From the Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801
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ABSTRACT |
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The immunosuppressant rapamycin, in complex with
its cellular receptor FKBP12, targets the cellular protein
FKBP12-rapamycin-associated protein/mammalian target of
rapamycin/rapamycin and FKBP12 target 1 (FRAP/mTOR/RAFT1) and
inhibits/delays G1 cell cycle progression in
mammalian cells. As a member of the novel phosphatidylinositol kinase-related kinase family, FRAP's kinase activity is essential for
its signaling function. The FKBP12-rapamycin binding (FRB) domain in
FRAP is also speculated to play an important role in FRAP function and
signaling. However, the biochemical and physiological functions of FRB,
as well as the mechanism for rapamycin inhibition, have been unclear.
The present study focuses on investigation of FRB's role and the
functional relationship between FRB domain and kinase domain in FRAP.
Microinjection of purified FRB protein into human osteosarcoma MG63
cells results in a drastic blockage of the G1 to S cell
cycle progression; such a dominant negative effect is reversed by a
point mutation (Trp2027 Mammalian cell proliferation is regulated by extracellular
mitogens via multiple signal transduction pathways. One such pathway leads to the up-regulation of protein synthesis, which is essential for
G1 progression of the cell cycle (1-3). At least two
proteins involved in regulating the translational machinery have been
found to lie downstream of this pathway: the p70 S6 kinase
(p70s6k)1 (4, 5)
and an eIF4E binding protein (4E-BP1) (3, 6-8). The immunosuppressant
rapamycin inhibits this pathway at a point upstream of
p70s6k (9, 10) and 4E-BP1 (11-14); this inhibition
requires the presence of the cellular protein FKBP12 (15) and results
in selective reduction of protein synthesis (16-19) and G1
arrest in a variety of mammalian cells (15), as well as in the yeast Saccharomyces cerevisiae (20, 21).
A major player in the rapamycin-sensitive pathway has been identified
as the cellular target of rapamycin-FKBP12 complex, designated FRAP
(22), RAFT1 (23), or mTOR (24). FRAP belongs to the novel family of
phosphatidylinositol kinase (PIK)-related kinases which include
Ataxia telangiectasia mutated. Members of this
family are involved in a range of essential cellular functions, including cell cycle progression, cell cycle checkpoints, DNA repair,
and DNA recombination (25-28). A kinase domain with sequence homology
to lipid and protein kinases has been found at the C termini of all
members in this family, and the kinase activity is crucial for the
functions of these proteins. The FRAP protein is a 289-kDa single
polypeptide containing the characteristic C-terminal kinase domain, the
activity of which has been shown to be toward serine/threonine residues
(10, 14, 29, 30) and it is required for signaling to p70s6k
and 4E-BP1 (10, 14, 31). However, it is currently unclear how FRAP
receives upstream signals and transduces them to the downstream
components. Phosphatidylinositol 3-kinase and protein kinase B/Akt have
been implicated to relay extracellular mitogenic stimuli to FRAP (32,
33), whereas evidence has also been presented to suggest that FRAP may
regulate a nutrient (amino acid)-responsive pathway that is a
prerequisite, but not an overlap, of the mitogenic pathway (34).
Further investigation is required to reconcile these observations.
In vitro phosphorylations of both p70s6k and
4E-BP1 by FRAP have been reported (14, 30); however, definitive
evidence is still needed to demonstrate which protein (if either) is
the physiological substrate for FRAP.
The FKBP12-rapamycin binding (FRB) domain in FRAP has been identified
as an 11-kDa segment located N-terminal to the kinase domain (35, 36)
(Fig. 1). A serine residue, Ser2035, within this domain is
crucial for the interaction between FRAP and FKBP12-rapamycin;
substitutions at this site with any residues that have larger side
chains than serine abolish formation of the ternary complex (35).
Consistently, full-length FRAP bearing these mutations displays
rapamycin resistance in Jurkat cells (10). Similarly, in the yeast
homologues of FRAP, TOR1 and TOR2, mutations at an equivalent serine
residue were found by genetic analysis to confer rapamycin resistance
(37-39). Crystal structure of the FRB domain indicates a well defined
four-helical bundle with its N and C termini close together (40),
implying a potential role as a functional module.
