The Type II Transforming Growth Factor-beta Receptor Autophosphorylates Not Only on Serine and Threonine but Also on Tyrosine Residues*

(Received for publication, January 23, 1997, and in revised form, March 26, 1997)

Sean Lawler Dagger , Xin-Hua Feng , Ruey-Hwa Chen §, E. Miko Maruoka , Christoph W. Turck , Irene Griswold-Prenner and Rik Derynck par

From the Department of Growth and Development and Department of Anatomy, Programs in Cell Biology and Developmental Biology, University of California, San Francisco, California 94143-0640 and the  Howard Hughes Medical Institute, Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, California 94143-0724

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The type I and type II receptors for transforming growth factor-beta (TGF-beta ) are structurally related transmembrane serine/threonine kinases, which are able to physically interact with each other at the cell surface. To help define the initial events in TGF-beta signaling, we characterized the kinase activity of the type II TGF-beta receptor. A recombinant cytoplasmic domain of the receptor was purified from Escherichia coli and baculovirus-infected insect cells. Anti-phosphotyrosine Western blotting demonstrated that the type II receptor kinase can autophosphorylate on tyrosine. Following an in vitro kinase reaction, the autophosphorylation of the cytoplasmic domain and phosphorylation of exogenous substrate was shown by phosphoamino acid analysis to occur not only on serine and threonine but also on tyrosine. The dual kinase specificity of the receptor was also demonstrated using immunoprecipitated receptors expressed in mammalian cells and in vivo 32P labeling showed phosphorylation of the receptor on serine and tyrosine. In addition, the kinase activity of the cytoplasmic domain was inhibited by the tyrosine kinase inhibitor tyrphostin. Tryptic mapping and amino acid sequencing of in vitro autophosphorylated type II receptor cytoplasmic domain allowed the localization of the sites of tyrosine phosphorylation to positions 259, 336, and 424. Replacement of all three tyrosines with phenylalanines strongly inhibited the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an autoregulatory role for the kinase activity of this receptor. These results demonstrate that the type II TGF-beta receptor can function as a dual specificity kinase and suggest a role for tyrosine autophosphorylation in TGF-beta receptor signaling.


INTRODUCTION

The importance of protein phosphorylation in various signaling events that regulate cell proliferation has been well documented. Most mitogenic growth factors interact with transmembrane tyrosine kinases or receptors that associate with cytoplasmic tyrosine kinases, which, as a result of ligand-induced autophosphorylation, trigger signaling cascades that involve multiple phosphorylation events (1, 2). Initiated by tyrosine phosphorylation, these cascades involve several serine/threonine kinases as well as a dual specificity kinase, MAP1 kinase kinase, which activates its substrate MAP kinase by phosphorylation on both tyrosine and threonine residues (3, 4).

In contrast to many mitogenic growth factors, transforming growth factor-beta (TGF-beta ) induces an antiproliferative effect in many cell types, including epithelial, endothelial and hematopoietic cells (5-7). In addition to its growth modulatory activity, TGF-beta has a wide range of effects on extracellular matrix synthesis, cell-substrate adhesion, cell differentiation, and migration (5-8). TGF-beta , which exists as three isoforms encoded by separate genes (9, 10), is considered a prototype for the many structurally related members of the TGF-beta superfamily, which play important roles in diverse cell differentiation and developmental processes.

Until the recent cloning and characterization of the cell surface receptors for TGF-beta , little was known about the mechanisms of signal transduction by this growth factor or related factors. Cross-linking studies had previously shown the presence of several cell surface TGF-beta binding proteins (for reviews, see Refs. 11-13), with most cells expressing three types of high affinity cell surface binding components known as types I, II, and III receptors. Studies on mutant cell lines lacking functional type I or type II receptors showed that these two receptor types mediate most if not all TGF-beta responses and that both receptor types are required for full responsiveness to TGF-beta (11-15). The type III receptor, also known as betaglycan, is not required for TGF-beta signaling but may contribute to presentation of the ligand to the type II receptor (16, 17).

The type I and type II TGF-beta receptors are structurally related transmembrane kinases with a cytoplasmic segment consisting largely of a kinase domain, which has a predicted specificity for serine and threonine (reviewed in 11-13). In fact, the serine/threonine kinase activity of these receptors has been experimentally verified both in vitro (18-20) and in vivo (21, 22). In addition to the TGF-beta type II receptor, several other type II receptors for TGF-beta related ligands have been characterized, including two types of type II activin receptors (18, 23, 24), a Caenorhabditis elegans type II receptor that binds BMP-2 and BMP-4 (25), a mammalian BMP-2/4 type II receptor (26, 27), and a Drosophila type II receptor that binds the related Dpp gene product (28, 29). A number of type I receptors have also been cloned (20, 30-38). They are generally smaller than the type II receptors, have a defined cysteine pattern in their extracellular domains, and contain a highly conserved SGSGSGLP sequence immediately upstream of the cytoplasmic kinase domain. In contrast to the type II receptors, which define their own specificity of ligand binding, many type I receptors have their specificity of ligand binding largely determined by the coexpressed type II receptor. For example, the type I receptors Tsk7L (39) and TSR1 (31) bind TGF-beta or activin depending on the coexpressed type II receptor. On the other hand, the ALK-5/R4 receptor is primarily a functional type I receptor for TGF-beta (32, 34).

The type I and type II receptors cooperate in signal transduction, and both receptor types are required for full responsiveness to TGF-beta (20, 40-43). Type II and type I receptors physically interact with each other, and such heteromeric complex formation is required for efficient ligand binding to type I receptors (20, 21, 39, 40, 44). The type II and type I receptors also exist as homomeric receptor complexes at the cell surface (45, 46). These findings led to the proposal that the two receptor types form a heteromeric, probably tetrameric, type II/type I receptor complex (12, 47, 48), which mediates TGF-beta signaling. In this complex, the cytoplasmic domains of the type II receptor are constitutively phosphorylated on serine and threonine, due to ligand-independent autophosphorylation and to phosphorylation by other cytoplasmic kinases (21, 22). Furthermore, the type II receptor kinase phosphorylates the cytoplasmic domain of the type I receptor on serine and threonine (21, 22) and the phosphorylation of both types of cytoplasmic domain contributes to the stability of the heteromeric complex (44). The existence of multiple type II receptors with defined ligand binding specificity and various type I receptors with an ability to bind different ligands, depending on the nature of the co-expressed type II receptor, suggests the existence of a complex signaling system in which combinatorial interactions may provide a substantial degree of flexibility in the cellular responses to TGF-beta and related factors.

To gain insight into the initial events in the signaling of TGF-beta and related factors, we have further characterized the kinase activity of the type II TGF-beta receptor. Using the cloned type II TGF-beta and activin receptors, it has previously been shown that their kinase domains are able to autophosphorylate on serine and threonine (18-22, 49). However, a detailed comparison of kinase domain sequences of these and related receptors indicates some structural similarities with tyrosine kinases (50).2 In addition, endogenous activin type II receptor purified from mammalian cells exhibited not only serine and threonine but also tyrosine kinase activity (51). In contrast, the recombinant type II receptors for activin and TGF-beta have been reported to only have serine and threonine kinase activity (18-22, 49). Because of this apparent contradiction, we have studied the autophosphorylation activity of the type II TGF-beta receptors. We show that the cytoplasmic domain of this receptor phosphorylates itself and exogenous substrates not only on serine and threonine but also on tyrosine residues. We have also localized the autophosphorylated tyrosine residues in the cytoplasmic domain of the type II receptor. Replacement of these tyrosines by phenylalanines strongly inhibits the kinase activity of the type II receptor. Our results establish the type II TGF-beta receptor as a dual specificity kinase, which is autophosphorylated not only on serine and threonine but also on tyrosine, and suggest a dual specificity activity for other members of this receptor kinase family.


EXPERIMENTAL PROCEDURES

Plasmid Construction and Mutagenesis

Plasmid pGST-IIK was designed to express in Escherichia coli the C-terminal 374 amino acids of the type II TGF-beta receptor cytoplasmic domain as a glutathione S-transferase (GST) fusion protein. The corresponding coding region of the human type II TGF-beta receptor cDNA was amplified using the polymerase chain reaction (PCR), incorporating flanking EcoRI restriction sites. The PCR primers used were 5'-GGGGCCGAATTCCGGCAGCAGAAGCTGAGTTC-3' and 5'-GGGGCCGAATTCGAGCTATTTGGTAGTGTTTAGG-3'. The EcoRI fragment was then ligated into the EcoRI site of pGEX2T (Pharmacia Biotech Inc.), thus generating pGST-IIK and the sequence of this insert was verified using the Sequenase kit (U. S. Biochemical Corp.).

