture in methionine- and cysteine-free DMEM for the final 4 h. The labeled culture media were incubated overnight with 100 µl of gelatin-Sepharose (Pharmacia) in the presence of 0.5% Triton X-100. The beads were washed once in Tris-buffered saline (50 mM Tris-HCl, pH 7.4, 150 mM NaCl), once in 50 mM Tris-HCl (pH 7.4), 0.5 M NaCl, and once in Tris-buffered saline. The fibronectin was eluted by boiling in SDS sample buffer in the presence of 10 mM DTT. The samples were analyzed by SDS-7% polyacrylamide gel electrophoresis and Bio-Imaging Analyzer.

GST Fusion Proteins

The GST fusion protein constructs were transformed into JM109 bacteria. Overnight cultures were diluted 1:8 in fresh medium, and after shaking for 2 h, isopropylthiogalactopyranoside (final concentration, 0.5 mM) was added. After another 3 h of shaking at 30 °C, the cells were lysed in PBS containing 1% Triton X-100, 1% Tween-20, 1% sodium deoxycholate, and 1 mM DTT, sonicated for 1 min, and centrifuged for 5 min. The supernatants were incubated with glutathione-Sepharose beads (glutathione-Sepharose 4B; Pharmacia) (5:1, v/v) for 1 h at 4 °C. After extensive washing in PBS, the beads were subjected to phosphorylation assays.

Protein Kinase Assay

25 µl of glutathione-Sepharose beads that attached GST fusion proteins were washed once with kinase buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, 10 mM MnCl(2), 0.5 mM DTT, 0.05% Triton X-100) and added with 25 µl of kinase buffer containing 1 µCi of [-P]ATP (Amersham Corp.). The beads were incubated for 15 min at 4 °C. Proteins were resolved on SDS-10% polyacrylamide gel under reducing conditions and analyzed by Bio-Imaging Analyzer.


RESULTS

Generation of a TbetaR-I Deletion Mutant and Its Binding Ability to TGF-beta

Based on the sequence comparison among the type I receptors, the cytoplasmic juxtamembrane region preceding the kinase domain of TbetaR-I can be divided into two subregions (Fig. 1). The C-terminal half of the juxtamembrane region (leucine 177 to valine 206), which is composed of 30 amino acids and rich in serine and threonine residues (three serines and four threonines), contains the highly conserved SGSGSG core sequence and other conserved amino acids among the type I receptors. Therefore, this region was previously designated GS domain (or type I box). Recent findings revealed that phosphorylation of several serine and threonine residues in the GS domain are essential for the TGF-beta signaling(22) . The N-terminal half remnant (asparagine 150 to threonine 176) of the juxtamembrane region is of interest because this region is also rich in serine and threonine residues (two serines and three threonines out of 27 amino acids), and in contrast to the GS domain, the sequences of the corresponding region in the type I receptors are very divergent (Fig. 1A), so that this region might be involved in the specification of the downstream substrates that mediate diverse responses triggered by the proteins in the TGF-beta superfamily. If this region is essential in TbetaR-I, its deletion should prevent signaling. We deleted the 32 amino acids of TbetaR-I in this region, yielding TbetaR-I/JD1(Delta150-181) (Fig. 1B). The wild-type TbetaR-I and mutant TbetaR-I/JD1(Delta150-181) in pMEP4, a Zn-inducible vector, were stably transfected into a TbetaR-I-defective Mv1Lu cell line, R4-2(29, 30, 33) . The expression of the exogenous receptors and their complex formation with the endogenous TbetaR-II were tested by affinity cross-linking of the cells using I-TGF-beta1 followed by immunoprecipitating the ligand-receptor complexes with anti-TbetaR-II antiserum. Fig. 2shows that the TbetaR-I/JD1(Delta150-181) (65-kDa component), like the wild-type TbetaR-I (70-kDa component), was able to bind TGF-beta in a Zn-inducible manner and formed a physiological complex with TbetaR-II (90-kDa component). Migration of the affinity labeled TbetaR-I/JD1(Delta150-181) that was slightly faster than that of the wild-type TbetaR-I complex was observed (Fig. 2) as expected from its shortened structure. A faint band observed in the uninduced wild-type TbetaR-I-transfectant may be ascribed to the leaky expression of the pMEP4 vector(27) .


