©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of the Transforming Growth Factor- Type I Receptor with Farnesyl-protein Transferase- (*)

(Received for publication, September 25, 1995; and in revised form, October 17, 1995)

Masahiro Kawabata (1) (2) Takeshi Imamura (3) Kohei Miyazono (2) (3) Michael E. Engel (1) Harold L. Moses (1)(§)

From the  (1)Vanderbilt Cancer Center, Nashville, Tennessee 37232-6838, (2)Department of Biochemistry, The Cancer Institute, Tokyo, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan, and (3)Ludwig Institute for Cancer Research, Box 595, Biomedical Center, S-751 24 Uppsala, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor-beta1 (TGF-beta1) is the prototype of a large family of molecules that regulate a variety of biological processes. The type I (TbetaR-I) and type II (TbetaR-II) receptors for TGF-beta1 are transmembrane serine/threonine kinases, forming a heteromeric signaling complex. Recent studies have shown that TbetaR-II is a constitutively active kinase and phosphorylates TbetaR-I upon ligand binding, suggesting that TbetaR-I is the effector subunit of the receptor complex, which transduces signals to intracellular targets. This model has been further confirmed by the identification of constitutively active TbetaR-I that mediates TGF-beta1-specific cellular responses in the absence of ligand and TbetaR-II. To investigate signaling by TGF-beta1, we have sought to isolate proteins that interact with the cytoplasmic region of TbetaR-I. One of the proteins identified was the alpha subunit of farnesyl-protein transferase (FTalpha) that modifies a series of peptides including Ras. TbetaR-I specifically interacts with FTalpha in the yeast two-hybrid system. Glutathione S-transferase-TbetaR-I fusion proteins bind FTalpha translated in vitro. TbetaR-I also phosphorylates FTalpha. We further show that the constitutively active TbetaR-I interacted with FTalpha very strongly whereas an inactive form of TbetaR-I did not. These results suggest that FTalpha may be one of the substrates of the activated TbetaR-I kinase.


INTRODUCTION

Transforming growth factor-beta1 (TGF-beta1) (^1)is a multifunctional cytokine that is involved in a variety of biological processes such as cell cycle progression, differentiation, adhesion, migration, extracellular matrix deposition, and immunoregulation(1, 2) . However, the mechanisms whereby TGF-beta1 exerts such pleiotropic effects have been elusive. TGF-beta1 binds to its specific receptors on the cell surface. Three distinct classes of receptors have been identified(2) . The type III (TbetaR-III) receptor termed betaglycan is involved in presenting ligand to other signaling receptors(3) . The type I (TbetaR-I) and type II (TbetaR-II) receptors belong to a family of transmembrane serine/threonine kinases that include receptors for other TGF-beta1-related molecules such as activins, bone morphogenic proteins, and Müllerian inhibiting substance(2) . TbetaR-II binds ligand without TbetaR-I but requires TbetaR-I to transduce signals. TbetaR-I, on the other hand, requires TbetaR-II to bind ligand(4) . In the case of TGF-betas and activins, the type II receptors determine ligand binding specificities(5) , and the type I receptors specify cellular responses to ligand(6) , suggesting that TbetaR-I is the downstream component of the receptor system.

Recently a model for activation of the TGF-beta receptor system has been proposed(7) . TbetaR-II is a constitutively active kinase. Upon ligand binding TbetaR-II recruits and phosphorylates TbetaR-I. The transphosphorylation sites in TbetaR-I reside between the transmembrane and the kinase domain(8) . This region contains a short peptide stretch called the GS domain, which is highly conserved among the type I receptors of the serine/threonine kinase family. Mutations of certain serines and threonines in this domain impair phosphorylation and signaling activity of TbetaR-I(8) , indicating that phosphorylation of the GS domain by TbetaR-II has a crucial role in TGF-beta signaling. Replacement of threonine 204, between the core GS domain and the kinase domain, with aspartic acid resulted in constitutively active TbetaR-I (8) . This mutant has elevated in vitro kinase activity and signals both anti-proliferative and transcriptional responses in the absence of ligand and TbetaR-II. These results further support the hypothesis that TbetaR-I transmits signals by phosphorylating intracellular substrates.