Expression plasmid pVL1393-(His)6IIK was constructed to express the cytoplasmic kinase domain of the type II receptor in the baculovirus expression system. The same receptor cDNA fragment as in pGST-IIK was ligated into the EcoRI site of the baculovirus expression vector pVL1393(His)6 to allow expression of the cytoplasmic domain with an N-terminal (His)6 extension. pVL1393(His)6 was constructed by inserting oligonucleotide linkers encoding the sequence Met-Ser-(His)6 into the BamHI and EcoRI cut plasmid pVL1393 (Invitrogen). The linkers used were 5'-GATCCTATAAATATGTCGCATCATCATCATCATCATGGTTCCATGG-3' and 5'-AATTCCATGGAACCATGATGATGATGATGATGCGACATATTTATAG-3'.

Plasmid pIIR-myc (46) expresses the full-length human type II TGF-beta receptor with a C-terminal Myc epitope tag when transfected into mammalian cells.

In vitro mutagenesis of the cytoplasmic domain was done using the Sculptor kit (Amersham) according to the manufacturer's recommendations. The PCR product used in the construction of pGST-IIK was subcloned into M13mp18 to mutagenize the sequence encoding the cytoplasmic domain of the type II receptor. The mutated inserts were then ligated back into pGEX2T to generate the GST-fusion proteins in E. coli.

Type II TGF-beta Receptor Kinase Domain Expression and Purification

The GST-IIK fusion protein was prepared as described (52). Briefly, 1 ml of an overnight culture of E. coli DH5alpha cells transformed with pGST-IIK was used to inoculate 1 liter of LB medium containing 50 µg/ml ampicillin. The culture was grown to an A600 nm of 1.0, and expression of the fusion protein was induced with 0.2 mM isopropyl-1-thio-beta -D-galactopyranoside. After an additional 5 h, the culture was harvested by centrifugation and the cells, resuspended in 30 ml of NETN (100 mM NaCl, 5 mM EDTA, 20 mM Tris, pH 7.4, 0.5% Nonidet P-40) containing protease inhibitors, were lysed by one freeze-thaw cycle followed by sonication for 2 min. The lysate was centrifuged, and 200 µl of glutathione-Sepharose 4B (Pharmacia) was added to the cleared supernatant and incubated in suspension for 1 h. Following adsorption, the beads were pelleted by centrifugation, washed three times with 30 ml of NETN, and resuspended at a concentration of 50% in NETN.

To express the (His)6-tagged cytoplasmic domain in insect cells, pVL1393-(His)6IIK was cotransfected with PharMingen Baculogold linearized baculovirus DNA into SF9 insect cells. Plaque purification and recombinant virus screening were carried out as described (53). For the production of fusion protein, cells were harvested 48-52 h after infection, pelleted, and lysed by resuspension in insect cell lysis buffer (10 mM HEPES, pH 7.4, 10 mM NaCl, 1 mM EDTA). After 20 min on ice, the suspension was cleared for 10 min in a microcentrifuge. The expressed fusion protein was purified by absorption through its (His)6-sequence using Co2+-chelate affinity chromatography. Briefly, the cleared cell lysate was incubated with Co2+-Sepharose 6B beads in 20 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10% glycerol for 30 min at 4 °C. The beads were washed in the same buffer containing 15 mM imidazole. The adsorbed protein was then eluted in buffer containing 100 mM imidazole and stored at -20 °C.

Phosphotyrosine Western Blotting

GST-IIK bound to glutathione-Sepharose was resuspended and boiled in 2 × sample buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 10% beta -mercaptoethanol, 0.1% bromphenol blue, 1 mM dithiothreitol, 10 mM EDTA), separated by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose was then blocked at 4 °C overnight using TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 1% gelatin (Bio-Rad). The nitrocellulose was washed with TBST, and the anti-phosphotyrosine monoclonal antibody PY20 (Zymed) was added at a final concentration of 1 µg/ml in TBST and incubated for 2 h at room temperature. The blot was washed three times in TBST before addition of an alkaline phosphatase-conjugated goat anti-mouse antibody (Promega) at 0.2 µg/ml in TBST. After 1 h at room temperature, the blot was washed again and tyrosine-phosphorylated proteins were visualized with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Kirkegaard and Perry Laboratories).

Transient Expression and Immunoprecipitation of Myc-tagged Type II Receptor

Plasmid pIIR-myc, which drives the expression of a Myc-tagged human type II receptor (46), was transiently transfected (54) into 293 cells using 25 µg of DNA/10-cm diameter plate. Cells were metabolically labeled using [35S]Cys and [35S]Met, and the 35S-labeled type II receptor was immunoprecipitated as described (46). The anti-Myc monoclonal antibody 9E10 was obtained from Dr. J. M. Bishop (University of California, San Francisco). Anti-phosphotyrosine immunoprecipitations were carried out using the PY20 (Zymed) and 4G10 (Upstate Biotechnology, Inc.) antibodies according to the manufacturers' recommendations.

In Vitro Kinase Assays and Kinase Inhibitor Studies

Ten µl (0.1-0.5 µg) of GST-IIK fusion protein, bound to glutathione-Sepharose and washed in 500 µl of kinase buffer (25 mM HEPES, pH 7.4, 10 mM MnCl2), was used for each reaction. Kinase reactions were carried out for 15 min in a final volume of 30 µl of kinase buffer. When appropriate, histone 2B (Boehringer Mannheim) was added as substrate at a final concentration of 100 ng/µl. The kinase reactions using purified (His)6-IIK protein were carried out similarly. Kinase assays were also carried out on full-length receptors expressed in mammalian cells. 293 cells were transfected with pIIR-myc and lysed, and the receptors were immunoprecipitated 72 h after transfection. The immune complexes absorbed to protein A-Sepharose were then washed with GST kinase buffer (33% glycerol, 0.1% Triton X-100, 25 mM HEPES, pH 7.4, 10 mM MnCl2, 1 mM NaVO4). The kinase reactions were initiated by addition of ATP to 10 µM and 1 µl of [gamma -32P]ATP (DuPont NEN; 3000 Ci/mmol) and allowed to proceed for 5 min at room temperature. Kinase inhibitors (Life Technologies, Inc.) were incorporated when appropriate and used according to the manufacturer's guidelines and as described previously (55). The kinase reactions were terminated by addition of an equal volume of 2 × sample buffer. The samples were boiled for 5 min prior to electrophoretic separation by SDS-PAGE. The gel was then fixed, dried, and autoradiographed.

Phosphoamino Acid Analysis

Kinase reactions were done as described above and the 32P-phosphorylated products were transferred to polyvinylidene difluoride membranes (Bio-Rad) after separation by SDS-PAGE. The reaction products visualized by autoradiography were cut from the membrane and subjected to phosphoamino acid analysis at 100 °C as described (56).

Peptide Mapping and Sequencing

100 µg of affinity-purified (His)6-IIK fusion protein was phosphorylated in vitro as described above in a total volume of 100 µl in the presence of unlabeled ATP at a concentration of 1 mM. In parallel, 1-5 µg of (His)6-IIK fusion protein was autophosphorylated in the presence of [gamma -32P]ATP. After completion of the assay, 400 µl of 50 mM ammonium bicarbonate, 20 mM EDTA containing 1 µg of modified trypsin, which does not undergo autoproteolysis (Promega), was added to the mixture which contained approximately 200,000 cpm of the 32P-labeled reaction product. Digestion was carried out for 2 h at 37 °C as described (57), and the reaction products were separated by SDS-PAGE, transferred to nitrocellulose, and treated with trypsin. To resolve the tryptic peptides by HPLC, the protein digest was loaded onto a reverse phase C18 column (25 cm × 4.6 mm, Vydac, Hesperia CA) and peptides were eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid/water over 1 h. The elution profile of the 32P-labeled peptides was determined by Cerenkov counting of individual fractions. 100 µl of each fraction was dried down and subjected to phosphoamino acid analysis. Peptide peaks that contained phosphotyrosine were sequenced by Edman degradation on a protein sequencer (model 492; Appied Biosystems, Inc., Foster City, CA) to establish their sequence and the location of the phosphorylated tyrosines. These peaks were usually a mixture, but allowed us to establish the sequences of individual peptides. The amount of radioactivity corresponding to each cycle of Edman degradation and comparison with predicted sequences of the tryptic peptides allowed us to assign the phosphorylation to particular amino acids. Finally, the peptides corresponding to the established tyrosine-phosphorylated sequence were synthesized, and their elution positions on the C18 column confirmed the sequence identity of the tyrosine-phosphorylated tryptic peptides.