Figure 1: Schematic illustration of the TbetaR-I mutants. A, sequence alignment of the cytoplasmic juxtamembrane region of different type I receptors(12) . Serine and threonine residues are shown in bold. The amino acid positions of TbetaR-I are indicated. The GS domain (type I box) is underlined. The SGSGSG core sequence is indicated by a bracket. TM, transmembrane domain. B, TbetaR-I mutant constructs. The open circles indicate serine and threonine residues within the cytoplasmic juxtamembrane region of TbetaR-I. The closed circles indicate serine and threonine residues altered to alanine and valine residues, respectively. The amino acid positions deleted or altered are indicated in parentheses. WT, wild-type TbetaR-I; TSR-I, TGF-beta superfamily receptor type I.




Figure 2: Binding of I-TGF-beta1 to wild-type TbetaR-I and its mutant derivatives. Parental Mv1Lu cells or R4-2 cells transfected with wild-type TbetaR-I and its mutant derivatives were pretreated with or without 100 µM ZnCl(2) for 6 h, followed by affinity cross-linking with I-TGF-beta1 using disuccinimidyl suberate. Cross-linked complexes were immunoprecipitated with an antiserum against TbetaR-II. Immune complexes were analyzed by SDS-gel electrophoresis under reducing conditions and Bio-Imaging Analyzer. Cross-linked complexes of TbetaR-II and TbetaR-I/JD1(Delta150-181) are indicated by arrows. Cross-linked complexes of wild-type TbetaR-I, TbetaR-I/JM123(S165A/S172A/T176V), TbetaR-I/JM1(S165A), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V) are indicated as TbetaR-I with an arrow. WT, wild-type TbetaR-I.



Signaling Activity of TbetaR-I/JD1(Delta150-181)

The signaling activities of TbetaR-I/JD1(Delta150-181) were determined by testing its ability to rescue biological responses to TGF-beta in R4-2 cells. We first examined the induction of PAI-1 and fibronectin because these responses in the parent Mv1Lu cells are well characterized, and they were the representatives among the various matrix proteins induced by TGF-beta(34) . In Mv1Lu cells, synthesis of PAI-1 was increased by the treatment with TGF-beta but not in R4-2 cells transfected with the vector alone (Fig. 3A). When R4-2 cells were transfected with the wild-type TbetaR-I or TbetaR-I/JD1(Delta150-181), the cells produced PAI-1 upon treatment with TGF-beta in the presence of ZnCl(2) (Fig. 3A). Fibronectin production by TGF-beta was restored also in R4-2 cells transfected with the wild-type TbetaR-I but much less potently in the cells transfected with TbetaR-I/JD1(Delta150-181) (Fig. 3B). PAI-1 and fibronectin production were not stimulated in the absence of ZnCl(2) (data not shown), indicating that the signals for the induction of PAI-1 and fibronectin were rescued by the exogenous receptors.


Figure 3: Extracellular matrix protein responses in R4-2 cells transfected with wild-type TbetaR-I and its mutant derivatives. A, stimulation of PAI-1 production by TGF-beta1. Subconfluent cultures of Mv1Lu or R4-2 cells transfected with the indicated receptor cDNAs were incubated with medium containing 100 µM of ZnCl(2) for 5 h. Cells were then incubated with (+) or without(-) 50 ng/ml of TGF-beta1 for 2 h and were labeled with a [S]methionine and [S]cysteine mixture. Induced PAI-1 was visualized by SDS-gel electrophoresis and Bio-Imaging Analyzer. PAI-1 was observed as a characteristic 45-kDa band. B, stimulation of fibronectin production by TGF-beta1. Cells were incubated with medium containing 100 µM of ZnCl(2) for 5 h. Cells were then incubated with (+) or without(-) 50 ng/ml of TGF-beta1 for 20 h and were labeled with a [S]methionine and [S]cysteine mixture for the last 4 h. Fibronectin secreted into the media was purified by adsorption to gelatin-Sepharose and analyzed by SDS-gel electrophoresis and Bio-Imaging Analyzer. WT, wild-type TbetaR-I.