We employed a yeast two-hybrid system (9) to identify proteins that interact with the cytoplasmic region of TbetaR-I. One of the identified clones encoded farnesyl-protein transferase-alpha (FTalpha), a component of an enzyme that modifies various proteins such as Ras. TbetaR-I was shown to interact with and phosphorylate FTalpha in vitro. The constitutively active form of TbetaR-I interacted with FTalpha more strongly than the wild type, and an inactive form of TbetaR-I did not bind FTalpha at all. These results indicate that FTalpha may be one of the substrates of activated TbetaR-I.


EXPERIMENTAL PROCEDURES

Screening and Interaction Assay

A HeLa cDNA expression library was screened exactly as described(10) . Briefly, the yeast strain, EGY48(9) , was transformed with the reporter, pSH18-34(9) , and the bait, pEGR4(10) , which contains the cytoplasmic region of the rat TbetaR-I also called R4(11) . The library was then introduced into EGY48. The transformants were grown on appropriate media, and positive clones were selected depending on beta-galactosidase activity and leucine prototrophy. Prey plasmids were rescued from EGY48, amplified in bacteria, and sequenced. Interaction assays using the interaction trap (9) were done as described before(10) .

Plasmids

Construction of pEGR4 and pEGIIR containing the cytoplasmic region of TbetaR-II was described(10) . The wild type and mutant forms of the human TbetaR-I, called ALK5(12) , were made using the polymerase chain reaction (PCR) and inserted into the yeast expression vector as follows. The internal EcoRI site in the human TbetaR-I was removed with the peptide sequence unchanged. The cytoplasmic region (amino acids 148-503) was then amplified with an EcoRI site and a XhoI site attached at the 5`- and 3`-ends, respectively, and subsequently inserted into pJG4-5(9), yielding pJG-TbetaR-I. The bait plasmid was constructed by excising the EcoRI-XhoI fragment from pJG-TbetaR-I and subcloning at the same restriction enzyme sites of pEG202 (9) (pEG-TbetaR-I). TbetaR-I mutants were constructed similarly. TbetaR-I(DeltaJM) lacks the juxtamembrane region (amino acids 148-204) of the wild type. TbetaR-I(T200V), TbetaR-I(T204D), and TbetaR-I(K232R) have valine instead of threonine 200, aspartic acid instead of threonine 204, and arginine instead of lysine 232, respectively. The cytoplasmic region of TbetaR-II (amino acids 192-567) was subcloned into pJG4-5 by PCR. The yeast expression plasmids of farnesyl transferase-alpha and -beta (FTbeta) were constructed by subcloning the entire coding region at the EcoRI and XhoI sites of pJG4-5 and pEG202. All of the PCR products were sequenced. Glutathione S-transferase (GST) fusion protein expression plasmids were made by subcloning the EcoRI-XhoI insert in pJG4-5 into pGEX-4T-1 (Pharmacia Biotech Inc.). pcDNA3-FTalpha, the in vitro expression plasmid of FTalpha, was constructed by subcloning the insert of pJG-FTalpha at the EcoRI and XhoI sites of pcDNA3 (Invitrogen). The detail of the subcloning procedures including the primer sequences can be obtained upon request.

In Vitro Binding Assay

GST fusion proteins were prepared as described (13) except that the induction with isopropyl thio-beta-D-galactoside was done at 30 °C for 8 h. To synthesize the FTalpha proteins in vitro, the reticulocyte lysate system (Promega) was used. pcDNA3-FTalpha was transcribed with T7 RNA polymerase and translated in the presence of [S]methionine. Equal amounts of GST fusion proteins (approximately 3.5 µg) bound to GST beads (50 µl of 50% slurry), and 20 µl of in vitro translation product were mixed with 500 µl of NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), incubated at 4 °C for 2 h, washed with NETN buffer 4 times, and subjected to SDS-PAGE (10% polyacrylamide gel) and autoradiography.

In Vitro Phosphorylation Assay

GST fusion proteins were released from GST beads by incubation with 15 mM reduced glutathione at 4 °C for 15 min. The concentration of the released proteins was assayed according to the Bradford method (Bio-Rad). In the autophosphorylation assay, 0.44 µg of released GST or GST-TbetaR fusion proteins was incubated in 30 µl of the kinase buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 10 mM MnCl(2), 10 mM dithiothreitol, 0.05% Triton X-100) with 10 µCi of [P-]ATP at room temperature for 30 min. In the transphosphorylation assay, the same amounts of GST-TbetaR fusion proteins were mixed with 0.4 µg of released GST proteins or GST-FTalpha proteins, preincubated at 4 °C for 30 min in the kinase buffer, and incubated in the presence of [P-]ATP as in the autophosphorylation assay. Cleavage of GST fusion proteins was accomplished by incubation with 10 µg of thrombin in 200 µl of the cleavage buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM CaCl(2), 1 mM dithiothreitol) at room temperature for 1 h. 0.6 µg of cleaved FTalpha proteins was used in the phosphorylation reaction. Samples were subjected to SDS-PAGE (10% polyacrylamide gel) and autoradiography.