In Vivo 32P Labeling of the Receptor and Phosphoamino Acid Analysis

293 cells were transiently transfected with plasmid pIIR-myc as described above, and the transfected cells were labeled with 1 mCi/ml of [32P]phosphate in Dulbecco's modified Eagle's medium, 3 g/liter glucose without phosphate, for 12 h. The cells were then lysed, and the type II receptors were immunoprecipitated using the anti-Myc antibody as described above for 35S-labeled receptors. Following SDS-PAGE, the 32P-labeled receptors were transferred to polyvinylidene difluoride membranes, visualized by autoradiography, and processed for phosphoaminoacid analysis as outlined above.

Functional Assays

Plasmid p800Luc contains a TGF-beta -responsive promoter for plasminogen activator inhibitor type I (PAI-1), which controls the expression of luciferase (58). This reporter plasmid was used to score TGF-beta -induced gene expression in transient transfection assays. Plasmid pCAL2, which contains a TGF-beta -responsive cyclin A promoter (-516 to +245), is a reporter plasmid to score TGF-beta -induced growth inhibition (59). Plasmid pRKbeta Gal, which expresses beta -galactosidase under the control of the cytomegalovirus promoter, was used to normalize for transfection efficiency (59).

The reporter assays were performed following transient co-expression of TGF-beta receptor and reporter expression plasmids in Mv1Lu cells as described (59). Briefly, cells (~3 × 106) were harvested and electroporated at 960 microfarads and 750 V/cm in a Bio-Rad electroporation apparatus, typically using 10 µg of p800luc or pCAL2, 10 µg of pRKbeta GAL, 10 µg of receptor expression plasmid(s), and 10 µg of pBluescript II SK+ (carrier DNA). After electroporation, cells were allowed to recover for 4 h in Eagle's minimal essential medium with Earle's BSS supplemented with NEAA and 10% fetal bovine serum. The cells were then treated with or without 10 ng/ml TGF-beta 1 in the same medium but containing 0.2% fetal bovine serum. After 24 h (for PAI-1 assay) or 48 h (for cyclin A assay), the cells were harvested and lysed in reporter lysis buffer (Promega) and cell lysates were assayed for luciferase and beta -galactosidase activities. The luciferase assay was carried out using Analytic Luminescence Laboratory's assay reagents, and beta -galactosidase was assayed in Galacto Light Plus kit (Tropix). Both luciferase and beta -galactosidase activity was measured in luminometer Monolight 2010. The luciferase activity, which reflects the promoter activity of cyclin A or PAI-1, was normalized to beta -galactosidase to account for transfection efficiency.


RESULTS

Expression of the Cytoplasmic Domain of the Type II TGF-beta Receptor in E. coli and Insect Cells

To study the kinase activity of the type II TGF-beta receptor, we expressed its cytoplasmic domain in E. coli and insect cells. In E. coli, the sequence of the C-terminal 374 amino acids of the type II TGF-beta receptor including the kinase domain, was expressed as a fusion to an N-terminal GST segment that confers high affinity of the fusion protein for glutathione-Sepharose (52). The affinity-purified protein consisted of about 90% full-length GST-IIK fusion protein with its degradation products as major contaminants (Fig. 1A).


Fig. 1. Expression of type II TGF-beta receptor cytoplasmic domain in E. coli and baculovirus-infected insect cells. A, expression of GST-IIK, the GST-fused cytoplasmic domain of the type II TGF-beta receptor, in E. coli. Lane 1, total lysate of GST-IIK expressing E. coli; lane 2, affinity-purified GST-IIK; lane 3, affinity-purified GST. B, expression of (His)6-IIK, the cytoplasmic domain of the type II receptor fused to an N-terminal (His)6 tag. Lane 1, total cell lysate; lane 2, affinity-purified (His)6-IIK. Proteins are stained with Coomassie Blue.
[View Larger Version of this Image (73K GIF file)]

The cytoplasmic domain of the type II TGF-beta receptor was also expressed in baculovirus-infected insect cells. For this purpose, we constructed an expression vector encoding the C-terminal 374 amino acids of the type II TGF-beta receptor preceded by an N-terminal (His)6 tag. This fusion protein, (His)6-IIK, was purified from infected insect cell lysates by absorption chromatography based on the high affinity of the (His)6 sequence for Co3+-chelate resin (Fig. 1B).

The two types of purified fusion proteins were used to characterize the kinase activity of the type II receptor in vitro, as discussed below. Whereas both proteins had similar properties, the (His)6-IIK protein from insect cells had a higher specific kinase activity (data not shown). This difference in specific activities is likely due to the fact that a large fraction of the GST-IIK protein expressed in E. coli may have been obtained as an inactive protein in inclusion bodies.

Auto- and Substrate Phosphorylation on Serine, Threonine, and Tyrosine

Initially, the presence of phosphotyrosine in the type II receptor cytoplasmic domain was tested by Western blot analyses using the anti-phosphotyrosine monoclonal antibody PY20 (Zymed). GST-IIK fusion protein reacted with the anti-phosphotyrosine antibody, whereas the degradation products containing the GST sequence did not (Fig. 2, lanes 1). The tyrosine-phosphorylated GST-IIK migrated slightly slower than the non-tyrosine phophorylated protein (data not shown), as often observed with differentially phosphorylated proteins. Since E. coli lacks detectable tyrosine kinase activity, any phosphotyrosine should have resulted from its intrinsic kinase activity. This was confirmed by testing a kinase-inactive version of GST-IIK in which the lysine at position 277 in the ATP binding site was replaced by arginine. This mutated fusion protein prepared in a similar way to GST-IIK was not phosphorylated on tyrosine as assessed by Western blot analysis (Fig. 2, lanes 2), indicating that tyrosine phosphorylation was dependent on the kinase activity of the cytoplasmic domain of the type II receptor.


Fig. 2. The cytoplasmic domain of the type II TGF-beta receptor reacts with anti-phosphotyrosine. A, anti-phosphotyrosine Western blot of total E. coli lysates containing GST-IIK (lane 1) or kinase-inactive GST-IIK in which Lys277 was replaced by Arg (lane 2). Kinase-active GST-IIK, but not kinase-inactive K277R GST-IIK, reacts with anti-phosphotyrosine. B, cell lysates of E. coli expressing GST-IIK (lane 1) or kinase-inactive K277R GST-IIK (lane 2), stained with Coomassie Blue, illustrating equal expression levels of GST-IIK (arrowhead). The results presented in panels A and B were derived using different gels.
[View Larger Version of this Image (59K GIF file)]

Purified GST-IIK protein and baculovirus-derived (His)6-IIK were subjected to in vitro kinase assays in the presence of [gamma -32P]ATP. As illustrated with GST-IIK, the fusion protein displayed kinase activity and autophosphorylated (Fig. 3A, lane 1). Autophosphorylated GST-IIK protein could be immunoprecipitated using anti-phosphotyrosine antibody 4G10 (Fig. 3A, lane 2), further documenting the ability of the cytoplasmic domain to autophosphorylate on tyrosine. GST-IIK or (His)6-IIK, 32P-labeled in autophosphorylation reactions, was subjected to phosphoamino acid analysis. In addition to phosphoserine as predominant phosphoamino acid and phosphothreonine, phosphotyrosine was clearly detected (Fig. 3B), confirming the dual specificity of the type II TGF-beta receptor kinase in vitro.