To evaluate whether TbetaR-I/JD1(Delta150-181) is able to restore TGF-beta antiproliferative effect, DNA synthesis assay was performed by measuring the incorporation of [^3H]thymidine into the DNA (Fig. 4, A and B). Upon treatment with TGF-beta, [^3H]thymidine incorporation into the DNA of Mv1Lu cells was inhibited dose-dependently up to 97% (Fig. 4A), whereas TGF-beta had no effect on the [^3H]thymidine incorporation in the R4-2 cells transfected with the vector alone. When R4-2 cells transfected with the wild-type TbetaR-I were treated with TGF-beta in the presence of ZnCl(2), [^3H]thymidine incorporation into the DNA was inhibited by 65-75%, whereas only a marginal inhibition was observed in the absence of ZnCl(2). In contrast, R4-2 cells transfected with TbetaR-I/JD1(Delta150-181) were refractory to TGF-beta growth inhibition in the presence or the absence of ZnCl(2) (Fig. 4, A and B). These results suggested that the N-terminal half of the cytoplasmic juxtamembrane domain of TbetaR-I was not required for signaling a PAI-1 response, whereas it was essential for signaling growth inhibitory activity.


Figure 4: Antiproliferative response in R4-2 cells transfected with wild-type TbetaR-I and its mutant derivatives. Cells were incubated in DMEM containing 0.2% FBS with or without 100 µM ZnCl(2) for 5 h. Then the cells were exposed to various concentrations of TGF-beta1 for 16 h and pulsed with [^3H]thymidine, and ^3H-radioactivity incorporated into the DNA was determined in a liquid scintillation counter. A, TGF-beta1 dose-response curve for growth inhibition. Closed squares, parent Mv1Lu cells; open squares, R4-2 cells transfected vector alone. The wild-type TbetaR-I-transfected R4-2 cells were treated with ZnCl(2) (closed circles) or without ZnCl(2) (open circles); the TbetaR-I/JD1(Delta150-181)-transfected R4-2 cells were treated with ZnCl(2) (closed triangles) or without ZnCl(2) (open triangles). These experiments were performed three times with similar results. B, inhibition of [^3H]thymidine incorporation in R4-2 cells expressing wild-type TbetaR-I and its mutant derivatives. Cells were pretreated with 100 µM ZnCl(2) followed by incubation with 15 ng/ml of TGF-beta1 and processed as described above. The data are plotted as the average percentage of inhibition ± standard deviation. WT, wild-type TbetaR-I.



Serine 172 and Threonine 176 Are Essential for Signaling Growth Inhibitory Activity

The inability of TbetaR-I/JD1(Delta150-181) to mediate a growth inhibitory signal raised the possibility that the N-terminal half of the cytoplasmic juxtamembrane domain of TbetaR-I contains a site for interaction with downstream component that transduces a signal specific for growth inhibition. Alternatively, such a deletion might change the structural conformation, yielding a receptor that is unable to transduce signals even if the substrate interaction sites were preserved. To address these questions, missense mutations instead of deletion were introduced into certain serine and threonine residues in the TbetaR-I juxtamembrane region that was deleted in TbetaR-I/JD1(Delta150-181). As an initial attempt, serine 165, serine 172, and threonine 176 were chosen because these serine and threonine residues were rather conserved among the type I receptors for the TGF-beta superfamily (Fig. 1), especially in ActR-IB, which transduces growth inhibition and PAI-1 signals by activin A(28) . Serine and threonine residues were mutated simultaneously or individually to alanine and valine residues, respectively, resulting in four different expression constructs including TbetaR-I/JM123(S165A/S172A/T176V), TbetaR-I/JM1(S165A), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V). These constructs were stably transfected into R4-2 cells, and their expression, TGF-beta binding, and physical association with TbetaR-II were examined by affinity cross-linking with I-TGF-beta1 followed by immunoprecipitation using anti-TbetaR-II antiserum (Fig. 2). All the different receptor mutants were expressed on the cell surface and bound TGF-beta in complex with TbetaR-II in a Zn-dependent manner.