Quantitative beta-Galactosidase Assay

beta-Galactosidase assay in liquid culture was done essentially as described(13) . Four independent colonies of each transformation were first grown in appropriate selection media, and then the prey proteins were induced with galactose. beta-Galactosidase activity was measured with o-nitrophenyl-beta-D-galactoside as substrate.


RESULTS

To search for proteins that interact with TbetaR-I, we used the interaction trap screen developed by Brent and co-workers(9) . A HeLa cell cDNA library was screened with the cytoplasmic region of the rat TbetaR-I (11) as the bait. One of the clones encoded T-ALK, a novel type II serine/threonine kinase receptor(10) , which was later shown to be the bone morphogenic protein type II receptor(14, 15) . Another subset of clones encoded FKBP12, a binding protein for FK506 and rapamycin (data not shown). Ten clones encoded varying portions of the alpha subunit of farnesyl transferase.

Farnesyl transferase is a heterodimeric enzyme composed of an alpha and beta subunit(16) . Prey (pJG-FTalpha or pJG-FTbeta) and bait (pEG-FTalpha or pEG-FTbeta) plasmids containing the entire coding region of FTalpha or FTbeta were constructed. FTalpha and FTbeta interacted very strongly in the yeast assay (data not shown) as shown in vivo(17) . To test the specificity of the interaction between TbetaR-I and FTalpha, pJG-FTalpha was introduced into EGY48 with pEG202, the bait vector, or a panel of unrelated baits. None of these were positive in the interaction assay (Fig. 1A). In addition, FTalpha did not interact with TbetaR-II, and FTbeta did not associate with TbetaR-I. Thus the interaction between TbetaR-I and FTalpha was specific in the yeast system.


Figure 1: Interaction of FTalpha with TbetaR-I. A, various combinations of a bait and a prey with the reporter plasmid were introduced into EGY48. Four independent colonies from each transformation were assayed on dextrose and galactose X-Gal plates. The results on galactose X-Gal plates are shown. A blue color represents a positive interaction. In the first seven cases, the prey is FTalpha and the baits are shown in the figure. pEG202 is the bait vector; Cdc2, Ftz, Max, and bicoid are unrelated baits (a gift of R. Finley). In the last case, the combination of the TbetaR-I bait and the FTbeta prey was tested. B, interaction of the FTalpha clones with TbetaR-I was compared. pJG-FTalpha* expresses the entire coding region of FTalpha (379 amino acids). CL69** includes part of the 5`-noncoding region. The other clones have a deletion in the N-terminal region.



Only one of the FTalpha clones (CL69) contained the entire coding region whereas the others lacked part of the 5`-coding sequence (Fig. 1B). To compare the strength of interactions, all of the rescued FTalpha plasmids and pJG-FTalpha were tested in the interaction trap. The beta-galactosidase production by the clones missing up to the first 45 amino acids of FTalpha was almost the same on X-Gal plates whereas the colony of CL74 starting from the 59th amino acid exhibited a lighter blue color. The color of CL24 starting from the 75th amino acid remained almost white (Fig. 1B). These data suggest that the first N-terminal 45 amino acids of FTalpha are dispensable for its interaction with TbetaR-I. Interestingly, deletion of 51 amino acids at the N terminus of FTalpha has been reported to allow normal stabilization of FTbeta and production of enzyme activity, but deletion of 106 amino acids abolished both functions(17) .

To confirm the specific interaction of FTalpha and TbetaR-I in another system, we used GST fusion proteins of the cytoplasmic region of the TGF-beta receptors and FTalpha translated in vitro. When FTalpha was translated in a reticulocyte system, two bands were obtained. A minor band with a faster mobility is probably a degradate of the entire protein since FTalpha is very unstable in mammalian cells(17) . FTalpha bound to GST-TbetaR-I but not to GST itself (Fig. 2). A minimal level of binding to GST-TbetaR-II was observed in this assay.


Figure 2: In vitro binding of FTalpha with TbetaR-I. GST or GST-TbetaR fusion proteins bound to GST beads and FTalpha proteins translated in vitro were mixed, washed, and analyzed by SDS-PAGE. Products from the in vitro translation reaction are shown as input. Arrows represent two bands obtained in the translation of FTalpha.