Fig. 3. In vitro autophosphorylation of the type II receptor cytoplasmic domain produced in E. coli. A, affinity-purified GST-IIK was subjected to in vitro phosphorylation in the presence of [gamma -32P]ATP and separated by SDS-PAGE. 32P-Autophosphorylated GST-IIK is marked with an arrowhead (lane 1), whereas the lower band corresponds to a degradation product of the fusion protein. The 32P-autophosphorylation reaction mixture was then subjected to immunoprecipitation using the 4G10 anti-phosphotyrosine antibody (lane 2) or an anti-hepatitis B surface antigen monoclonal antibody (lane 3). B, 32P-autophosphorylated GST-IIK from lane 1 was subjected to phosphoaminoacid analysis revealing the presence of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y).
[View Larger Version of this Image (60K GIF file)]

Finally, the kinase specificity of the type II TGF-beta receptor cytoplasmic domain was tested using exogenous substrates. Histone 2B, enolase, poly(Glu-Tyr), and casein were efficiently phosphorylated by both the GST-IIK and (His)6-IIK fusion proteins (Fig. 4A), and phosphoamino acid analysis of 32P-labeled histone 2B revealed its phosphorylation primarily on serine but also on threonine and tyrosine (Fig. 4B), a pattern similar to that seen in autophosphorylation reactions.


Fig. 4. In vitro substrate phosphorylation by the type II receptor cytoplasmic domain. A, affinity-purified (His)6IIK was subjected to in vitro kinase reaction in the presence of [gamma -32P]ATP in the absence of exogenous substrate (lane 1, autophosphorylation) or presence of histone 2B (lane 2), enolase (lane 3), poly(Glu-Tyr) ((E,Y)n; lane 4), or casein (lane 5). The 32P-labeled reaction products were separated by SDS-PAGE. The positions of the autophosphorylated receptor cytoplasmic domain (RII) and phosphorylated substrates are marked. B, affinity-purified GST-IIK was 32P-phosphorylated in vitro in the absence (lane 1) or presence (lane 2) of histone 2B as substrate. The position of autophosphorylated GST-IIK (RII) and histone 2B are shown. Histone 2B phosphorylated in vitro by the type II receptor cytoplasmic domain was then subjected to phosphoamino acid analysis (panel 3) (S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine).
[View Larger Version of this Image (43K GIF file)]

The Type II TGF-beta Receptor Immunoprecipitated from Transfected Mammalian Cells Phosphorylates Serine, Threonine, and Tyrosine Residues

The experiments described above indicate that the recombinant type II TGF-beta receptor cytoplasmic domain has dual kinase specificity in vitro. To verify that the full-length type II receptor expressed in mammalian cells had a similar kinase specificity, we performed immunoprecipitations of Myc epitope-tagged type II receptors, transiently expressed in transfected 293 cells. An expression vector for the human type II TGF-beta receptor with the C-terminal epitope tag (46) was transfected into 293 cells. These cells have, based on cross-linking of cell surface receptors with 125I-TGF-beta , low endogenous levels of type II TGF-beta receptors (30). The immunoprecipitated receptor had the expected molecular mass of about 69 kDa (Fig. 5, lane 2) and was autophosphorylated in an in vitro kinase assay in the presence of [gamma -32P]ATP (Fig. 5, lane 4). Similar immunoprecipitations carried out using cells transfected with untagged receptor (Fig. 5, lanes 1 and 3) or a kinase-deficient receptor point mutant (Ref. 60 and data not shown; see below) or using untransfected cells (data not shown) revealed no detectable levels of phosphorylation and demonstrated that the observed kinase activity was due to the immunoprecipitated type II receptor.


Fig. 5. Immunoprecipitation and in vitro autophosphorylation of the type II receptor expressed in 293 cells. Cells were transfected with an expression plasmid for untagged (lanes 1 and 3) or Myc-tagged (lanes 2 and 4) type II receptor. Immunoprecipitations were carried out using an anti-Myc monoclonal antibody. In lanes 1 and 2, the cells were metabolically 35S-labeled prior to immunoprecipitation and SDS-PAGE, whereas in lanes 3 and 4, the cells were unlabeled and subjected to immunoprecipitations followed by in vitro 32P-kinase reaction and SDS-PAGE. RII marks the type II receptor, which has size heterogeneity due to differences in glycosylation (46), and several degradation products including one prominent one.
[View Larger Version of this Image (51K GIF file)]

Phosphoamino acid analysis of autophosphorylated Myc-tagged type II receptor showed the presence of predominantly phosphoserine with less phosphothreonine and phosphotyrosine (Fig. 6, panel 1), a pattern consistent with the activity of the cytoplasmic domain expressed in E. coli or baculovirus-infected cells. Histone 2B could also be phosphorylated by the immunoprecipitated type II receptor kinase. Again, phosphoamino acid analysis showed that the 32P-labeled histone 2B contained phosphorylated serine, threonine and tyrosine (Fig. 6, panel 2), further documenting the dual kinase specificity of the type II TGF-beta receptor.


Fig. 6. Phosphoamino acid analysis of immunoprecipitated Myc-tagged type II receptor subjected to in vitro 32P-autophosphorylation (panel 1), and of histone 2B phosphorylated in vitro by the immunoprecipitated Myc-tagged type II receptor (panel 2) (S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine).
[View Larger Version of this Image (69K GIF file)]

The Type II TGF-beta Receptor Is Phosphorylated on Tyrosine in Vivo

The type II TGF-beta receptor is known to be constitutively autophosphorylated on serine and threonine (21, 22). We thus determined whether the receptor expressed in vivo can also be phosphorylated on tyrosine. The transfected Myc-tagged type II receptor was 32P-labeled in vivo and immunoprecipitated using the tag-specific antibody (Fig. 7, left lane). Phosphoamino acid analysis of the gel-purified type II receptor band revealed the presence of a low level of phosphotyrosine, in addition to the abundant phosphoserine (Fig. 7), thus confirming the dual kinase specificity of the receptor in vivo. Parallel experiments using the kinase-inactive point mutant of the type II receptor expressed in transfected cells, revealed a much lower level of phosphorylation of the receptor band, presumably due to phosphorylation by cytoplasmic kinases (21, 49), resulting in a much lower level of phosphoserine and no detectable tyrosine phosphorylation (data not shown).


Fig. 7. In vivo tyrosine phosphorylation of the type II TGF-beta receptor expressed in transfected 293 cells. The left panel shows the immunoprecipitated, Myc-tagged type II receptor, 32P-labeled in vivo. Phosphoamino acid analysis of in vivo 32P-labeled receptor (right panel) shows the presence of phosphotyrosine (Y), in addition to a large amount of phosphoserine (S).
[View Larger Version of this Image (54K GIF file)]

Sensitivity of the Kinase Activity to Tyrphostin and Other Kinase Inhibitors

The type II receptor kinase was tested for its sensitivity to a panel of kinase inhibitors. The kinase activity of the immunoprecipitated Myc-tagged type II receptor was partially inhibited by staurosporine, an inhibitor of many serine/threonine kinases, and methyl 2,5-dihydroxycinnamate, but not by several other kinase inhibitors, such as genistein and lavendustin A (Fig. 8A). Interestingly, the kinase activity of the type II receptor was strongly inhibited by tyrphostin, a competitive inhibitor of substrate binding to some tyrosine kinases (Fig. 8B). The latter result further supports the finding that the receptor phosphorylates on tyrosine and suggests that, in contrast to most or all serine/threonine kinases, the active site of the enzyme can accommodate a tyrosine as substrate.


