(Received for publication, August 21, 1995; and in revised form, November 20, 1995)
From the
Proteins in the transforming growth factor- (TGF-
)
superfamily exert their effects by forming heteromeric complexes of
their type I and type II serine/threonine kinase receptors. The type I
and type II receptors form distinct subgroups in the serine/threonine
kinase receptor family based on the sequences of the kinase domains and
the presence of a highly conserved region called the GS domain (or type
I box) located just N-terminal to the kinase domain in the type I
receptors. Recent studies have revealed that upon TGF-
binding
several serine and threonine residues in the GS domain of TGF-
type I receptor (T
R-I) are phosphorylated by TGF-
type II
receptor (T
R-II) and that the phosphorylation of GS domain is
essential for TGF-
signaling. Here we investigated the role of
cytoplasmic juxtamembrane region located between the transmembrane
domain and the GS domain of T
R-I by mutational analyses using
mutant mink lung epithelial cells, which lack endogenous T
R-I.
Upon transfection, wild-type T
R-I restored the TGF-
signals
for growth inhibition and production of plasminogen activator
inhibitor-1 (PAI-1) and fibronectin. A deletion mutant,
T
R-I/JD1(
150-181), which lacks the juxtamembrane region
preceding the GS domain, bound TGF-
in concert with T
R-II and
transduced a signal leading to production of PAI-1 but not growth
inhibition. Recombinant receptors with mutations that change serine 172
to alanine (S172A) or threonine 176 to valine (T176V) were similar to
wild-type T
R-I in their abilities to bind TGF-
, formed
complexes with T
R-II, and transduced a signal for PAI-1 and
fibronectin. Similar to T
R-I/JD1(
150-181), however,
these missense mutant receptors were impaired to mediate a growth
inhibitory signal. These observations indicate that serine 172 and
threonine 176 of T
R-I are dispensable for extracellular matrix
protein production but essential to the growth inhibition by TGF-
.
The cell growth and differentiation in a multicellular organism
are critically regulated by members of transforming growth factor-
(TGF-
) (
)superfamily including TGF-
,
activin/inhibin, bone morphogenetic protein (BMP),
Müllerian inhibiting substance, and glial cell
line-derived neurotrophic factor. TGF-
is a prototype in this
superfamily of structurally related molecules and regulates cell
proliferation, extracellular matrix formation, migration, adhesion, and
many other cellular functions important for development and homeostasis
(reviewed in (1, 2, 3, 4) ).
Certain members of the TGF- superfamily exert their biological
actions through heteromeric complexes of two types (type I and type II)
of transmembrane receptors with a serine/threonine kinase domain in
their cytoplasmic
region(5, 6, 7, 8) . To date, more
than 15 receptor serine/threonine kinases have been cloned in flies
through humans (reviewed in (4) and (9) -12).
Among them six different type I receptors have been identified in
mammals(5, 8, 13, 14, 15, 16, 17, 18, 19, 20) ,
including one TGF-
type I receptor (T
R-I), two activin type I
receptors (ActR-I and ActR-IB), two BMP type I receptors (BMPR-IA and
BMPR-IB), and one additional type I receptor called activin
receptor-like kinase-1 (also termed TGF-
superfamily receptor type
I or R3) that has recently been shown to mediate certain signals in
response to BMP-7 (osteogenic protein-1). (
)The type I
receptors have similar sizes (502-532 amino acid residues) and
60-90% amino acid sequence identities to each other in their
kinase domains. In addition, type I receptors contain a conserved
sequence known as the GS domain (also called type I box) in their
cytoplasmic juxtamembrane region(10, 11) . Type I
receptors are more similar to each other than they are to the known
type II receptors, including TGF-
type II receptor (T
R-II)
and two activin type II receptors (ActR-II and ActR-IIB), and thus form
a subgroup of mammalian type I receptors in the family of receptor
serine/threonine kinases.