The biochemical function of FRB and the mechanism for
rapamycin-inhibition of FRAP are currently unclear. Although it has been a common speculation that rapamycin may inhibit the kinase activity of FRAP, micromolar concentrations of rapamycin required to
inhibit FRAP activity in vitro (10, 14, 33) still need to be
reconciled with the low IC50 observed in vivo
(subnanomolar) and the high affinity of FKBP12-rapamycin-FRAP binding
(35). Only 15-50% activity of FRAP toward p70s6k and
4E-BP1 in vitro was inhibited by nanomolar concentrations of
rapamycin (30). In addition, FRAP bound to rapamycin-FKBP12 is found to
retain a robust autokinase activity (41). The absence of FRB domain in
other PIK-related kinases also seems to imply that this domain is
probably not required for the intrinsic catalytic activity
of the kinase.
To understand FRAP function and the mechanism for rapamycin inhibition
of its pathway, we have probed the physiological function of FRB
in vivo and examined the functional relationship between FRB
and the kinase domain in FRAP. Here we report experimental evidence
suggesting that the kinase activity of FRAP requires a functional FRB,
which may provide a binding site for an upstream activator; this
putative function of FRB is necessary for G1 progression in
mammalian cells.
Tissue Culture and Bacterial Strain--
Both human osteosarcoma
MG63 cells and human embryonic kidney 293 cells were maintained in DMEM
containing 10% fetal bovine serum (FBS) and penicillin/streptomycin at
37 °C with 5% CO2. Escherichia coli strain
BL21 (F DNA Constructs and Site-specific Mutagenesis--
To express FRB
as GST fusions, the cDNA encoding FRB from amino acid 2015 to 2114 was inserted into the vector pGEX-2T (Amersham Pharmacia Biotech).
Point mutations of FRB were introduced by PCR using primers carrying
the desired mutations: the Tyr2105 Protein Purification--
The expressions of GST-FRB wild-type
and mutant proteins were induced in BL21 at room temperature to ensure
solubility, whereas GST-FKBP12 and GST-4E-BP1 fusion proteins were
induced at 37 °C. Purification and thrombin cleavage of the fusion
proteins were carried out following standard procedures (Amersham
Pharmacia Biotech). After cleavage and removal of most GST proteins by
glutathione-Sepharose, the FRB proteins were further purified using a
Sephacryl S-100 column (2.5 cm × 100 cm). The entire procedure
was performed in phosphate-buffered saline (PBS) buffer containing 1 mM DTT. The purified FRB proteins were concentrated to
4.5-6 mg/ml, and SDS-PAGE analysis indicated homogeneity of at least
98%.
FKBP12-Rapamycin Binding Assays--
To assess FKBP12-rapamycin
binding capacity of the FRB mutants, GST-FKBP12 and rapamycin were
pre-mixed in 1 ml of binding buffer (PBS with 1 mM DTT and
0.3% Triton X-100) at 1 µM each, followed by addition of
1 µM FRB protein and brief incubation on ice.
Glutathione-Sepharose (20 µl) was then added to the mixture and
rocked at 4 °C for 1 h. The matrix was subsequently washed three times in 1 ml of binding buffer and boiled in SDS sample buffer
and loaded on a 15% SDS gel.
Microinjection and Cell Staining--
MG63 cells were plated
onto coverslips at ~30% confluence and starved in serum-free medium
for 48 h. The starved cells were microinjected with various
proteins at 4.5-5 mg/ml in PBS containing 1 mM DTT and 1 mg/ml fluorescein-conjugated dextran (Mr 70,000; Molecular Probes). At least 100 cells were injected each time. Upon completion of injection, the coverslips were transferred to DMEM
containing 10% FBS and incubated at 37 °C in 5% CO2
for 25 h before staining. Two to 12 h prior to fixation,
bromodeoxyuridine (BrdUrd) was added to the media to a final
concentration of 1 µM. The injected cells were fixed in
3.7% formaldehyde for 15 min, followed by permeabilization in 0.1%
Triton X-100 for 10 min and incubation in 4 N HCl for
10 min. The cells were then incubated with a monoclonal anti-BrdUrd
antibody (Sigma) for 30 min and subsequently with a
rhodamine-conjugated anti-mouse IgG antibody (Jackson Immunoresearch).