Fig. 8. Inhibition of type II receptor kinase activity by various kinase inhibitors (A) and by tyrphostin (B). A, affinity-purified (His)6IIK was subjected to in vitro 32P-autophosphorylation in the presence of the following kinase inhibitors: lane 1, methyl 2,5-dihydroxycinnamate (300 µM); lane 2, lavendustin A (2 µM); lane 3, genistein (180 µM); lane 4, RCAM-lysozyme (2 µM); lane 5, tyrphostin (240 µM); lane 6, hydroxydihydroxylbenzylaminobenzoic acid (0, 3 µM); lane 7, herbimycin A (50 µg/ml); lane 8, bisindolylmaleimide (1 µM); lane 9, H7 (100 µM); lane 10, staurosporine (1 µM); lane 11, HA1004 (100 µM); lane 12, control Me2SO (10%); lane 13, no kinase inhibitors. B, in vitro autophosphorylation of (His)6IIK was carried out in the presence of increasing concentrations of tyrphostin. Lane 1, no inhibitor; lane 2, 1 µM tyrphostin; lane 3, 10 µM tyrphostin; lane 4, 100 µM tyrphostin; lane 5, 1 mM tyrphostin.
[View Larger Version of this Image (41K GIF file)]

Localization of the Phosphorylated Tyrosines in the Type II Receptor Cytoplasmic Domain

To evaluate the biological importance of the autophosphorylation of the type II TGF-beta receptor on tyrosine, we identified the phosphorylated tyrosine residues in the cytoplasmic domain. For this purpose, purified (His)6-IIK protein was autophosphorylated in vitro. Whereas most of the protein was phosphorylated using unlabeled ATP, a fraction was phosphorylated with [gamma -32P]ATP in a separate reaction. The autophosphorylated (His)6-IIK protein was digested with trypsin, and tryptic peptides, obtained following HPLC separation, were assayed for the presence of phosphotyrosine using phosphoamino acid analysis. The three HPLC-fractionated peptide peaks, which contained phosphotyrosine, were then subjected to Edman degradation. As outlined under "Experimental Procedures," the combination of cold and radioactive sequencing, confirmed by the HPLC migration of corresponding chemically synthesized peptides, allowed the localization of the autophosphorylated tyrosines at positions 259, 336, and 424 in individual peptides of the cytoplasmic domain of the receptor (Fig. 9). The presence of phosphoserine and/or phosphothreonine in these phosphoamino acid analyses is due to contaminating phosphorylated peptides.


Fig. 9. Phosphoamino acid analysis and sequence of tryptic peptides. A tryptic digest of autophosphorylated type II receptor cytoplasmic domain was separated by HPLC. Phosphoamino acid analysis was carried out for each peak fraction, and three of them, shown here, were found to contain phosphotyrosine (Y). Edman degradation of these peak fractions revealed the phosphotyrosine containing tryptic peptide sequence shown. Each of these peptides contained a single tyrosine, which therefore corresponds to a phosphotyrosine at the indicated position. However, the phosphotyrosine containing peptide was usually contaminated with another peptide that comigrated on HPLC, which explains the presence of phosphoserine or phosphothreonine, even though some of the sequences shown did not contain serine or threonine. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine.
[View Larger Version of this Image (37K GIF file)]

Effect of Mutagenesis of the Phosphorylated Tyrosines on the Kinase Activity

To assess the effect of tyrosine autophosphorylation of the type II receptor cytoplasmic domain on the kinase activity, we constructed mutants of the GST-IIK fusion protein, in which the three tyrosines that were autophosphorylated in vitro were individually replaced by phenylalanines. Furthermore, we made a mutant GST-IIK protein in which all three tyrosines were replaced by phenylalanine. All mutated fusion proteins, as well as the kinase-defective version of GST-IIK with the Lys to Arg replacement in the ATP binding site, were purified from E. coli lysates. Equal quantities of wild type and mutant GST-IIK proteins were subjected to anti-phosphotyrosine Western blot analysis (Fig. 10A). Consistent with our observations in Fig. 2A, wild type GST-IIK was autophosphorylated on tyrosine (Fig. 10A, lane 1), whereas the kinase-inactive point mutant was not (Fig. 10A, lane 6). The Tyr to Phe mutation at position 336 did not greatly affect tyrosine autophosphorylation (Fig. 10A, lane 3), whereas the Tyr mutations at positions 259 and 424 diminished the reactivity of the GST-IIK fusion protein with anti-phosphotyrosine (Fig. 10A, lanes 2 and 4). Finally, replacement of all three tyrosines by phenylalanines abolished the reactivity with anti-phosphotyrosine (Fig. 10A, lane 5), similarly to the kinase-defective point mutant of GST-IIK (Fig. 10A, lane 6).


Fig. 10. Tyrosine phosphorylation of the type II receptor mutants in which the autophosphorylated tyrosines are replaced by phenylalanines. A, anti-phosphotyrosine Western blot of affinity-purified GST-IIK fusion proteins mutated at specific residues and expressed in E. coli. Lane 1, wild type GST-IIK; lane 2, Y259F mutation of GST-IIK; lane 3, Y336F mutation of GST-IIK; lane 4, Y424F mutation of GST-IIK; lane 5, triple mutation Y259F/Y336F/Y424F in GST-IIK; lane 6, K277R kinase-inactive mutant of GST-IIK. The top panel shows the anti-phosphotyrosine Western blot, whereas the lower panel illustrates equal loading of the fusion proteins stained with Coomassie Blue. B, in vitro 32P-autophosphorylation of affinity-purified GST-IIK fusion proteins mutated at specific positions. Equivalent amounts of purified fusion proteins were subjected to an in vitro kinase reaction in the presence of [gamma -32P]ATP prior to gel electrophoresis. The lanes contain the specific GST-IIK mutations as in A. C, expression of full-size type II receptors mutated at specific residues in transfected 293 cells and in vitro 32P-autophosphorylation. The Myc-tagged type II receptor was immunoprecipitated from transfected cells and subjected to in vitro kinase reaction prior to SDS-PAGE. The multiple bands reflect heterogeneity in glycosylation and degradation products (the full-size receptor is marked with an arrowhead). Western blot using the anti-Myc epitope antibody shows approximate quantitation of the expressed receptors in the total cell lysate. Only the full-size receptor reacts with the antibody. The lanes and specific mutations in the cytoplasmic domains of the receptors are as shown in A.
[View Larger Version of this Image (28K GIF file)]

This decreased tyrosine phosphorylation could in principle be due to a specific decrease in the number of tyrosine phosphorylation sites resulting from the mutation of the tyrosine while maintaining the kinase activity, or could result from a generally impaired kinase activity, which is largely on serine and threonine. To distinguish between these possibilities, we evaluated the kinase activity of the different GST-IIK proteins in autophosphorylation assays in the presence of [gamma -32P]ATP (Fig. 10B). The single tyrosine mutation at position 336 did not affect the kinase activity of the cytoplasmic domain. In contrast, the single mutations at positions 259 and 424 decreased the kinase activity, which is consistent with the result of the anti-phosphotyrosine Western blot, and the triple tyrosine mutation resulted in a greatly impaired kinase activity similar to the Lys to Arg mutation in the kinase-defective GST-IIK (Fig. 10B). Finally, to verify the effect of these mutations on the kinase activity of the type II receptor made by mammalian cells, we expressed the wild type and the point-mutated kinase-defective type II receptor as well as the mutated receptor with the three tyrosines replaced by phenylalanine in 293 cells. In vitro autophosphorylation assays of the immunoprecipitated receptors confirmed that the receptor with the triple tyrosine mutation did not have detectable kinase activity, similarly to the kinase-inactive point mutant (Fig. 10C, lanes 5 and 6). Whereas the mutation of Tyr259 decreased receptor autophosphorylation (Fig. 10C, lane 2) consistent with the results using the E. coli-derived cytoplasmic domain, we did not observe a major decrease in kinase activity as a result of the Tyr424 mutation (Fig. 10C, lane 4). The basis of the discrepancy between the latter result and the decreased activity resulting from the same mutation in the E. coli-derived fusion protein is unclear. Finally, we were unable to efficiently express the Tyr336-mutated receptor in 293 cells (Fig. 10C, lane 3). In summary, the results obtained using fusion proteins produced in E. coli suggest that autophosphorylation on Tyr259 and Tyr424 is functionally important for the kinase activity of the type II TGF-beta receptor. The results using full-size receptors expressed in mammalian cells support this notion but are not totally in agreement, in part due to the discrepancy with the Tyr424 mutation and due to the influence of these point mutations on the expression levels.

Role of Tyrosine Autophosphorylation in Type II Receptor Signaling

To evaluate the role of tyrosine autophosphorylation of the type II TGF-beta receptor in vivo, we first evaluated the effect of tyrphostin on TGF-beta signaling in Mv1Lu cells that have endogenous type II and type I receptors. As a measure of the TGF-beta signaling ability, we used the PAI-1 luciferase reporter assay in which the luciferase gene is expressed under control of the TGF-beta -inducible PAI-1 promoter (58). As is apparent from Fig. 11, the TGF-beta -induced expression from the PAI-1 reporter was strongly inhibited in a dose-dependent way by increasing concentrations of tyrphostin. This inhibition of signaling by the receptors is consistent with the inhibition of the type II receptor kinase by tyrphostin in vitro (Fig. 8).