TGF- initiates the signaling of its
multiple responses through formation of a heteromeric complex of
T
R-I and T
R-II. TGF-
binds directly to T
R-II that
is a constitutively active kinase, which then recruits T
R-I into
the complex. T
R-II in the complex then phosphorylates the GS
domain of T
R-I, resulting in propagation of further downstream
signals(21, 22) . The catalytic activities of the
kinases of T
R-I and T
R-II are indispensable for signaling (22, 23, 24, 25) . Mutational
analyses altering serine and threonine residues in the T
R-I GS
domain have revealed that phosphorylation of certain serines and
threonines by T
R-II is essential for TGF-
signaling, although
its signaling activity does not appear to depend on the phosphorylation
of any particular serine or threonine residue in the TTSGSGSG sequence
of the GS domain(22, 26, 27) . In addition,
recent identification of a constitutively active form of T
R-I that
does not require T
R-II and TGF-
for signaling suggested that
T
R-I acts as a downstream signaling molecule of
T
R-II(27) .
Despite the functional importance of the GS
domain for initiating intracellular signals, little is known about how
the signals are propagated after phosphorylation of the GS domain.
Based on the knowledge of receptor tyrosine kinases, one possible
mechanism could be that the phosphorylated serine and/or threonine
residues in the GS domain may act as the binding sites for the
intracellular substrate to be activated by the TR-I kinase. This
hypothesis is attractive to explain the signaling mechanism for certain
common effects induced by the members of TGF-
superfamily because
the GS domain of the known type I receptors is highly
conserved(10, 11, 12) . On the other hand,
amino acid sequences of the GS domain of the type I receptors might be
too similar to each other to confer specificities to the signals that
mediate a wide variety of responses induced by the TGF-
superfamily. In fact, a T
R-I chimeric receptor substituting the GS
domain of ActR-I for that of T
R-I still transduces the
TGF-
-induced antiproliferative signal, which is not mediated
through intact ActR-I (27, 28) . Thus, certain
region(s) other than the GS domain in the type I receptors may also be
important for diverse signaling activities of the proteins in the
TGF-
superfamily.
In the present study we focused on the role
of the TR-I juxtamembrane region preceding the GS domain, and
serine 172 and threonine 176 within this region were found to be
essential for signaling a TGF-
antiproliferative response but not
plasminogen activator inhibitor-1 (PAI-1) and fibronectin induction.
Identification of such cytoplasmic regions important only for a limited
response may suggest that at least two different signals are specified
through different cytoplasmic parts of T
R-I.
Expression vectors for bacterial expression of wild-type
TR-I glutathione S-transferase (GST) fusion protein
(GST-WT), its deletion mutant GST-JD1(
150-181), and missense
mutants GST-JM1(S165A), GST-JM2(S172A), GST-JM3(T176V), and
GST-JM123(S165A/S172A/T176V) were obtained by insertion of
PCR-generated fragments of the corresponding cytoplasmic regions of
T
R-I into pGEX-4T-1 (Pharmacia) using their stable expression
plasmids as templates with RIS1-sma or RISdel2-sma as sense primers and
RIAS-not as an antisense primer. PCR conditions were 1 min at 94
°C, 1 min at 54 °C, and 1 min at 72 °C for 25 cycles. The
resulting PCR products for the GST fusion protein constructs were
digested with SmaI and NotI and ligated in-frame into
pGEX-4T-1. The structures of PCR-amplified region of the recombinants
were all confirmed by sequencing using a Sequenase DNA sequencing kit
(U. S. Biochemical Corp.).
