(Received for publication, September 25, 1995; and in revised form, October 17, 1995)
From the
Transforming growth factor-1 (TGF-
1) is the prototype
of a large family of molecules that regulate a variety of biological
processes. The type I (T
R-I) and type II (T
R-II) receptors
for TGF-
1 are transmembrane serine/threonine kinases, forming a
heteromeric signaling complex. Recent studies have shown that
T
R-II is a constitutively active kinase and phosphorylates
T
R-I upon ligand binding, suggesting that T
R-I is the
effector subunit of the receptor complex, which transduces signals to
intracellular targets. This model has been further confirmed by the
identification of constitutively active T
R-I that mediates
TGF-
1-specific cellular responses in the absence of ligand and
T
R-II. To investigate signaling by TGF-
1, we have sought to
isolate proteins that interact with the cytoplasmic region of
T
R-I. One of the proteins identified was the
subunit of
farnesyl-protein transferase (FT
) that modifies a series of
peptides including Ras. T
R-I specifically interacts with FT
in the yeast two-hybrid system. Glutathione S-transferase-T
R-I fusion proteins bind FT
translated in vitro. T
R-I also phosphorylates FT
. We
further show that the constitutively active T
R-I interacted with
FT
very strongly whereas an inactive form of T
R-I did not.
These results suggest that FT
may be one of the substrates of the
activated T
R-I kinase.
Transforming growth factor-1 (TGF-
1) (
)is a
multifunctional cytokine that is involved in a variety of biological
processes such as cell cycle progression, differentiation, adhesion,
migration, extracellular matrix deposition, and
immunoregulation(1, 2) . However, the mechanisms
whereby TGF-
1 exerts such pleiotropic effects have been elusive.
TGF-
1 binds to its specific receptors on the cell surface. Three
distinct classes of receptors have been identified(2) . The
type III (T
R-III) receptor termed betaglycan is involved in
presenting ligand to other signaling receptors(3) . The type I
(T
R-I) and type II (T
R-II) receptors belong to a family of
transmembrane serine/threonine kinases that include receptors for other
TGF-
1-related molecules such as activins, bone morphogenic
proteins, and Müllerian inhibiting
substance(2) . T
R-II binds ligand without T
R-I but
requires T
R-I to transduce signals. T
R-I, on the other hand,
requires T
R-II to bind ligand(4) . In the case of
TGF-
s and activins, the type II receptors determine ligand binding
specificities(5) , and the type I receptors specify cellular
responses to ligand(6) , suggesting that T
R-I is the
downstream component of the receptor system.
Recently a model for
activation of the TGF- receptor system has been
proposed(7) . T
R-II is a constitutively active kinase.
Upon ligand binding T
R-II recruits and phosphorylates T
R-I.
The transphosphorylation sites in T
R-I reside between the
transmembrane and the kinase domain(8) . This region contains a
short peptide stretch called the GS domain, which is highly conserved
among the type I receptors of the serine/threonine kinase family.
Mutations of certain serines and threonines in this domain impair
phosphorylation and signaling activity of T
R-I(8) ,
indicating that phosphorylation of the GS domain by T
R-II has a
crucial role in TGF-
signaling. Replacement of threonine 204,
between the core GS domain and the kinase domain, with aspartic acid
resulted in constitutively active T
R-I (8) . This mutant
has elevated in vitro kinase activity and signals both
anti-proliferative and transcriptional responses in the absence of
ligand and T
R-II. These results further support the hypothesis
that T
R-I transmits signals by phosphorylating intracellular
substrates.
We employed a yeast two-hybrid system (9) to
identify proteins that interact with the cytoplasmic region of
TR-I. One of the identified clones encoded farnesyl-protein
transferase-
(FT
), a component of an enzyme that modifies
various proteins such as Ras. T
R-I was shown to interact with and
phosphorylate FT
in vitro. The constitutively active form
of T
R-I interacted with FT
more strongly than the wild type,
and an inactive form of T
R-I did not bind FT
at all. These
results indicate that FT
may be one of the substrates of activated
T
R-I.
To search for proteins that interact with TR-I, we used
the interaction trap screen developed by Brent and
co-workers(9) . A HeLa cell cDNA library was screened with the
cytoplasmic region of the rat T
R-I (11) as the bait. One
of the clones encoded T-ALK, a novel type II serine/threonine kinase
receptor(10) , which was later shown to be the bone morphogenic
protein type II receptor(14, 15) . Another subset of
clones encoded FKBP12, a binding protein for FK506 and rapamycin (data
not shown). Ten clones encoded varying portions of the
subunit of
farnesyl transferase.
Farnesyl transferase is a heterodimeric enzyme
composed of an and
subunit(16) . Prey (pJG-FT
or pJG-FT
) and bait (pEG-FT
or pEG-FT
) plasmids
containing the entire coding region of FT
or FT
were
constructed. FT
and FT
interacted very strongly in the yeast
assay (data not shown) as shown in vivo(17) . To test
the specificity of the interaction between T
R-I and FT
,
pJG-FT
was introduced into EGY48 with pEG202, the bait vector, or
a panel of unrelated baits. None of these were positive in the
interaction assay (Fig. 1A). In addition, FT
did
not interact with T
R-II, and FT
did not associate with
T
R-I. Thus the interaction between T
R-I and FT
was
specific in the yeast system.