Fig. 11. Effect of tyrphostin on PAI-1 luciferase expression. Mink lung epithelial cells lacking the type II TGF-beta receptor (Mv1Lu-DR26 cells) were transfected with reporter plasmids p800Luc and pRKbeta Gal and treated with the indicated concentrations of tyrphostin in the presence or absence of TGF-beta . Twenty-four hours later, cells were lysed and the luciferase activities were measured and normalized for beta -galactosidase activity. The data were obtained in two identical experiments with each point determined in triplicate. The vertical bars show the standard deviations.
[View Larger Version of this Image (22K GIF file)]

We also evaluated the ability of the triple mutated type II receptor, in which the three tyrosine targets of autophosphorylation were replaced by phenylalanine, in comparison with the wild type type II receptor. Thus, expression plasmids for both receptors were transiently transfected at two concentrations (0.5 or 5 µg) in DR26 mutant Mv1Lu cells, which lack type II receptors. To measure TGF-beta responsiveness in these transient transfection assays, we used both the PAI-1 luciferase assay which scores TGF-beta -induced gene expression (58), and the cyclin A reporter assay in which decreased luciferase expression from the cyclin A promoter correlates with growth inhibition (59). As shown in Fig. 12, transfection of wild type type II receptor restored TGF-beta responsiveness in Mv1Lu-DR26 cells in both assays, and the response was somewhat higher at 5 µg of transfected plasmid than at 0.5 µg. Surprisingly, the triple Tyr mutant of the type II receptor also allowed TGF-beta signaling as assessed in both assays, although the PAI-1 response at 5 µg of transfected plasmid was reproducibly lower than for wild type receptor, and the cyclin A response of the triple mutant was lower at 0.5 µg of transfected plasmid.


Fig. 12. Signaling activities of wild type and mutant type II receptor with three tyrosines replaced by phenylalanine. Mv1Lu-DR26 cells lacking endogenous functional type II receptor were transfected with the reporter plasmids p800Luc or pCAL2, and pRKbeta Gal, and treated with the indicated concentrations of TGF-beta . The luciferase activities were determined as in Fig. 11. A, PAI-1-luciferase expression assay. B, cyclin A-luciferase expression assay.
[View Larger Version of this Image (30K GIF file)]


DISCUSSION

The receptors for TGF-beta and related proteins constitute a distinct family of transmembrane proteins with predicted serine/threonine kinase activity (reviewed in Refs. 12 and 13). The experiments described here demonstrate that the type II TGF-beta receptor phosphorylates not only on serine and threonine but also on tyrosine residues, and should therefore be considered as a dual specificity kinase. The evidence for this conclusion is obtained from a variety of experiments. The kinase domain expressed as a fusion protein in E. coli or in insect cells autophosphorylates not only on serine and threonine but also on tyrosine. Because E. coli does not have detectable intrinsic tyrosine kinase activity and since an inactivated point mutant of the kinase did not react with anti-phosphotyrosine antibody, we conclude that the tyrosine phosphorylation resulted from autophosphorylation of the receptor kinase. In addition, the cytoplasmic domain phosphorylates exogenous substrates such as histone 2B on serine, threonine, and tyrosine. We also showed that the receptor immunoprecipitated from transfected mammalian cells phosphorylates on serine, threonine, and tyrosine residues, similarly to the cytoplasmic domain expressed in E. coli or insect cells. Furthermore, phosphoamino acid analysis of the in vivo 32P-labeled, transfected receptor revealed phosphoserine and a small amount of phosphotyrosine, thus supporting the significance of this dual kinase specificity in vivo. This phosphorylation did not require the presence of ligand (data not shown), which is in agreement with the constitutive activity and autophosphorylation of the type II TGF-beta receptor (21, 22). Finally, the kinase activity of the receptor can be inhibited by tyrphostin, a competitive inhibitor of tyrosine phosphorylation, suggesting that this kinase, unlike standard serine/threonine kinases, can accommodate a tyrosine residue as substrate at its active site.

The amino acid sequences of the kinases of the TGF-beta receptor family predict, based on specific motifs (18, 19, 50, 61), a kinase specificity for serine and threonine. Accordingly, previous results using recombinant receptor proteins have demonstrated that the type II receptors for TGF-beta or activin as well as the Tbeta RI/ALK-5/R4 type I receptor autophosphorylate on serine and threonine (19-22, 49). Whereas phosphothreonine is the major phosphorylated amino acid following in vitro assays (19), the type II TGF-beta receptor autophosphorylates in vivo primarily on serine (21, 22). Interestingly, the endogenous activin receptor purified from cells has been reported to have dual kinase specificity (51), but this was not confirmed in a study using the cloned type II activin receptor (49). The results described here demonstrate that the type II TGF-beta receptor is indeed a dual specificity kinase. This discrepancy between the current and previous results is probably due to the low levels of phosphotyrosine compared with phosphoserine and phosphothreonine, combined with the lability of phosphotyrosine at high temperatures during the hydrolysis reaction of the phosphorylated cytoplasmic domain. Accordingly, we performed the hydrolyses for the phosphoamino acid analyses at 100 °C instead of the more standard 110 °C. Furthermore, the ability of the receptor kinase to autophosphorylate on tyrosine in vitro may be considerably attenuated because the tyrosines are already phosphorylated when expressed in E. coli, insect cells, or mammalian cells.

A number of reports have suggested or demonstrated the dual specificity of various kinases (Refs. 62 and 63; reviewed in Ref. 64). In general, their levels of tyrosine phosphorylation are low compared with serine and threonine, and dual specificity is usually only demonstrated in autophosphorylation reactions. Indeed, an in vivo function for dual kinase specificity has to date only been demonstrated for the MAP kinase kinases (3, 4). Based on their primary structure, dual specificity kinases appear to be indistinguishable from serine/threonine kinases and map throughout the kinase family tree (50). Accordingly, the type II and type I receptors for TGF-beta superfamily members have been classified as serine/threonine kinase receptors based on their sequence and kinase activity (19, 20, 49). However, they also show similarity in their kinase domains with tyrosine kinases (50). For example, a CW motif in subdomain XI that is highly conserved in tyrosine kinases is also conserved in the type II and type I receptors. The current report, together with the sequence conservation of the kinase domains, suggests that perhaps all these receptors are dual specificity kinases with the ability to autophosphorylate on tyrosine(s).

Sequencing of tryptic phosphopeptides led to the localization of three autophosphorylated tyrosines in the type II TGF-beta receptor: Tyr259 in kinase subdomain I, Tyr336 in subdomain V, and Tyr424 in subdomain VIII. Tyr259 is conserved among the type II TGF-beta receptors from different species, but not among other type II receptors (data not shown). Replacement of this tyrosine by phenylalanine decreased the kinase activity of the cytoplasmic domain expressed in E. coli, but not in mammalian cells. The basis for this reproducible discrepancy is unclear. This tyrosine is in the ATP-binding site of the kinase, and phosphorylation of residues in this region is known to be an important factor in the inhibition of kinase activity of cyclin-dependent kinases (65, 66). Tyr336 is well conserved among not only the different type II but also the type I receptors. In addition to tyrosine, phenylalanine is also found in the corresponding position in other type II receptors. Accordingly, replacement of Tyr336 by phenylalanine did not affect the kinase activity of the type II TGF-beta receptor. Finally, Tyr424 is absolutely conserved in all type II and type I receptors characterized so far. Its replacement by phenylalanine strongly decreased the kinase activity of the type II receptor. This tyrosine is located two amino acids upstream from the signature sequence APE in kinase subdomain VIII. The sequence between subdomain VII and the APE sequence represents a target for regulatory phosphorylations in several kinases and can function as an activation loop (67). For example, phosphorylation of Thr197 in this loop of protein kinase A (68) and Thr183 and Tyr185 in the corresponding sequence in MAP kinase (69) contribute to activation of these kinases. Based on structural information, phosphorylation of this loop alters the conformation and increases the activity of the kinase (70, 71). Similarly, the dual specificity kinase glycogen synthetase kinase 3 undergoes tyrosine autophosphorylation on the corresponding residue in subdomain VIII and this phosphorylation enhances its kinase activity (72). Thus, for both the MAP kinases and for glycogen synthetase kinase, tyrosine phosphorylation upstream from the APE sequence enhances the kinase activity and plays an autoregulatory role. Likewise, the conserved Tyr424 is located closely upstream from the APE motif in subdomain VIII of the type II TGF-beta receptor, and its replacement by phenylalanine strongly inhibits the kinase activity. Taken together, the autophosphorylation of the tyrosines in the kinase domain of the type II TGF-beta receptor, and possibly all serine/threonine kinase receptors, illustrates the dual specificity of the kinase activity and suggests an autoregulatory role similar to that seen in various other kinases. The putative regulatory role of these tyrosines may explain the strong inhibition of replacement of all three tyrosines on the kinase activity of this receptor.