Figure 1:
Schematic illustration of the TR-I
mutants. A, sequence alignment of the cytoplasmic
juxtamembrane region of different type I receptors(12) . Serine
and threonine residues are shown in bold. The amino acid
positions of T
R-I are indicated. The GS domain (type I box) is underlined. The SGSGSG core sequence is indicated by a bracket. TM, transmembrane domain. B,
T
R-I mutant constructs. The open circles indicate serine
and threonine residues within the cytoplasmic juxtamembrane region of
T
R-I. The closed circles indicate serine and threonine
residues altered to alanine and valine residues, respectively. The
amino acid positions deleted or altered are indicated in parentheses. WT, wild-type T
R-I; TSR-I,
TGF-
superfamily receptor type I.
Figure 2:
Binding of I-TGF-
1 to
wild-type T
R-I and its mutant derivatives. Parental Mv1Lu cells or
R4-2 cells transfected with wild-type T
R-I and its mutant
derivatives were pretreated with or without 100 µM ZnCl
for 6 h, followed by affinity cross-linking with
I-TGF-
1 using disuccinimidyl suberate. Cross-linked
complexes were immunoprecipitated with an antiserum against T
R-II.
Immune complexes were analyzed by SDS-gel electrophoresis under
reducing conditions and Bio-Imaging Analyzer. Cross-linked complexes of
T
R-II and T
R-I/JD1(
150-181) are indicated by arrows. Cross-linked complexes of wild-type T
R-I,
T
R-I/JM123(S165A/S172A/T176V), T
R-I/JM1(S165A),
T
R-I/JM2(S172A), and T
R-I/JM3(T176V) are indicated as
T
R-I with an arrow. WT, wild-type
T
R-I.
Figure 3:
Extracellular matrix protein responses in
R4-2 cells transfected with wild-type TR-I and its mutant
derivatives. A, stimulation of PAI-1 production by TGF-
1.
Subconfluent cultures of Mv1Lu or R4-2 cells transfected with the
indicated receptor cDNAs were incubated with medium containing 100
µM of ZnCl
for 5 h. Cells were then incubated
with (+) or without(-) 50 ng/ml of TGF-
1 for 2 h and
were labeled with a [
S]methionine and
[
S]cysteine mixture. Induced PAI-1 was
visualized by SDS-gel electrophoresis and Bio-Imaging Analyzer. PAI-1
was observed as a characteristic 45-kDa band. B, stimulation
of fibronectin production by TGF-
1. Cells were incubated with
medium containing 100 µM of ZnCl
for 5 h.
Cells were then incubated with (+) or without(-) 50 ng/ml of
TGF-
1 for 20 h and were labeled with a
[
S]methionine and
[
S]cysteine mixture for the last 4 h.
Fibronectin secreted into the media was purified by adsorption to
gelatin-Sepharose and analyzed by SDS-gel electrophoresis and
Bio-Imaging Analyzer. WT, wild-type
T
R-I.
To
evaluate whether TR-I/JD1(
150-181) is able to restore
TGF-
antiproliferative effect, DNA synthesis assay was performed
by measuring the incorporation of [
H]thymidine
into the DNA (Fig. 4, A and B). Upon treatment
with TGF-
, [
H]thymidine incorporation into
the DNA of Mv1Lu cells was inhibited dose-dependently up to 97% (Fig. 4A), whereas TGF-
had no effect on the
[
H]thymidine incorporation in the R4-2
cells transfected with the vector alone. When R4-2 cells
transfected with the wild-type T
R-I were treated with TGF-
in
the presence of ZnCl
, [
H]thymidine
incorporation into the DNA was inhibited by 65-75%, whereas only
a marginal inhibition was observed in the absence of ZnCl
.
In contrast, R4-2 cells transfected with
T
R-I/JD1(
150-181) were refractory to TGF-
growth
inhibition in the presence or the absence of ZnCl
(Fig. 4, A and B). These results
suggested that the N-terminal half of the cytoplasmic juxtamembrane
domain of T
R-I was not required for signaling a PAI-1 response,
whereas it was essential for signaling growth inhibitory activity.
Figure 4:
Antiproliferative response in R4-2
cells transfected with wild-type TR-I and its mutant derivatives.