Transient Transfection, FRAP Kinase Assays, and ATP
Binding--
HEK293 cells were seeded onto 60-mm plates (1 × 106 cells/plate) 12-16 h prior to transfection. Six
microliters of SuperFect reagent (Qiagen) was used to transfect 2 µg
of DNA for each plate. At 24 h after transfection, the cells were
lysed in lysis buffer (20 mM sodium phosphate, pH 7.2, 1 mM Na3VO4, 25 mM NaF,
25 mM Kinase Domain Defined by Homology May Not Be Sufficient for
Activity of FRAP--
To examine the primary structure requirement for
FRAP kinase activity, we have generated a series of N-terminal deletion
mutants of FRAP (Fig. 1A).
These truncated mutant proteins were transiently expressed in HEK293
cells, immunoprecipitated, and assayed for their autophosphorylation
activities. Surprisingly, a fragment (1362C) much larger than the
predicted kinase domain was required for such activity (Fig.
1B). This fragment includes the FRB domain and a stretch of
upstream sequences in addition to the C-terminal kinase domain. The
kinase domain alone (2061C) was inactive, and so were the fragments
containing both the kinase and FRB domains without additional upstream
sequences (1819C and 1967C) (Fig. 1B). It was conceivable
that the loss of autophosphorylation might be due to deletion of the
autophosphorylation site(s). The autophosphorylation site is currently
unknown. However, a loss of 4E-BP1 phosphorylation (data not shown) was
observed simultaneously with the loss of autophosphorylation,
suggesting that the catalytic activity was abolished in these deletion
mutants. Although we cannot rule out the possibility of protein
misfolding in these mutants, at least 1819C was still able to bind
FKBP12-rapamycin (data not shown). These observations implicate
potential involvement of FRB and/or upstream sequences in regulation of
the FRAP catalytic activity.
Microinjection of FRB Protein Prevents Cell Cycle Progression into
S Phase--
Given the obvious significance of FRB domain in FRAP's
signaling function as indicated by rapamycin effect, and considering the implications of the kinase activity data, we decided to investigate the function of FRB with a more direct approach. Taking advantage of
the large quantities of soluble FRB protein purified from E. coli (35), we examined the effect of introducing excess exogenous FRB into cultured cells by microinjection. Human osteosarcoma MG63 cell
line has been chosen for these experiments because of the previously
demonstrated rapamycin effect and well characterized cell cycle in this
cell line (42). Cells synchronized at G0 by serum
starvation re-entered the cell cycle upon serum stimulation and
completed S phase (DNA synthesis) in 25 h (data not shown). As
shown in Fig. 2, cells microinjected with
4.5 mg/ml FRB protein no longer entered S phase, as indicated by the
lack of DNA synthesis. This effect was apparently not an artifact of
high protein concentrations injected and was specific to the FRB
protein, as injection of a nonspecific protein (rabbit IgG) at the same
concentration or the indicator fluorescein-dextran alone had no effect
on the cell cycle (Fig. 2). The statistical significance of the FRB
effect is indicated by data summarized in Table
I; more than 80% of FRB injected cells
were arrested at G1. The effect of FRB was concentration-dependent; injection of the protein at less
than 4 mg/ml concentrations arrested a lower percentage of cells (data not shown). Due to the variation in injection flow rate, injected cells
received various amounts of protein even when a constant concentration
(4.5 mg/ml) of FRB was used. Normal cell cycle progression was almost
always observed in cells with lower amounts of injected protein (as
indicated by the fluorescence intensity), again suggesting a
concentration dependence.