We next tested whether TbetaR-I phosphorylates FTalpha in vitro using GST fusion proteins (Fig. 3A). GST-TbetaR-I (lane 2, 67 kDa) and GST-TbetaR-II (lane 3, 70 kDa) showed autophosphorylation activity whereas no kinase activity was detected in GST itself (lane 1) or GST-FTalpha alone (lane 5). A mutant of human TbetaR-I (K232R), in which lysine 232 was changed to arginine, was constructed. This mutation completely abolished its signaling and kinase activities (7) (lane 4). When GST-TbetaR-I was mixed with GST-FTalpha, a new band that corresponds to GST-FTalpha (71 kDa) appeared above the GST-TbetaR-I autophosphorylated band (lane 6). GST-K232R did not phosphorylate itself or FTalpha (lane 9). It was not clear whether GST-FTalpha was phosphorylated by GST-TbetaR-II (lane 8) since a faint band of a similar size of GST-FTalpha was detected in GST-TbetaR-II alone (lane 3). However, phosphorylation of FTalpha as intense as in TbetaR-I was not observed. It is possible that TbetaR-I could phosphorylate the GST portion of the GST-FTalpha fusion proteins. When GST itself was mixed with GST-TbetaR-I, there was no phosphorylation of GST (27 kDa) (lane 7). A possibility that TbetaR-I may phosphorylate GST through its interaction with FTalpha still remained. Therefore the FTalpha portion (44 kDa) was cleaved from the GST-FTalpha fusion proteins with thrombin (36 kDa) (Fig. 3B). Again FTalpha alone did not show any kinase activity (lane 1). When the cleaved FTalpha was mixed with GST-TbetaR-I, a doublet-phosphorylated band the size of FTalpha appeared (lane 2) but not in GST-K232R (lane 4). Similar but much fainter bands were detected in GST-TbetaR-II (lane 3). These data show that TbetaR-I can phosphorylate FTalpha. TbetaR-I did not phosphorylate FKBP12 under the same condition (data not shown).


Figure 3: In vitro phosphorylation of FTalpha by TbetaR-I. In vitro phosphorylation was done using GST fusion proteins. A, eluted GST-FTalpha fusion proteins were phosphorylated by GST-TbetaR fusion proteins. Lane 1, GST; lane 2, GST-TbetaR-I; lane 3, GST-TbetaR-II; lane 4, GST-K232R; lane 5, GST-FTalpha; lane 6, GST-TbetaR-I plus GST-FTalpha; lane 7, GST-TbetaR-I plus GST; lane 8, GST-TbetaR-II plus GST-FTalpha; lane 9, GST-K232R plus GST-FTalpha. A fast migrating band designated by an arrow was recognized by anti-ALK5 antibodies (data not shown), suggesting that the band represents a degradate of GST-TbetaR-I. Molecular weight markers are shown on the right. B, FTalpha proteins cleaved from GST-FTalpha proteins were used. Lane 1, FTalpha; lane 2, GST-TbetaR-I plus FTalpha; lane 3, GST-TbetaR-II plus FTalpha; lane 4, GST-K232R plus FTalpha. A band designated by an arrow is the same as in A.



Recently Massagué and co-workers reported mutants of TbetaR-I with various levels of signaling activity(8) . One of the mutants, in which threonine 204 was replaced with aspartic acid (T204D), showed an elevated level of in vitro kinase activity and functioned as a constitutively active TbetaR-I in vivo. Another mutation of threonine 200 to valine (T200V) yielded completely inactive TbetaR-I. We tested the interaction of FTalpha with the wild type and mutant forms of TbetaR-I using the interaction trap. In the X-Gal plate assay, colonies transformed with T204D showed an intense blue color whereas colonies of T200V remained completely white. The K232R transformants exhibited a faint blue color while the wild type and DeltaJM colonies showed intensity between K232R and T204D (data not shown). A quantitative beta-galactosidase assay was performed (Table 1). As in plate assay, T204D showed the strongest interaction and T200V did not interact with FTalpha. K232R interacted more strongly than T200V but less efficiently than the wild type. DeltaJM interacted more efficiently than the wild type but not as strongly as T204D. FKBP12 did not interact with DeltaJM but did interact with the wild type and all of the other mutants (data not shown). These results indicate that the activated TbetaR-I binds FTalpha more efficiently than the wild type or loss of function mutant TbetaR-I.