Figure 1:
Interaction of FT with T
R-I. A, various combinations of a bait and a prey with the reporter
plasmid were introduced into EGY48. Four independent colonies from each
transformation were assayed on dextrose and galactose X-Gal plates. The
results on galactose X-Gal plates are shown. A blue color represents a positive interaction. In the first seven cases, the
prey is FT
and the baits are shown in the figure. pEG202 is the
bait vector; Cdc2, Ftz, Max, and bicoid are unrelated baits (a gift of
R. Finley). In the last case, the combination of the T
R-I bait and
the FT
prey was tested. B, interaction of the FT
clones with T
R-I was compared. pJG-FT
* expresses the entire
coding region of FT
(379 amino acids). CL69** includes part of the
5`-noncoding region. The other clones have a deletion in the N-terminal
region.
Only one of the FT clones (CL69)
contained the entire coding region whereas the others lacked part of
the 5`-coding sequence (Fig. 1B). To compare the
strength of interactions, all of the rescued FT
plasmids and
pJG-FT
were tested in the interaction trap. The
-galactosidase production by the clones missing up to the first 45
amino acids of FT
was almost the same on X-Gal plates whereas the
colony of CL74 starting from the 59th amino acid exhibited a lighter
blue color. The color of CL24 starting from the 75th amino acid
remained almost white (Fig. 1B). These data suggest
that the first N-terminal 45 amino acids of FT
are dispensable for
its interaction with T
R-I. Interestingly, deletion of 51 amino
acids at the N terminus of FT
has been reported to allow normal
stabilization of FT
and production of enzyme activity, but
deletion of 106 amino acids abolished both functions(17) .
To confirm the specific interaction of FT and T
R-I in
another system, we used GST fusion proteins of the cytoplasmic region
of the TGF-
receptors and FT
translated in vitro.
When FT
was translated in a reticulocyte system, two bands were
obtained. A minor band with a faster mobility is probably a degradate
of the entire protein since FT
is very unstable in mammalian
cells(17) . FT
bound to GST-T
R-I but not to GST
itself (Fig. 2). A minimal level of binding to GST-T
R-II
was observed in this assay.
Figure 2:
In vitro binding of FT with
T
R-I. GST or GST-T
R fusion proteins bound to GST beads and
FT
proteins translated in vitro were mixed, washed, and
analyzed by SDS-PAGE. Products from the in vitro translation
reaction are shown as input. Arrows represent two bands
obtained in the translation of FT
.
We next tested whether TR-I
phosphorylates FT
in vitro using GST fusion proteins (Fig. 3A). GST-T
R-I (lane 2, 67 kDa) and
GST-T
R-II (lane 3, 70 kDa) showed autophosphorylation
activity whereas no kinase activity was detected in GST itself (lane 1) or GST-FT
alone (lane 5). A mutant of
human T
R-I (K232R), in which lysine 232 was changed to arginine,
was constructed. This mutation completely abolished its signaling and
kinase activities (7) (lane 4). When GST-T
R-I was
mixed with GST-FT
, a new band that corresponds to GST-FT
(71
kDa) appeared above the GST-T
R-I autophosphorylated band (lane
6). GST-K232R did not phosphorylate itself or FT
(lane
9). It was not clear whether GST-FT
was phosphorylated by
GST-T
R-II (lane 8) since a faint band of a similar size
of GST-FT
was detected in GST-T
R-II alone (lane 3).
However, phosphorylation of FT
as intense as in T
R-I was not
observed. It is possible that T
R-I could phosphorylate the GST
portion of the GST-FT
fusion proteins. When GST itself was mixed
with GST-T
R-I, there was no phosphorylation of GST (27 kDa) (lane 7). A possibility that T
R-I may phosphorylate GST
through its interaction with FT
still remained. Therefore the
FT
portion (44 kDa) was cleaved from the GST-FT
fusion
proteins with thrombin (36 kDa) (Fig. 3B). Again
FT
alone did not show any kinase activity (lane 1). When
the cleaved FT
was mixed with GST-T
R-I, a
doublet-phosphorylated band the size of FT
appeared (lane
2) but not in GST-K232R (lane 4). Similar but much
fainter bands were detected in GST-T
R-II (lane 3). These
data show that T
R-I can phosphorylate FT
. T
R-I did not
phosphorylate FKBP12 under the same condition (data not shown).