These observations imply that the free FRB protein acts as a dominant
negative factor that inhibits FRAP's normal function, which is
presumably essential for G1 progression. Assuming the observed FRB effect is specific, one simple explanation is that this
domain in FRAP functions as a binding site for either an upstream
activator or a downstream effector; an excess of free FRB titrates off
this factor and therefore inhibits FRAP signaling. To confirm the
specificity of the FRB effect as well as to characterize the
structure-function relationship in FRB, we set out to construct and
examine the following FRB mutants.
FKBP12-Rapamycin Binding of Mutant FRB Proteins--
Serine 2035 in FRB has been shown to be crucial for rapamycin binding; all
mutations at this site containing larger side chains abolish the
formation of FKBP12-rapamycin-FRB complex (35). It is obviously of
great interest to study the cell cycle effect of FRB mutated at
Ser2035. However, given that full-length FRAP carrying
Ser2035 mutations can signal to p70s6k and
4E-BP1 normally (10, 14, 31), this mutant is expected to retain
wild-type functions other than rapamycin binding. Using the crystal
structure of FKBP12-rapamycin-FRB ternary complex (40) as a guide, we
designed two other mutants of FRB (Fig. 3A) that could potentially
interfere with the presumed effector/activator binding. Tyrosine 2105 is located at the interface between FRB and FKBP12 in the complex and
forms interactions with both rapamycin and FKBP12 (40); mutation at
this residue may disrupt FRB's normal function if the putative
effector binding is molecularly similar to FKBP12-rapamycin binding.
Another site under consideration was tryptophan 2027; a
Trp2027
Ser2035 Trp2027, but Not Ser2035, Is Crucial for
FRB's Cellular Function--
The various FRB mutant proteins analyzed
above were microinjected into MG63 cells under the same conditions as
described earlier, and the cell cycle progression into S phase was
examined in the injected cells. Similar to the wild-type protein, both
Ser2035 Mutation at Trp2027 Abolishes FRAP Kinase
Activity--
To establish a direct link between the observed FRB
effect and FRAP function, and to investigate the functional
relationship between the FRB domain and the kinase domain in FRAP, we
have examined the effect of the Trp2027
We further examined the ATP binding capacity of these mutant FRAP
proteins using ATP-agarose. As shown in Fig.
6, wild-type and all mutant proteins
bound the ATP matrix equally well; addition of free ATP abolished the
matrix binding of both the wild-type and Trp2027 Rapamycin blocks/delays G1 cell cycle progression in
various mammalian cells and S. cerevisiae, presumably
through inhibition of a pathway leading to translational regulation. In
yeast, rapamycin depletes the pool of polysomes (45) and abolishes
protein translation via inhibition of the targets of rapamycin-FKBP12,
TOR1 and TOR2. These two proteins have been shown to be absolutely
required for G1 progression (20, 21, 39, 45). The mammalian
homolog FRAP is believed to function similarly, as deduced from
rapamycin inhibition of G1 progression. However, the
rapamycin effect in mammalian cells is less straightforward. Different
cell lines display varied sensitivity to rapamycin in their growths;
only translation of a small subset of mammalian mRNAs is affected
by rapamycin (16-19), and to a lesser extent; rapamycin inhibition of
p70s6k does not always correlate with cell cycle blockage
(46). Our observations of a dominant negative effect on the
G1 cell cycle elicited by microinjection of the FRB domain
in FRAP provide direct evidence linking FRAP function to G1 progression.