Cells were incubated in DMEM containing 0.2% FBS with or without 100
µM ZnCl
for 5 h. Then the cells were exposed
to various concentrations of TGF-
1 for 16 h and pulsed with
[
H]thymidine, and
H-radioactivity
incorporated into the DNA was determined in a liquid scintillation
counter. A, TGF-
1 dose-response curve for growth
inhibition. Closed squares, parent Mv1Lu cells; open
squares, R4-2 cells transfected vector alone. The wild-type
T
R-I-transfected R4-2 cells were treated with ZnCl
(closed circles) or without ZnCl
(open
circles); the T
R-I/JD1(
150-181)-transfected
R4-2 cells were treated with ZnCl
(closed
triangles) or without ZnCl
(open triangles).
These experiments were performed three times with similar results. B, inhibition of [
H]thymidine
incorporation in R4-2 cells expressing wild-type T
R-I and
its mutant derivatives. Cells were pretreated with 100 µM ZnCl
followed by incubation with 15 ng/ml of
TGF-
1 and processed as described above. The data are plotted as
the average percentage of inhibition ± standard deviation. WT, wild-type T
R-I.
To test the signaling
activities of these missense mutant forms of TR-I, the transfected
cells were subjected to the analyses for extracellular matrix
production and growth inhibition by TGF-
. In PAI-1 and fibronectin
assays, like wild-type T
R-I, all the constructs analyzed including
T
R-I/JM123(S165A/S172A/T176V), T
R-I/JM1(S165A),
T
R-I/JM2(S172A), and T
R-I/JM3(T176V) restored responsiveness
to TGF-
(Fig. 3, A and B). With regard to
TGF-
antiproliferative effect, the T
R-I/JM1(S165A) construct
mediated a growth inhibitory effect comparable with that mediated by
the wild-type T
R-I, whereas T
R-I/JM123(S165A/S172A/T176V),
T
R-I/JM2(S172A), and T
R-I/JM3(T176V) were unable to restore
this activity (Fig. 4B). More than five different
clones for T
R-I/JM1(S165A), T
R-I/JM2(S172A), and
T
R-I/JM3(T176V) were subjected to the growth inhibition assay,
which gave essentially the same results (data not shown).
Figure 5:
Kinase activity of wild-type TR-I and
its mutant derivatives in vitro. Glutathione-Sepharose beads
that attached the indicated GST fusion proteins were incubated with
kinase buffer containing 1 µCi of
[
-
P]ATP for 15 min at 4 °C. Proteins
were resolved on an SDS-polyacrylamide gel under reducing conditions
and analyzed by Bio-Imaging Analyzer. WT, wild-type
T
R-I.
Recent studies on transmembrane serine/threonine kinases have
disclosed that certain members of TGF- superfamily exert their
multiple effects through binding to unique sets of heteromeric
complexes between type I and type II receptors. In the case of
TGF-
, T
R-II is a constitutively active kinase and capable of
binding TGF-
in the absence of T
R-I(22) , whereas
T
R-I requires T
R-II for the ligand binding. The T
R-I
kinase appears to be activated by formation of a hetero-oligomeric
complex composed of TGF-
, T
R-II, and T
R-I. In the
complex, several serine and threonine residues in the GS domain of
T
R-I become phosphorylated by T
R-II, and the phosphorylation
of GS domain is essential for TGF-
signaling(22, 26, 27) ; however, the
functional role of phosphorylated serine and threonine residues in the
GS domain as well as the mechanism of signaling after the
phosphorylation are largely unknown. In addition, functional importance
of the T
R-I cytoplasmic region other than the GS domain remains to
be elucidated.