Whereas the ability of the type II receptor to autophosphorylate on tyrosines is clearly illustrated and the tyrosine to phenylalanine mutations inhibit the kinase activity in vitro, the role of the tyrosine autophosphorylation is less unambiguous. Clearly, the inhibition of the signaling activity by tyrphostin is consistent with the in vitro inhibitory effect on the kinase activity of the type II receptor. However, the triple tyrosine mutant of the type II receptor has signaling activity in reporter assays, and this activity is only slightly lower than that of the wild type receptor. This result thus indicates that the triple mutated type II receptor is biologically active. However, these data have to be interpreted with caution, since a primary role of the type II receptor is to phosphorylate and activate the type I receptor, which has an effector role in signaling. Therefore, a low level of activity of the type II receptor may be sufficient to allow signaling by the heteromeric receptor complex. In addition, we tested only two responses, and other responses may show a greater sensitivity to the impaired type II receptor kinase activity.

By analogy with tyrosine kinase receptors (2), the tyrosine autophosphorylation also raises the possibility that, in addition to an autoregulatory role of the kinase activity, the sites of tyrosine phosphorylation may also act as docking sites for signaling proteins. However, this is unlikely since the three tyrosine phosphorylation sites are located in functional kinase domains and not in flanking domains or inserts as is the case for the known tyrosine kinase receptor docking sites. Another possibility is that the type II and/or type I receptor phosphorylate target proteins on tyrosine. However, TRIP-1, which can associate with the type II receptor in vivo, is only phosphorylated on serine and threonine (60). Similarly, Smad2 and Smad3, downstream mediators of TGF-beta signaling, which associate with the heteromeric receptor complex, are also serine- and threonine-phosphorylated (73, 74). Future studies will have to determine whether any substrate proteins are tyrosine-phosphorylated in vivo by the type II TGF-beta receptor or by related receptors and will hopefully reveal the biological significance of the tyrosine autophosphorylation in the regulation of receptor activity in vivo.


FOOTNOTES

*   This work was supported by Grant CA63101 from the NCI, National Institutes of Health (to R. D.), by postdoctoral fellowships from the Tobacco-Related Diseases Program and the Leukemia Society of America (to R.-H. C.) and the American Cancer Society (to X.-H. F.), and by a National Institutes of Health postdoctoral training grant of the Cardiovascular Research Institute (to I. G.-P.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Current address: MRC Protein Phosphorylation Unit, Biochemistry Dept., Dundee University, Dundee DD1 4HN, Scotland.
§   Current address: Institute of Molecular Medicine, National Taiwan University, School of Medicine, Taipei, Taiwan.
par    To whom correspondence should be addressed: Dept. of Growth and Development, University of California, San Francisco, CA 94143-0640. Tel.: 415-476-7322; Fax: 415-476-1499; E-mail: derynck{at}itsa.ucsf.edu.
1   The abbreviations used are: MAP, mitogen-activated protein; TGF-beta , transforming growth factor-beta ; GST, glutathione S-transferase; PCR, polymerase chain reaction; TBST, Tris-buffered saline with Tween 20; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.
2   S. Lawler, X.-H. Feng, R.-H. Chen, E. M. Maruoka, C. W. Turck, I. Griswold-Prenner, and R. Derynck, unpublished observations.