To test the signaling activities of these missense mutant forms of TbetaR-I, the transfected cells were subjected to the analyses for extracellular matrix production and growth inhibition by TGF-beta. In PAI-1 and fibronectin assays, like wild-type TbetaR-I, all the constructs analyzed including TbetaR-I/JM123(S165A/S172A/T176V), TbetaR-I/JM1(S165A), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V) restored responsiveness to TGF-beta (Fig. 3, A and B). With regard to TGF-beta antiproliferative effect, the TbetaR-I/JM1(S165A) construct mediated a growth inhibitory effect comparable with that mediated by the wild-type TbetaR-I, whereas TbetaR-I/JM123(S165A/S172A/T176V), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V) were unable to restore this activity (Fig. 4B). More than five different clones for TbetaR-I/JM1(S165A), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V) were subjected to the growth inhibition assay, which gave essentially the same results (data not shown).

Kinase Activity of TbetaR-I and Its Mutant Derivatives in Vitro

The differences among TbetaR-I and its mutant derivatives in their ability to restore responsiveness to TGF-beta might be due to altered catalytic activity of their receptor kinase. To address this issue, kinase activity was determined by expressing the cytoplasmic regions of TbetaR-I and its mutants as GST fusion proteins in Escherichia coli and testing their kinase activities in vitro. The protein products of wild-type TbetaR-I (GST-WT) and all the mutant constructs including GST-JD1(Delta150-181), GST-JM123(S165A/S172A/T176V), GST-JM1(S165A), GST-JM2(S172A), and GST-JM3(T176V) became phosphorylated (Fig. 5). These observations indicate that all the mutant constructs of TbetaR-I used in these experiments were active as kinases at least in vitro.


Figure 5: Kinase activity of wild-type TbetaR-I and its mutant derivatives in vitro. Glutathione-Sepharose beads that attached the indicated GST fusion proteins were incubated with kinase buffer containing 1 µCi of [-P]ATP for 15 min at 4 °C. Proteins were resolved on an SDS-polyacrylamide gel under reducing conditions and analyzed by Bio-Imaging Analyzer. WT, wild-type TbetaR-I.




DISCUSSION

Recent studies on transmembrane serine/threonine kinases have disclosed that certain members of TGF-beta superfamily exert their multiple effects through binding to unique sets of heteromeric complexes between type I and type II receptors. In the case of TGF-beta, TbetaR-II is a constitutively active kinase and capable of binding TGF-beta in the absence of TbetaR-I(22) , whereas TbetaR-I requires TbetaR-II for the ligand binding. The TbetaR-I kinase appears to be activated by formation of a hetero-oligomeric complex composed of TGF-beta, TbetaR-II, and TbetaR-I. In the complex, several serine and threonine residues in the GS domain of TbetaR-I become phosphorylated by TbetaR-II, and the phosphorylation of GS domain is essential for TGF-beta signaling(22, 26, 27) ; however, the functional role of phosphorylated serine and threonine residues in the GS domain as well as the mechanism of signaling after the phosphorylation are largely unknown. In addition, functional importance of the TbetaR-I cytoplasmic region other than the GS domain remains to be elucidated.