In the present communication, we studied the role of
the N-terminally flanking region of the TR-I GS domain by mutating
this region and testing its ability to restore the signaling activity
in T
R-I-defective R4-2 cells. When expressed in R4-2
cells, like wild-type T
R-I, the deletion mutant
T
R-I/JD1(
150-181) and other missense mutants including
T
R-I/JM123(S165A/S172A/T176V), T
R-I/JM1(S165A),
T
R-I/JM2(S172A), and T
R-I/JM3(T176V) were all cross-linked
with radioiodinated TGF-
and co-immunoprecipitated with T
R-II (Fig. 2), indicating that this region in T
R-I is
dispensable at least for its expression and binding to TGF-
on the
cell surface and forming a complex with T
R-II.
The signaling
activities of these mutant TR-I constructs were tested for some of
the most characteristic responses to TGF-
; i.e. PAI-1 and
fibronectin induction and growth inhibition. Wild-type T
R-I and
all the missense mutants restored PAI-1 and fibronectin responses in
R4-2 cells (Fig. 3, A and B), indicating
that serine 165, serine 172, and threonine 176 of T
R-I are not
needed to transduce a signal for PAI-1 and fibronectin induction.
Antiproliferative response was also restored by the wild-type
TR-I and one of the receptor mutants, T
R-I/JM1(S165A);
however, the other mutants including T
R-I/JD1(
150-181),
T
R-I/JM123(S165A/S172A/T176V), T
R-I/JM2(S172A), and
T
R-I/JM3(T176V) were unable to rescue this response (Fig. 4, A and B). Because the wild-type
T
R-I and all the mutant T
R-I were similar in their activities
to bind TGF-
, form a complex with T
R-II, and phosphorylate
themselves in vitro, the differences in their ability to
restore the antiproliferative response does not seem to be at the level
of ligand-receptor complex formation or basal kinase activity. Rather,
T
R-I/JM123(S165A/S172A/T176V), T
R-I/JM2(S172A), and
T
R-I/JM3(T176V) are likely to be impaired in interacting with a
specific substrate that transduces antiproliferative response but not
PAI-1 and fibronectin responses.
From our present data, it is not
easy to deduce the mechanistic significance of serine 172 and threonine
176 of TR-I in TGF-
signaling. Although it was reported that
TGF-
-induced phosphorylation of these residues was not detected in vivo(22) , it is still possible that T
R-II
may phosphorylate these residues as minor phosphorylation site(s).
Alternatively, these residues might be constitutively phosphorylated
even in the absence of TGF-
, which would not be detected as the
ligand-induced phosphorylation sites. It is also possible that serine
172 and threonine 176 in T
R-I may not be themselves
phosphorylated, but their integrity is essential to maintain the proper
conformation of T
R-I to interact with its substrates.
It was
reported that whereas Mv1Lu cells expressing SV40 T-antigen were
refractory to the antiproliferative effect of TGF-, TGF-
induced the expressions of junB mRNA and extracellular matrix
proteins including PAI-1, fibronectin, and thrombospondin in these
cells(34, 35) . In addition, we have previously shown
that growth inhibition and extracellular matrix production by TGF-
are sensitive and insensitive, respectively, to phorbol 12-myristate
13-acetate in prostatic carcinoma cells(36) . These
observations have suggested that the signals induced by TGF-
that
lead to growth inhibition and to extracellular matrix production should
differ at a certain step within the signaling cascade from the receptor
to the nucleus. In this regard, the present study is of particular
importance. T
R-I mutants including
T
R-I/JM123(S165A/S172A/T176V), T
R-I/JM2(S172A), and
T
R-I/JM3(T176V) had signaling activity for extracellular matrix
protein responses but not growth inhibition. Although other responses
including expressions of junB and thrombospondin should be
determined, identification of such mutant forms of T
R-I strongly
suggests that the signals for growth inhibition and extracellular
matrix production are diverged closely at the receptor level. In
conclusion, serine 172 and threonine 176 within the T
R-I
juxtamembrane region preceding the GS domain are essential for
signaling the TGF-
antiproliferative response and might be
involved in the interaction with the downstream substrate responsible
for growth inhibition.