DISCUSSION

We screened a human cDNA library to identify proteins that bind to the cytoplasmic region of TbetaR-I. One of the clones was a novel type II serine/threonine kinase receptor (10) showing that the two-hybrid system is useful in isolating one of the subunits of a membrane-bound receptor complex. The second clone was FKBP12. FKBP12 was reported to specifically associate with TbetaR-I in vitro(18) . However, its role in TGF-beta signaling is yet unknown. Here we report FTalpha as another TbetaR-I binding protein.

Farnesyl transferase is a heterodimeric enzyme composed of alpha and beta subunits(16) . Coexpression of both subunits seems to be necessary for stabilization and activity of the enzyme(17, 19, 20) . The holoenzyme attaches a farnesyl isoprenoid to a cysteine residue(16, 21) . The CAAX (where A is an aliphatic amino acid and X is any amino acid) sequence is the target motif found at the C-terminal end of all Ras proteins and many other isoprenylated proteins(17, 21) . The alpha subunit may perform the catalytic function (17) , but the precise enzymatic mechanism is still elusive. Farnesylation plays a pivotal role in the subcellular localization of a variety of proteins including Ras(21, 22) . Heterologous proteins like protein A (23) and Raf (24) can be targeted to the plasma membrane by adding the C-terminal sequence of K-Ras. Membrane localization is critical for the activity of Ras. Inhibition of farnesylation leads to suppression of Ras-induced responses such as Xenopus oocyte maturation(25) , cytoskeletal disorganization(26) , cell growth(25) , and tumorigenesis in vivo(27) . In Caenorhabditis elegans, the multivulva phenotype resulting from an activated let-60 Ras mutation was suppressed by FT inhibitors (28) .

A number of investigations have suggested a relationship between TGF-beta and Ras. TGF-beta1 treatment caused a rapid increase in GTP-bound Ras in several cell lines(29, 30) . In another report, however, microinjection of oncogenic Ha-Ras proteins overcame TGF-beta1-mediated growth inhibition and TGF-beta1 decreased the GTP-bound form of Ras(31) . Although the effect of TGF-beta1 treatment on Ras status may vary depending on the context(31) , the latter results are consistent with the fact that Ras is mitogenic whereas TGF-beta1 predominantly inhibits cell growth. Furthermore, TGF-beta1 inhibited the coupling of Ras to the activation of phosphatidylcholine hydrolysis(32) . TGF-beta1 partially antagonized transformed phenotypes, including loss of organized actin cytoskeleton, caused by Ras transformation(33) . Intriguingly, farnesyl transferase inhibition also caused actin stress fiber formation and morphological reversion in Ras-transformed cells(26) . Thus TGF-beta1 negatively regulates Ras under certain conditions. It was also shown that cellular Ras activity is required for passage through the G(1) phase in Balb/c 3T3 cells while TGF-beta1 inhibits cell growth if added at any point in G(1)(34) . These findings suggest that Ras may be directly involved in TGF-beta signaling.

In the present report, we show that TbetaR-I not only binds but phosphorylates FTalpha in vitro. FTalpha binds preferentially to an activated form of TbetaR-I. These results indicate that FTalpha may be phosphorylated by TbetaR-I upon ligand binding. Phosphorylation of FTalpha has not yet been reported. It is important to study whether TGF-beta1 induces phosphorylation of FTalpha and modulates its activity in vivo.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant CA 42572 (to H. L. M.) and grants-in-aid from the Ministry of Education, Science and Culture of Japan (to M. K. and K. M.). 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: Vanderbilt Cancer Center, 649 Medical Research Bldg. II, Nashville, TN 37232-6838. Tel.: 615-936-1782; Fax: 615-936-1790.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; TbetaR, TGF-beta receptor; FT, farnesyl transferase; ALK, activin receptor-like kinase; PCR, polymerase chain reaction; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; X-Gal, 5-bromo-4-chloro-3-indoyl beta-D-galactoside.


ACKNOWLEDGEMENTS

We thank R. Finley and R. Brent for plasmids, strains, and the cDNA library used in the interaction trap, X.-F. Wang and P. Donahoe for the rat TbetaR-I cDNA, H. Y. Lin for the human TbetaR-II cDNA, C. Omer for the human FTalpha and FTbeta cDNAs, J. Pietenpol for valuable suggestions, and R. Serra for critical reading of the manuscript.


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