As for all members of the PIK-related kinase family, the kinase
activity of FRAP is essential for its function (10, 14, 31). The FRB
domain located N-terminal to the kinase domain has been speculated to
play an important role in FRAP signaling since it is the target site
for rapamycin. Although rapamycin inhibition of FRAP kinase activity
in vitro has been reported (10, 14, 33), more definitive
evidence was needed to link FRB directly to the kinase in FRAP, given
that high concentrations of rapamycin were required for such
inhibition, and that FRAP bound to FKBP12-rapamycin matrix was still
active (41). Our observation that a point mutation (Trp2027
It should be noted that the FRB domain and the kinase domain are highly
conserved in the yeast homologs TOR1 and TOR2, where TOR2 has two
separate functions: one shared with TOR1 (G1 function) (38,
47) and one unique (actin cytoskeleton function) (48, 49); both
functions require the kinase activity, but only the G1
function is sensitive to rapamycin (39). Our model of FRB's involvement in kinase activity does not seem to apply to TOR2's function on actin cytoskeleton. It is conceivable that the yeast proteins may differ from the mammalian homologue in their functions despite high sequence homology. However, it is also possible that the
regulation of TORs' G1 function mirrors that of FRAP,
whereas TOR2's actin cytoskeleton function is regulated differently.
For instance, the latter function may involve a lipid substrate rather than a protein substrate, thus differentiating two modes of regulation.
An alternative mode of regulation that would explain our current
observations equally well is an intramolecular interaction involving
FRB and another domain of FRAP (e.g. the kinase domain), rather than another factor. However, we have extensively examined potential interactions between FRB and FRAP or various domains/segments of FRAP (including FRB self-association) using co-immunoprecipitation and yeast two-hybrid methods, and no interaction has been detected. Therefore, although we cannot completely exclude the possibility of
intramolecular interactions, it is likely that the FRB domain in FRAP
interacts with another factor in vivo. In view of the recent
report that insulin stimulates mTOR (FRAP) kinase activity via the
protein kinase B pathway (33), the activator-binding model for FRB
function is particularly attractive as it may provide a molecular basis
for such stimulations. Several scenarios can be envisioned for the
mechanism of kinase regulation via FRB. For instance, activator binding
at FRB may elicit conformational changes of the kinase domain favorable
for catalysis or binding of a substrate; chemical modification
(e.g. phosphorylation) of FRB, rather than physical
interaction with the activator, may have an impact on the kinase
domain; the activator may also be some intracellular
component instead of a soluble factor, and localization of FRAP through
FRB may be required for kinase activity. Further investigations are
under way to examine these possible mechanisms of FRAP regulation.
Phe). The same mutation also
abolishes kinase activity of FRAP without affecting ATP binding, and
truncation studies suggest that upstream sequences including FRB are
required for kinase activity in vitro. Given these data, we
propose a model for FRAP function, in which the FRB domain is required
for activation of the kinase domain, possibly through the interaction
with an upstream activator. In addition, our observations provide
direct evidence linking FRAP function to G1 cell cycle progression.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
, ompT,
hsdSB(rB
mB+),
dcm, gal) was used for expression and isolation
of GST-FRB wild-type and mutant proteins.
Ala and
Trp2027
Phe mutations were introduced through the 3'
and 5' primers, respectively. The Ser2035
Ile mutant
has been described previously (35). Full-length FRAP cDNAs
containing Ser2035
Thr and Asp2357
Glu
mutations were isolated from corresponding pBJ5 constructs (10) and
inserted into the pCDNA3 vector via NotI sites. To construct full-length FRAP cDNA with Trp2027
Phe,
the 5' end 5.9-kilobase pair sequence was first removed by
KpnI digest; the mutation was then introduced into the 3'
1755-base pair fragment via ApaI and PflMI sites,
followed by re-insertion of the 5' KpnI fragment. FRAP
N-terminal truncation mutants 1362C, 1967C, and 2061C were constructed
by deleting FRAP sequences 5' of EcoRI, KpnI, and
ApaI site, respectively. FRAP 1819C fragment was
generated by PCR. pGEX-4E-BP1 construct was a generous gift from Dr.
Nahum Sonenberg's laboratory at McGill University.