In the present communication, we studied the role of the N-terminally flanking region of the TbetaR-I GS domain by mutating this region and testing its ability to restore the signaling activity in TbetaR-I-defective R4-2 cells. When expressed in R4-2 cells, like wild-type TbetaR-I, the deletion mutant TbetaR-I/JD1(Delta150-181) and other missense mutants including TbetaR-I/JM123(S165A/S172A/T176V), TbetaR-I/JM1(S165A), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V) were all cross-linked with radioiodinated TGF-beta and co-immunoprecipitated with TbetaR-II (Fig. 2), indicating that this region in TbetaR-I is dispensable at least for its expression and binding to TGF-beta on the cell surface and forming a complex with TbetaR-II.

The signaling activities of these mutant TbetaR-I constructs were tested for some of the most characteristic responses to TGF-beta; i.e. PAI-1 and fibronectin induction and growth inhibition. Wild-type TbetaR-I and all the missense mutants restored PAI-1 and fibronectin responses in R4-2 cells (Fig. 3, A and B), indicating that serine 165, serine 172, and threonine 176 of TbetaR-I are not needed to transduce a signal for PAI-1 and fibronectin induction.

Antiproliferative response was also restored by the wild-type TbetaR-I and one of the receptor mutants, TbetaR-I/JM1(S165A); however, the other mutants including TbetaR-I/JD1(Delta150-181), TbetaR-I/JM123(S165A/S172A/T176V), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V) were unable to rescue this response (Fig. 4, A and B). Because the wild-type TbetaR-I and all the mutant TbetaR-I were similar in their activities to bind TGF-beta, form a complex with TbetaR-II, and phosphorylate themselves in vitro, the differences in their ability to restore the antiproliferative response does not seem to be at the level of ligand-receptor complex formation or basal kinase activity. Rather, TbetaR-I/JM123(S165A/S172A/T176V), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V) are likely to be impaired in interacting with a specific substrate that transduces antiproliferative response but not PAI-1 and fibronectin responses.

From our present data, it is not easy to deduce the mechanistic significance of serine 172 and threonine 176 of TbetaR-I in TGF-beta signaling. Although it was reported that TGF-beta-induced phosphorylation of these residues was not detected in vivo(22) , it is still possible that TbetaR-II may phosphorylate these residues as minor phosphorylation site(s). Alternatively, these residues might be constitutively phosphorylated even in the absence of TGF-beta, which would not be detected as the ligand-induced phosphorylation sites. It is also possible that serine 172 and threonine 176 in TbetaR-I may not be themselves phosphorylated, but their integrity is essential to maintain the proper conformation of TbetaR-I to interact with its substrates.

It was reported that whereas Mv1Lu cells expressing SV40 T-antigen were refractory to the antiproliferative effect of TGF-beta, TGF-beta induced the expressions of junB mRNA and extracellular matrix proteins including PAI-1, fibronectin, and thrombospondin in these cells(34, 35) . In addition, we have previously shown that growth inhibition and extracellular matrix production by TGF-beta are sensitive and insensitive, respectively, to phorbol 12-myristate 13-acetate in prostatic carcinoma cells(36) . These observations have suggested that the signals induced by TGF-beta that lead to growth inhibition and to extracellular matrix production should differ at a certain step within the signaling cascade from the receptor to the nucleus. In this regard, the present study is of particular importance. TbetaR-I mutants including TbetaR-I/JM123(S165A/S172A/T176V), TbetaR-I/JM2(S172A), and TbetaR-I/JM3(T176V) had signaling activity for extracellular matrix protein responses but not growth inhibition. Although other responses including expressions of junB and thrombospondin should be determined, identification of such mutant forms of TbetaR-I strongly suggests that the signals for growth inhibition and extracellular matrix production are diverged closely at the receptor level. In conclusion, serine 172 and threonine 176 within the TbetaR-I juxtamembrane region preceding the GS domain are essential for signaling the TGF-beta antiproliferative response and might be involved in the interaction with the downstream substrate responsible for growth inhibition.