REFERENCES

  1. Fantl, W., Johnson, D. E., and Williams, L. T. (1993) Annu. Rev. Biochem. 62, 453-481 [CrossRef][Medline] [Order article via Infotrieve]
  2. Schlessinger, J., and Ullrich, A. (1994) Neuron 9, 383-391
  3. Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akuyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982 [Abstract]
  4. Seger, R., Ahn, D. G., Posada, J., Munar, E. S., Jensen, A. M., Cooper, J. A., Cobb, M. H., and Krebs, E. G. (1992) J. Biol. Chem. 267, 14373-14381 [Abstract/Free Full Text]
  5. Roberts, A. B., and Sporn, M. B. (1990) in Peptide Growth Factors and Their Receptors (Sporn, M. B., and Roberts, A. B., eds), pp. 419-472, Springer-Verlag, New York
  6. Roberts, A. B., and Sporn, M. B. (1993) Growth Factors 8, 1-9 [Medline] [Order article via Infotrieve]
  7. Derynck, R. (1994) in The Cytokine Handbook (Thompson, A., ed), pp. 319-342, Academic Press, Boston
  8. Kingsley, D. M. (1994) Genes Dev. 8, 133-146 [CrossRef][Medline] [Order article via Infotrieve]
  9. Derynck, R., Lindquist, P. B., Lee, A., Wen, D., Tamm, J., Graycar, J. L., Rhee, L., Mason, A. J., Miller, D. A., Coffey, R. J., Moses, H. L., and Chen, E. Y. (1988) EMBO J. 7, 3737-3743 [Abstract]
  10. ten Dijke, P., Hanson, P., Iwata, K. K., Pieler, C., and Foulkes, J. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 82, 4715-4719
  11. Derynck, R. (1994) Trends Biochem. Sci. 19, 548-553 [CrossRef][Medline] [Order article via Infotrieve]
  12. ten Dijke, P., Miyazono, K., and Heldin, C.-H. (1996) Curr. Opin. Cell Biol. 8, 139-145 [CrossRef][Medline] [Order article via Infotrieve]
  13. Massagué, J. (1996) Cell 85, 947-950 [Medline] [Order article via Infotrieve]
  14. Laiho, M., Weis, F. M. B., and Massagué, J (1990) J. Biol. Chem. 265, 18518-18524 [Abstract/Free Full Text]
  15. 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]
  16. Lopez-Casillas, F., Wrana, J. L., and Massagué, J. (1993) Cell 67, 785-795
  17. Moustakas, A., Lin, H. Y., Henis, Y. I., Plamondon, J., O'Connor-McCourt, M. D., and Lodish, H. F. (1993) J. Biol. Chem. 268, 22215-22218 [Abstract/Free Full Text]
  18. Mathews, L. S., and Vale, W. W. (1991) Cell 65, 973-982 [Medline] [Order article via Infotrieve]
  19. Lin, H. Y., Wang, X.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. M. (1992) Cell 68, 775-785 [Medline] [Order article via Infotrieve]
  20. 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]
  21. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massagué, J. (1994) Nature 370, 341-347 [CrossRef][Medline] [Order article via Infotrieve]
  22. Chen, F., and Weinberg, R. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1565-1569 [Abstract]
  23. Mathews, L. S., Vale, W. W., and Kintner, C. R. (1992) Science 255, 1702-1703 [Medline] [Order article via Infotrieve]
  24. Attisano, L., Wrana, J. L., Cheifetz, S., and Massagué, J. (1992) Cell 68, 97-108 [Medline] [Order article via Infotrieve]
  25. Estevez, M., Attisano, L., Wrana, J. L., Albert, P. S., and Massagué, J. (1994) Nature 365, 644-649
  26. Liu, F., Ventura, F., Doody, J., and Massagué, J. (1995) Mol. Cell. Biol. 15, 3479-3486 [Abstract]
  27. Rosenzweig, B. L., Imamura, T., Okadome, T., Cox, G. N., Yamashita, H., ten Dijke, P., Heldin, C.-H., and Miyazono, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7632-7636 [Abstract]
  28. Letsou, A., Arora, K., Wrana, J. L., Simin, K., Twombly, V., Jamal, J., Staehling-Hampton, K., Hoffman, F. M., Gelbart, W. M., Massagué, J., and O'Connor, M. B. (1995) Cell 80, 899-908 [Medline] [Order article via Infotrieve]
  29. Ruberte, E., Marty, T., Nellen, D., Affolter, M., and Basler, K. (1995) Cell 80, 889-897 [Medline] [Order article via Infotrieve]
  30. Ebner, R., Chen, R.-H., Shum, L., Lawler, S., Zionchek, T. F., Lee, A., Lopez, A. R., and Derynck, R. (1993) Science 260, 1344-1348 [Medline] [Order article via Infotrieve]
  31. Attisano, L., Carcámo, J., Ventura, F., Weis, F. M. B., Massagué, J., and Wrana, J. L. (1993) Cell 75, 671-680 [Medline] [Order article via Infotrieve]
  32. Franzén, P., ten Dijke, P., Ichijo, H., Heldin, C.-H., and Miyazono, K. (1993) Cell 75, 681-692 [Medline] [Order article via Infotrieve]
  33. Wrana, J. L., Tram, H., Attisano, L., Arora, K., Childs, S. R., Massagué, J., and O'Connor, M. B. (1994) Mol. Cell. Biol. 94, 944-950
  34. 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]
  35. Brummel, T. J., Twombly, V., Marques, G., Wrana, J. L., Newfeld, S. J., Attisano, L., Massagué, J., O'Connor, M. B., and Gelbart, W. M. (1994) Cell 78, 251-261 [Medline] [Order article via Infotrieve]
  36. Nellen, D., Affolter, M., and Basler, K. (1994) Cell 78, 225-237 [Medline] [Order article via Infotrieve]
  37. Penton, A., Chen, Y., Staehling-Hampton, K., Wrana, J. L., Attisano, L., Szidonya, J., Cassill, J., Massagué, J., and Hoffman, F. M. (1994) Cell 78, 239-250 [Medline] [Order article via Infotrieve]
  38. Xie, T., Finelli, A. L., and Padgett, R. W. (1994) Science 263, 1756-1759 [Medline] [Order article via Infotrieve]
  39. Ebner, R., Chen, R.-H., Lawler, S., Zionchek, T., and Derynck, R. (1993) Science 262, 900-902 [Medline] [Order article via Infotrieve]
  40. Wrana, J. L., Attisano, L., Carcamo, J., Zentalla, A., Doody, J., Laiho, M., Wang, X.-F., and Massagué, J. (1992) Cell 71, 1003-1014 [Medline] [Order article via Infotrieve]
  41. Wieser, R., Attisano, L., Wrana, J. L., and Massagué, J. (1993) Mol. Cell. Biol. 13, 7239-7247 [Abstract]
  42. Okadome, T., Yamashita, H., Franzén, P., Morén, A., Heldin, C.-H., and Miyazono, K. (1994) J. Biol. Chem. 269, 30753-30756 [Abstract/Free Full Text]
  43. Feng, X.-H., Filvaroff, E. H., and Derynck, R. (1995) J. Biol. Chem. 270, 24237-24245 [Abstract/Free Full Text]
  44. Chen, R.-H., Moses, H. L., Maruoka, E. M., Derynck, R., and Kawabata, M. (1995) J. Biol. Chem. 270, 12235-12241 [Abstract/Free Full Text]
  45. Henis, Y. I., Moustakas, A., Lin, H. Y., and Lodish, H. F. (1994) J. Cell Biol. 126, 139-154 [Abstract]
  46. Chen, R.-H., and Derynck, R. (1994) J. Biol. Chem. 269, 22868-22874 [Abstract/Free Full Text]
  47. Yamashita, H., ten Dijke, P., Franzén, P., Miyazono, K., and Heldin, C.-H. (1994) J. Biol. Chem. 269, 20172-20178 [Abstract/Free Full Text]
  48. Weis-Garcia, F. M. B., and Massagué, J. (1996) EMBO J. 15, 276-289 [Abstract]
  49. Mathews, L. S., and Vale, W. W. (1993) J. Biol. Chem. 268, 19013-19018 [Abstract/Free Full Text]
  50. Hanks, S. K., and Hunter, T. (1995) FASEB J. 9, 576-596 [Abstract/Free Full Text]
  51. Nakamura, T., Sugino, K., Kurosawa, N., Sawai, M., Takio, K., Eto, Y., Iwashita, S., Muramatsu, M., Titani, K., and Sugino, H. (1992) J. Biol. Chem. 267, 18924-18928 [Abstract/Free Full Text]
  52. Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67, 31-40 [CrossRef][Medline] [Order article via Infotrieve]
  53. O'Reilly, D. R,., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman and Co., New York
  54. Gorman, C., Padmanabhan, R., and Howard, B. H. (1983) Science 221, 551-553 [Medline] [Order article via Infotrieve]
  55. Shum, L., Reeves, S. A., Kuo, A. C., Fromer, E. S., and Derynck, R. (1994) J. Cell Biol. 125, 903-916 [Abstract]
  56. Kamps, M. A, and Sefton, B. M. (1988) Anal. Biochem. 176, 22
  57. van der Geer, P., Lao, K., Sefton, B. M., and Hunter, T. (1993) in Protein Phosphorylation: A Practical Approach (Hardie, G. D., ed), pp. 31-59, Oxford University Press, Oxford
  58. Keeton, M. R., Curriden, S. A., van Zonneveld, A. J., and Loskutoff, D. J. (1991) J. Biol. Chem. 266, 23048-23052 [Abstract/Free Full Text]
  59. Deleted in proofDeleted in proof
  60. Chen, R.-H., Miettinen, P. J., Maruoka, E. M., Choy, L., and Derynck, R. (1995) Nature 377, 548-552 [CrossRef][Medline] [Order article via Infotrieve]
  61. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42-52 [Medline] [Order article via Infotrieve]
  62. Duncan, P. I., Howell, B. W., Marius, R. M., Drmanic, S., Douville, E. M. J., and Bell, J. C. (1995) J. Biol. Chem. 270, 21524-21531 [Abstract/Free Full Text]
  63. Lauze, E., Stoelcker, B., Luca, F. C., Weiss, E., Schultz, A. R., and Winey, M. (1995) EMBO J. 14, 1655-1663 [Abstract]
  64. Lindberg, R. A., Quinn, A. M., and Hunter, T. (1992) Trends Biochem. Sci. 17, 114-119 [CrossRef][Medline] [Order article via Infotrieve]
  65. Atherton-Fessler, S., Liu, F., Gabrielli, B., Lee, M. S., Peng, C.-Y., and Piwnica-Worms, H. (1994) Mol. Biol. Cell 5, 989-1001 [Abstract]
  66. Kornbluth, S., Sebastian, B., Hunter, T., and Newport, J. (1994) Mol. Biol. Cell 5, 273-282 [Abstract]
  67. Taylor, S. S., and Radzio-Andzelm, E. (1994) Structure 2, 345-355 [Medline] [Order article via Infotrieve]
  68. Steinberg, R. A., Cauthron, R. D., Symcox, M. M., and Shuntoh, H. (1993) Mol. Cell. Biol. 13, 2332-2341 [Abstract]
  69. Payne, D. M., Rossamondo, A. J., Martino, P., Erickson, A. K., Her, J.-H., Shananowitz, J., Hunt, D. F., Weber, M. J., and Sturgill, T. W. (1991) EMBO J. 10, 885-892 [Abstract]
  70. Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N.-H., Taylor, S. S., and Sowadski, J. M. (1991) Science 253, 407-414 [Medline] [Order article via Infotrieve]
  71. Zhang, J., Zhang, F., Ebert, D., Cobb, M. H., and Goldsmith, E. J. (1995) Structure 3, 299-307 [Abstract]
  72. Wang, Q. M., Fiol, C. J., DePaoli-Roach, A. A., and Roach, P. J. (1994) J. Biol. Chem. 269, 14566-14574 [Abstract/Free Full Text]
  73. Zhang, Y., Feng, X.-H., Wu, R.-Y., and Derynck, R. (1996) Nature 383, 168-172 [CrossRef][Medline] [Order article via Infotrieve]
  74. Macias-Siva, M., Abdollah, S., Hoodless, P., Pirone, R., Attisano, L., and Wrana, J. L. (1996) Cell 87, 1215-1224 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.