-glycerophosphate, 2 mM EDTA, 2 mM EGTA, 0.3% Triton X-100, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride), immunoprecipitated using
M2 FLAG affinity gel (Sigma). The immune complexes were washed three
times with lysis buffer, two times with lysis buffer containing 0.5 M NaCl, and once with kinase buffer (40 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 20% glycerol, 0.1% Triton
X-100, 10 mM MnSO4, 1 mM DTT),
followed by addition of 20 µl of kinase reaction mixture containing
100 µM ATP, 10 µCi of [
-32P]ATP, and 2 µg of GST-4E-BP1 in the kinase buffer. The reaction was carried out
at 30 °C with rocking for 15 min and stopped by addition of 20 µl
of 2× SDS sample buffer. Samples were heated briefly before loaded on
SDS-PAGE for autoradiography and Western analysis. To assess ATP
binding activity of FRAP, transfection and cell lysis were carried out
as above, followed by addition of ATP-agarose (C8-linked; Sigma) and
rocking at 4 °C for 2 h. The matrix was then washed with lysis
buffer, eluted with SDS sample buffer, and analyzed by Western blotting.
RESULTS
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Fig. 1.
Kinase activity of FRAP N-terminal truncation
mutants. A, primary structures of four deletion mutants
of FRAP are presented diagrammatically. The kinase domain, FRB domain,
and the conserved C-terminal tail are indicated. B,
full-length and various truncation mutants of FRAP were transiently
expressed as FLAG-tagged proteins in HEK293 cells, immunoprecipitated,
and assayed for autokinase activity in vitro as described
under "Experimental Procedures." The reaction mixtures
were run on 7.5% SDS-PAGE and analyzed by Western blot and
autoradiography. Note that the 2061C protein (54 kDa) comigrated with
the IgG heavy chain; expression of this protein was confirmed in cell
lysate before immunoprecipitation (data not shown).
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Fig. 2.
Microinjection of FRB protein into MG63 cells
prevents G1 to S cell cycle progression. MG63 cells
synchronized by serum starvation for 48 h were microinjected with
4.5 mg/ml wild-type FRB protein (FRB) together with 1 mg/ml
fluorescin-dextran (Mr 70,000). As controls,
cells were injected with dextran alone (dextran) or with 5.0 mg/ml rabbit IgG (IgG). Injected cells were incubated in
DMEM with 10% fetal bovine serum for 25 h and in 10 µM bromodeoxyuridine for 2-12 h prior to fixation. The
cells were then stained using a monoclonal anti-BrdUrd antibody as
described under "Experimental Procedures" At least four
independent experiments were performed for each injection condition,
and each experiment yielded 50-200 successfully injected cells.
Representative results are shown here. Statistical data from these
experiments are summarized in Table I. Injected cells are identified by
fluorescein (green), and S phase entry is monitored through
BrdUrd incorporation detected by rhodamine-conjugated secondary
antibody (red).
Statistical data of cell cycle progression upon microinjection of
wild-type and mutant FRB proteins
Arg mutant FRAP was found unable to bind
rapamycin-FKBP12.2 Since this
residue is not situated in the vicinity of the rapamycin binding pocket
or FRB-FKBP12 interface (35), it is unlikely to be directly involved in
rapamycin binding. It is therefore possible that mutations at
Trp2027 may perturb the tertiary structure of FRB; such
mutants would be expected to abrogate the cellular function of FRB.
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Fig. 3.
In vitro
rapamycin-FKBP12 binding properties of mutant FRB proteins.
A, the complete sequence of the FRB protein (amino acids
2015-2114) used in this study is shown, and the residues subjected to
mutational studies are indicated. B, the binding assays were
carried out as described under "Experimental
Procedures," with purified wild-type and mutant FRB
proteins. In addition to binding assays in the absence or presence of
rapamycin, the pure protein for each FRB was loaded as a control (no
rapamycin, no GST-FKBP).
Ile, Tyr2105
Ala, and
Trp2027
Phe were separately introduced into the FRB
cDNA by site-directed mutagenesis using PCR. The mutant FRB
proteins were expressed and purified from E. coli and tested
for rapamycin binding in a pull-down assay using GST-FKBP12 (35). As
shown in Fig. 3B, Ser2035
Ile FRB did not
bind to rapamycin-GST-FKBP12, consistent with earlier observations (36,
43). The loss of binding activity in the Trp2027
Phe
mutant suggests a crucial role of this tryptophan residue in
maintaining the tertiary structure of FRB, since this residue is not
directly involved in rapamycin binding (40). The Tyr2105
Ala mutant FRB bound rapamycin-FKBP12 equally as well as did the
wild-type protein, which is somehow unexpected, as the crystal structure predicted a critical role for this residue (40). On the other
hand, there might be certain degree of affinity decrease resulted from
this mutation that was not detected by our qualitative binding assays,
which were carried out with concentrations much higher than the
wild-type dissociation constant (~5 nM; Ref. 35).