FOOTNOTES

*
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan and a grant from the Mochida Memorial Foundation For Medical and Pharmaceutical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, The Cancer Institute, Tokyo, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan. Tel.: 81-3-3918-0111, ext. 4504; Fax: 81-3-3918-0342.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; BMP, bone morphogenetic protein; TbetaR, TGF-beta receptor; ActR, activin receptor; BMPR, BMP receptor; PAI, plasminogen activator inhibitor; PCR, polymerase chain reaction; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DTT, dithiothreitol.

(^2)
H. Nishitoh, M. Saitoh, I. Asahina, S. Enomoto, T. K. Sampath, M. Takagi, and H. Ichijo, submitted for publication.


ACKNOWLEDGEMENTS

We thank J. Massagué, J. L. Wrana, and L. Attisano for pMEP4 vector, M. Laiho and J. Massagué for R4-2 cells, and H. Ohashi for TGF-beta1. We are grateful to C.-H. Heldin, P. ten Dijke, M. Takarada, and Y. Qu for valuable discussion. We are also grateful to M. Kawabata and K. Takeda for critical reading of the manuscript.


REFERENCES

  1. Lyons, R. M., and Moses, H. L. (1990) Eur. J. Biochem. 187, 467-473 [Abstract]
  2. Sporn, M. B., and Roberts, A. B. (1992) J. Cell Biol. 119, 1017-1021 [Medline] [Order article via Infotrieve]
  3. Massagué, J., Attisano, L., and Wrana, J. L. (1994) Trends Cell Biol. 4, 172-178 [CrossRef]
  4. Miyazono, K., ten Dijke, P., Ichijo, H., and Heldin, C.-H. (1994) Adv. Immunol. 55, 181-220 [Medline] [Order article via Infotrieve]
  5. Attisano, L., Cárcamo, J., Ventura, F., Weis, F. M. B., Massagué, J., and Wrana, J. L. (1993) Cell 75, 671-680 [Medline] [Order article via Infotrieve]
  6. Bassing, C. H., Yingling, J. M., Howe, D. J., Wang, T., He, W. W., Gustafson, M. L., Shah, P., Donahoe, P. K., and Wang, X.-F. (1994) Science 263, 87-89 [Medline] [Order article via Infotrieve]
  7. Ebner, R., Chen, R.-H., Lawler, S., Zioncheck, T., and Derynck, R. (1993) Science 262, 900-902 [Medline] [Order article via Infotrieve]
  8. Franzén, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C.-H., and Miyazono, K. (1993) Cell 75, 681-692 [Medline] [Order article via Infotrieve]
  9. Lin, H. Y., and Lodish, H. F. (1993) Trends Cell Biol. 3, 14-19 [CrossRef]
  10. Attisano, L., Wrana, J. L., López-Casillas, F., and Massagué, J. (1994) Biochim. Biophys. Acta 1222, 71-80 [Medline] [Order article via Infotrieve]
  11. Kingsley, D. M. (1994) Genes & Dev. 8, 133-146
  12. ten Dijke, P., Franzén, P., Yamashita, H., Ichijo, H., Heldin, C.-H., and Miyazono, K. (1994) Prog. Growth Factor Res. 5, 55-72 [Medline] [Order article via Infotrieve]
  13. Ebner, R., Chen, R.-H., Shum, L., Lawler, S., Zioncheck, T. F., Lee, A., Lopez, A. R., and Derynck, R. (1993) Science 260, 1344-1348 [Medline] [Order article via Infotrieve]
  14. He, W. W., Gustafson, M. L., Hirobe, S., and Donahoe, P. K. (1993) Dev. Dyn. 196, 133-142 [Medline] [Order article via Infotrieve]
  15. ten Dijke, P., Ichijo, H., Franzén, P., Schulz, P., Saras, J., Toyoshima, H., Heldin, C.-H., and Miyazono, K. (1993) Oncogene 8, 2879-2887 [Medline] [Order article via Infotrieve]
