(Received for publication, January 23, 1997, and in revised form, March 26, 1997)
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
The type I and type II receptors for transforming
growth factor- (TGF-
) 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-
signaling, we characterized the kinase activity of the type II TGF-
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-
receptor can function as a dual
specificity kinase and suggest a role for tyrosine autophosphorylation
in TGF-
receptor signaling.
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- (TGF-
) induces an antiproliferative effect in many cell
types, including epithelial, endothelial and hematopoietic cells
(5-7). In addition to its growth modulatory activity, TGF-
has a
wide range of effects on extracellular matrix synthesis, cell-substrate
adhesion, cell differentiation, and migration (5-8). TGF-
, which
exists as three isoforms encoded by separate genes (9, 10), is
considered a prototype for the many structurally related members of the
TGF-
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-, 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-
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-
responses and
that both receptor types are required for full responsiveness to
TGF-
(11-15). The type III receptor, also known as betaglycan, is
not required for TGF-
signaling but may contribute to presentation of the ligand to the type II receptor (16, 17).
The type I and type II TGF- 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-
type II receptor, several other type II
receptors for TGF-
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-
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-
(32, 34).
The type I and type II receptors cooperate in signal transduction, and
both receptor types are required for full responsiveness to TGF-
(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-
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-
and related
factors.
To gain insight into the initial events in the signaling of TGF- and
related factors, we have further characterized the kinase activity of
the type II TGF-
receptor. Using the cloned type II TGF-
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-
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-
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-
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.
Plasmid pGST-IIK was
designed to express in Escherichia coli the C-terminal 374 amino acids of the type II TGF- receptor cytoplasmic domain as a
glutathione S-transferase (GST) fusion protein. The
corresponding coding region of the human type II TGF-
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- 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-The GST-IIK fusion protein was prepared as described
(52). Briefly, 1 ml of an overnight culture of E. coli
DH5 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-
-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.
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%
-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).
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 StudiesTen
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
[-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.
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 Sequencing100 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
[-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.
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 AssaysPlasmid p800Luc contains a
TGF--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-
-induced gene expression in
transient transfection assays. Plasmid pCAL2, which contains a
TGF-
-responsive cyclin A promoter (
516 to +245), is a reporter
plasmid to score TGF-
-induced growth inhibition (59). Plasmid
pRK
Gal, which expresses
-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- 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 pRK
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-
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
-galactosidase activities. The luciferase assay was carried out
using Analytic Luminescence Laboratory's assay reagents, and
-galactosidase was assayed in Galacto Light Plus kit (Tropix). Both
luciferase and
-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
-galactosidase to
account for transfection efficiency.
To study the kinase activity
of the type II TGF- 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-
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).
The cytoplasmic domain of the type II TGF- 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-
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 TyrosineInitially, 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.
Purified GST-IIK protein and baculovirus-derived (His)6-IIK
were subjected to in vitro kinase assays in the presence of
[-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-
receptor kinase in vitro.
Finally, the kinase specificity of the type II TGF- 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.
The Type II TGF-
The experiments described above indicate that the
recombinant type II TGF- 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-
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-
, low endogenous levels of type II TGF-
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 [
-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.
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- receptor.
The Type II TGF-
The type II TGF- 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).
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.
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- 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
[
-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.
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).
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 [-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-
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.
To evaluate the role of tyrosine autophosphorylation of
the type II TGF- receptor in vivo, we first evaluated the
effect of tyrphostin on TGF-
signaling in Mv1Lu cells that have
endogenous type II and type I receptors. As a measure of the TGF-
signaling ability, we used the PAI-1 luciferase reporter assay in which the luciferase gene is expressed under control of the TGF-
-inducible PAI-1 promoter (58). As is apparent from Fig. 11, the
TGF-
-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).
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- responsiveness
in these transient transfection assays, we used both the PAI-1
luciferase assay which scores TGF-
-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-
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-
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.
The receptors for TGF- 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-
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-
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- 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-
or activin as well as the T
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-
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-
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-
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- receptor: Tyr259 in kinase subdomain I, Tyr336 in
subdomain V, and Tyr424 in subdomain VIII.
Tyr259 is conserved among the type II TGF-
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-
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-
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-
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- 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-
receptor or by related
receptors and will hopefully reveal the biological significance of the
tyrosine autophosphorylation in the regulation of receptor activity
in vivo.