Ile and Tyr2105
Ala mutants
caused G1 arrest in cells upon injection (Fig. 4; Table I). The conserved
Trp2027
Phe mutation, on the other hand, abolished
FRB's inhibitory effect on the cell cycle (Fig. 4; Table I). The
behavior of Ser2035
Ile is consistent with the fact
that full-length FRAP carrying this mutation can signal normally to
downstream components of the pathway (10, 14, 31), indicating that this
mutant most likely retains wild-type functions such as binding of an
activator/effector. The wild-type effect elicited by the
Tyr2105
Ala mutant confirms that this mutation does not
alter FRB's native structure, nor does it interfere with the putative
activator/effector binding. Results from the Trp2027
Phe mutant suggest that this tryptophan residue is crucial for FRB's
dominant negative effect, and is therefore instrumental for the
putative activator/effector binding by FRAP in vivo. This site may be directly involved in binding; alternatively, this residue
may be important for maintaining the tertiary structure of FRB, since a
mutation at this site abolishes rapamycin binding. The loss of the
dominant negative effect resulted from this single mutation strongly
supports the specificity and physiological relevance of the effect
caused by wild-type FRB, and rules out the possibility of an artifact
resulted from injecting high concentrations of proteins.
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Fig. 4.
Effect of mutant FRB proteins on MG63 cell
cycle progression. Serum-starved MG63 cells were microinjected
with various FRB mutant proteins, as described in Fig. 2 legends.
Protein concentrations injected were between 4.5 and 5.0 mg/ml. Three
to eight independent experiments were carried out using each protein,
and each experiment yielded 50-200 successfully injected cells.
Representative results are shown here. Statistical data from these
experiments are summarized in Table I.
Phe mutation on
kinase activity of FRAP. Trp2027
Phe is introduced into
full-length FRAP cDNA carrying Ser2035
Thr, which
has been shown not to affect FRAP's activity and function but to
confer rapamycin resistance (10, 14, 31). The mutant FRAP proteins
tagged with a FLAG epitope were transiently expressed in HEK293 cells,
immunoprecipitated, and subjected to kinase assays. As shown in Fig.
5, the Trp2027
Phe
mutation abolished FRAP's activity in both autophosphorylation and
phosphorylation of purified 4E-BP1, whereas the Ser2035
Thr mutation did not affect the kinase activity. A point mutation (Asp2357
Glu) at a conserved catalytic site also
inactivated FRAP kinase activity, consistent with previous reports (10,
30).
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Fig. 5.
Trp2027 Phe mutation
abolishes FRAP kinase activity. Wild-type and various mutants of
FRAP were transiently expressed as FLAG-tagged proteins in HEK293
cells. Immunoprecipitation and kinase assays were carried out as
described under "Experimental Procedures." Both
autophosphorylation and phosphorylation of GST-4E-BP1 were examined.
rap-insensitive, Ser2035
Thr;
kinase-dead, Asp2357
Glu;
FRB-defective, Trp2027
Phe. The latter
mutations are constructed on rap-insensitive background.
Phe
mutant proteins, confirming the specificity of the binding. In
comparison to protein kinase A, the Asp2357 site in the
kinase domain is thought to be crucial for catalysis but not to be
involved in ATP binding (44). As expected, Asp2357
Glu
mutant FRAP retains wild-type affinity for ATP (Fig. 6). The ATP
binding of the Trp2027
Phe mutant FRAP indicates that
inactivation of kinase activity by this mutation is not due to a direct
perturbation on the structure of the kinase domain. Taken together, an
attractive model for FRAP function emerges, where the FRB domain may
serve as a binding site for a positive regulator of FRAP kinase
activity, although it cannot be excluded that FRB may be an integral
part of the kinase domain. Since the kinase activity of FRAP has been
shown to be essential for FRAP signaling (10, 14, 31), the behavior of
Trp2027
Phe mutation provides a direct link between the
observed cell cycle arrest effect upon FRB microinjection and the
function of FRAP.