  16. Köenig, B. B, Cook, J. S., Wolsing, D. H., Ting, J., Tiesman, J. P., Correa, P. E., Olson, C. A., Pecquet, A. L., Ventura, F., Grant, R. A., Chen, G.-X., Wrana, J. L., Massagué, J., and Rosenbaum, J. S. (1994) Mol. Cell. Biol. 14, 5961-5974 [Abstract]
  17. Matsuzaki, K., Xu, J., Wang, F., McKeehan, W. L., Krummen, L., and Kan, M. (1993) J. Biol. Chem. 268, 12719-12723 [Abstract/Free Full Text]
  18. Suzuki, A., Thies, R. S., Yamaji, N., Song, J. J., Wozney, J. M., Murakami, K., and Ueno, N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10255-10259 [Abstract/Free Full Text]
  19. ten Dijke, P., Yamashita, H., Ichijo, H., Franzén, P., Laiho, M., Miyazono, K., and Heldin, C.-H. (1994) Science 264, 101-104 [Medline] [Order article via Infotrieve]
  20. Tsuchida, K., Mathews, L. S., and Vale, W. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11242-11246 [Abstract]
  21. Ventura, F., Doody, J., Liu, F., Wrana, J. L., and Massagué, J. (1994) EMBO J 13, 5581-5589 [Abstract]
  22. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massagué, J. (1994) Nature 370, 341-347 [CrossRef][Medline] [Order article via Infotrieve]
  23. Cárcamo, J., Zentella, A., and Massagué, J. (1995) Mol. Cell. Biol. 15, 1573-1581 [Abstract]
  24. Wieser, R., Attisano, L., Wrana, J. L., and Massagué, J. (1993) Mol. Cell. Biol. 13, 7239-7247 [Abstract]
  25. Wrana, J. L., Attisano, L., Cárcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X.-F., and Massagué, J. (1992) Cell 71, 1003-1014 [Medline] [Order article via Infotrieve]
  26. Franzén, P., Heldin, C.-H., and Miyazono, K. (1995) Biochem. Biophys. Res. Commun. 207, 682-689 [CrossRef][Medline] [Order article via Infotrieve]
  27. Wieser, R., Wrana, J. L., and Massagué, J. (1995) EMBO J. 14, 2199-2208 [Abstract]
  28. Cárcamo, J., Weis, F. M. B., Ventura, F., Wieser, R., Wrana, J. L., Attisano, L., and Massagué, J. (1994) Mol. Cell. Biol. 14, 3810-3821 [Abstract]
  29. Laiho, M., Weis, F. M. B., and Massagué, J. (1990) J. Biol. Chem. 265, 18518-18524 [Abstract/Free Full Text]
  30. Laiho, M., Weis, F. M. B., Boyd, F. T., Ignotz, R. A., and Massagué, J. (1991) J. Biol. Chem. 266, 9108-9112 [Abstract/Free Full Text]
  31. Frolik, C. A., Wakefield, L. M., Smith, D. M., and Sporn, M. B. (1984) J. Biol. Chem. 259, 10995-11000 [Abstract/Free Full Text]
  32. Ohtsuki, M., and Massagué, J. (1992) Mol. Cell. Biol. 12, 261-265 [Abstract]
  33. Boyd, F. T., and Massagué, J. (1989) J. Biol. Chem. 264, 2272-2278 [Abstract/Free Full Text]
  34. Laiho, M., Rönnstrand, L., Heino, J., Decaprio, J. A., Ludlow, J. W., Livingston, D. M., and Massagué, J. (1991) Mol. Cell. Biol. 11, 972-978 [Medline] [Order article via Infotrieve]
  35. Zentella, A., Weis, F. M. B, Ralph, D. A., Laiho, M., and Massagué, J. (1991) Mol. Cell. Biol. 11, 4952-4958 [Medline] [Order article via Infotrieve]
  36. Franzén, P., Ichijo, H., and Miyazono, K. (1993) Exp. Cell Res. 207, 1-7 [CrossRef][Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.