Figure 3:
In vitro phosphorylation of
FT by T
R-I. In vitro phosphorylation was done using
GST fusion proteins. A, eluted GST-FT
fusion proteins
were phosphorylated by GST-T
R fusion proteins. Lane 1,
GST; lane 2, GST-T
R-I; lane 3, GST-T
R-II; lane 4, GST-K232R; lane 5, GST-FT
; lane
6, GST-T
R-I plus GST-FT
; lane 7, GST-T
R-I
plus GST; lane 8, GST-T
R-II plus GST-FT
; lane
9, GST-K232R plus GST-FT
. A fast migrating band designated by
an arrow was recognized by anti-ALK5 antibodies (data not
shown), suggesting that the band represents a degradate of
GST-T
R-I. Molecular weight markers are shown on the right. B, FT
proteins cleaved from GST-FT
proteins were
used. Lane 1, FT
; lane 2, GST-T
R-I plus
FT
; lane 3, GST-T
R-II plus FT
; lane 4,
GST-K232R plus FT
. A band designated by an arrow is the
same as in A.
Recently Massagué and co-workers reported
mutants of TR-I with various levels of signaling
activity(8) . One of the mutants, in which threonine 204 was
replaced with aspartic acid (T204D), showed an elevated level of in
vitro kinase activity and functioned as a constitutively active
T
R-I in vivo. Another mutation of threonine 200 to valine
(T200V) yielded completely inactive T
R-I. We tested the
interaction of FT
with the wild type and mutant forms of T
R-I
using the interaction trap. In the X-Gal plate assay, colonies
transformed with T204D showed an intense blue color whereas colonies of
T200V remained completely white. The K232R transformants exhibited a
faint blue color while the wild type and
JM colonies showed
intensity between K232R and T204D (data not shown). A quantitative
-galactosidase assay was performed (Table 1). As in plate
assay, T204D showed the strongest interaction and T200V did not
interact with FT
. K232R interacted more strongly than T200V but
less efficiently than the wild type.
JM interacted more
efficiently than the wild type but not as strongly as T204D. FKBP12 did
not interact with
JM but did interact with the wild type and all
of the other mutants (data not shown). These results indicate that the
activated T
R-I binds FT
more efficiently than the wild type
or loss of function mutant T
R-I.
We screened a human cDNA library to identify proteins that
bind to the cytoplasmic region of TR-I. One of the clones was a
novel type II serine/threonine kinase receptor (10) showing
that the two-hybrid system is useful in isolating one of the subunits
of a membrane-bound receptor complex. The second clone was FKBP12.
FKBP12 was reported to specifically associate with T
R-I in
vitro(18) . However, its role in TGF-
signaling is
yet unknown. Here we report FT
as another T
R-I binding
protein.
Farnesyl transferase is a heterodimeric enzyme composed of
and
subunits(16) . Coexpression of both subunits
seems to be necessary for stabilization and activity of the
enzyme(17, 19, 20) . The holoenzyme attaches
a farnesyl isoprenoid to a cysteine residue(16, 21) .
The CAAX (where A is an aliphatic amino acid and X is any amino acid) sequence is the target motif found at the
C-terminal end of all Ras proteins and many other isoprenylated
proteins(17, 21) . The
subunit may perform the
catalytic function (17) , but the precise enzymatic mechanism
is still elusive. Farnesylation plays a pivotal role in the subcellular
localization of a variety of proteins including
Ras(21, 22) . Heterologous proteins like protein A (23) and Raf (24) can be targeted to the plasma
membrane by adding the C-terminal sequence of K-Ras. Membrane
localization is critical for the activity of Ras. Inhibition of
farnesylation leads to suppression of Ras-induced responses such as Xenopus oocyte maturation(25) , cytoskeletal
disorganization(26) , cell growth(25) , and
tumorigenesis in vivo(27) . In Caenorhabditis
elegans, the multivulva phenotype resulting from an activated let-60 Ras mutation was suppressed by FT inhibitors (28) .
A number of investigations have suggested a
relationship between TGF- and Ras. TGF-
1 treatment caused a
rapid increase in GTP-bound Ras in several cell
lines(29, 30) . In another report, however,
microinjection of oncogenic Ha-Ras proteins overcame
TGF-
1-mediated growth inhibition and TGF-
1 decreased the
GTP-bound form of Ras(31) . Although the effect of TGF-
1
treatment on Ras status may vary depending on the context(31) ,
the latter results are consistent with the fact that Ras is mitogenic
whereas TGF-
1 predominantly inhibits cell growth. Furthermore,
TGF-
1 inhibited the coupling of Ras to the activation of
phosphatidylcholine hydrolysis(32) . TGF-
1 partially
antagonized transformed phenotypes, including loss of organized actin
cytoskeleton, caused by Ras transformation(33) . Intriguingly,
farnesyl transferase inhibition also caused actin stress fiber
formation and morphological reversion in Ras-transformed
cells(26) . Thus TGF-
1 negatively regulates Ras under
certain conditions. It was also shown that cellular Ras activity is
required for passage through the G
phase in Balb/c 3T3
cells while TGF-
1 inhibits cell growth if added at any point in
G
(34) . These findings suggest that Ras may be
directly involved in TGF-
signaling.
In the present report, we
show that TR-I not only binds but phosphorylates FT
in
vitro. FT
binds preferentially to an activated form of
T
R-I. These results indicate that FT
may be phosphorylated by
T
R-I upon ligand binding. Phosphorylation of FT
has not yet
been reported. It is important to study whether TGF-
1 induces
phosphorylation of FT
and modulates its activity in vivo.