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Fig. 6.
ATP binding properties of mutant FRAP
proteins. Upper panel, ATP-agarose
(C8-linked) was incubated for 2 h at 4 °C with HEK293 cell
lysates containing transiently expressed FLAG-tagged wild-type and
mutant FRAP proteins. After extensive washing with lysis buffer, the
bound proteins were eluted by SDS sample buffer and analyzed by Western
using M2 FLAG antibody. Lower panel, same
procedures were carried out as above with or without 1 mM
free ATP added to the lysates before incubation with ATP-agarose.
rap-insensitive, Ser2035 Thr;
kinase-dead, Asp2357
Glu;
FRB-defective, Trp2027
Phe. The latter
mutations are constructed on rap-insensitive background.
DISCUSSION
Phe) in the FRB domain abolishes FRAP kinase activity, both in
autophosphorylation and in phosphorylation of 4E-BP1, provides compelling evidence for the involvement of FRB in the function of the
kinase domain. The dominant negative effect on the G1 cell cycle elicited by microinjection of the wild-type (but not
Trp2027
Phe) FRB suggests a plausible mechanism for FRB
function, in which the FRB domain interacts with an activator of FRAP
kinase in vivo and an excess of free FRB titrates off this
activator. The observed concentration dependence of FRB effect upon
microinjection is consistent with such a model; a threshold
concentration would be determined by both the cellular abundance of the
putative activator and the affinity of FRAP/activator interaction. The
different roles of the two critical residues, Trp2027 and
Ser2035, in the FRB domain have provided insight into the
interaction between FRB and the putative activator. Although serine
2035 has been shown to locate in the rapamycin binding cleft (35),
mutation at this site (Ser2035
Ile) does not seem to
change the physiological function of FRB, whereas Trp2027,
with no direct contact with rapamycin or FKBP12, is crucial for FRB
function. Thus, the presumed interaction with an activator and
rapamycin binding of FRB may not share the same molecular mechanisms.
Nonetheless, one can envision that the functional interaction between
FRB and the putative activator is abolished by the binding of
rapamycin-FKBP12 complex, due to either steric hindrance or
conformational change. The lack of complete rapamycin inhibition of
FRAP kinase activity in vitro may be due to artifacts of the
in vitro systems. Alternatively, partial inhibition may be
sufficient for bringing FRAP activity below a threshold level critical
for signaling. Finally, we cannot exclude the possibility that FRB has
yet another function, which is targeted by rapamycin.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Vladimir Gelfand, Carolina Tuma, and Steve Rogers for their generosity in providing technical assistance and instrument for the microinjection experiments, and for helpful discussions. We also thank Dr. Nahum Sonenberg and Dr. Anne-Claude Gingras for providing the plasmid pGEX-4E-BP1.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Dept. of Cell and
Structural Biology, University of Illinois, Urbana-Champaign, 601 S. Goodwin Ave., B107, Urbana, IL 61801. E-mail:
jchen{at}life.uiuc.edu.
The abbreviations used are: p70s6k, 70 kDa S6 kinase; FKBP12, FK506-binding protein; FRAP, FKBP12-rapamycin-associated protein; FRB, FKBP12-rapamycin-binding (domain); rap, rapamycin; RAFT1, rapamycin and FKBP12 target 1; mTOR, mammalian target of rapamycin; TOR1, TOR2, target of rapamycin 1 and 2; PIK, phosphatidylinositol kinase; 4E-BP1, eIF4E-binding protein 1; GST, glutathione S-transferase; PCR, polymerase chain reaction; DTT, dithiothreitol; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.
2 J. Chen, unpublished observation.
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REFERENCES |
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