(Received for publication, June 28, 1995; and in revised form, July 17, 1995)
From the
The immunosuppressant rapamycin prevents cell cycle progression in several mammalian cell lines and the yeast Saccharomyces cerevisiae. In mammalian cells, rapamycin binds to the small FK506-binding protein, FKBP12, allowing the drug-receptor complex to interact with the 289-kDa RAFT1/FRAP proteins. These proteins, along with their yeast homologs, TOR1/DRR1 and TOR2/DRR2, contain a C-terminal domain with amino acid homology to several phosphatidylinositol (PI) 4- and 3-kinases. However, no direct demonstration of kinase activity for this family of proteins has been reported. We now show that RAFT1, immunoprecipitated from rat brain and MG63 and HEK293 cells, contains PI 4-kinase activity and that rapamycin-FKBP12 has no effect on this activity. Thus, it is likely that, in vivo, rapamycin does not directly inhibit the PI 4-kinase activity and affects the RAFT1/FRAP protein through another mechanism.
The ability of rapamycin, a potent immunosuppressant drug, to
arrest a variety of mammalian cells and the yeast Saccharomyces
cerevisiae in the G stage of the cell
cycle(1, 2, 3, 4) has made it a
valuable tool for studying the intermediate signaling events that
convey mitogenic stimuli to the cell nucleus.
Rapamycin binds with low nanomolar affinity to the FK506-binding protein (FKBP12), a small soluble protein that is also the intracellular receptor for FK506, a structurally similar immunosuppressant(5, 6, 7) . Analogous to the mechanism of action of FK506, it is the drug-receptor complex that mediates the effects of the rapamycin, which include inhibition of the 70-kDa S6 kinase (8, 9) and of several cyclin-dependent kinases(2, 3, 4) . The immunosuppressant effects of FK506 derive from the binding of the FK506-FKBP12 complex to the calcium-activated phosphatase, calcineurin, and the resulting inhibition of its activity(10) . Although rapamycin binds to FKBP12, the complex does not interact with calcineurin. Instead, rapamycin-FKBP12 binds to a recently purified and molecularly cloned protein designated rapamycin and FKBP12 target-1 (RAFT1) in rats (11) and FKBP-rapamycin-associated protein (FRAP) in humans(12) . Several other groups have subsequently purified and/or cloned the same protein(13, 14, 15) .
RAFT1 is a
2549-amino acid protein with a predicted molecular mass of 289 kDa and
is thought to be a mammalian homolog of the products of two yeast
genes, TOR1/DRR1 and TOR2/DRR2, which when mutated
lead to dominant rapamycin resistance in
yeast(1, 16, 17) . The C-terminal 600-amino
acid domain of RAFT1 and the yeast TOR proteins has homology to several
PI ()kinases, including the p110 subunit of mammalian PI
3-kinase(18) ; the yeast PI 3-kinase VPS34(19) ; two
yeast PI 4-kinases, STT4 (20) and
PIK1(21, 22) ; and the recently cloned mammalian PI
4-kinase, PI4K
(23) . This homology to known lipid kinases
has lead to the suggestion that RAFT1 may also be a PI kinase and that
rapamycin exerts its effects by inhibiting this activity(11) .
However, no direct demonstration of kinase activity for RAFT1/FRAP or
the yeast TOR proteins has been reported.
In this study, we show that immunoprecipitates of endogenous RAFT1 from rat brain or tissue culture cells contain PI 4-kinase activity and that, surprisingly, rapamycin-FKBP12 has no effect on this activity.
Proteins separated by 6% SDS-polyacrylamide gel electrophoresis were
transferred to nitrocellulose on a semidry apparatus using Dunn
carbonate buffer(27) . Blots were blocked for 1 h in PBS with
5% milk, incubated overnight with 2 µg/ml antibody in PBS with 3%
BSA, washed 3 5 min in PBS with 5% milk, incubated for 1 h with
horseradish peroxidase-conjugated anti-rabbit secondary antibody,
washed as before, and detected with ECL (Amersham Corp.).
The inability to detect kinase activity
of recombinant RAFT1 might reflect an improper folding of the
overexpressed protein, an interference by the epitope tag, or the
absence of post-translational modifications or of another subunit
necessary for activity. To investigate the lipid kinase activity of
endogenous RAFT1, we developed antibodies to distinct portions of the
RAFT1 protein itself, using as immunogens three synthetic peptides
corresponding to regions at the N and C termini and within the central
portion of RAFT1 (Fig. 1A). The -64-RAFT1 antibody
recognizes a single band with an apparent molecular mass of 220 kDa in
extracts from brain and MG63 osteosarcoma cells as well as from control
HEK293 cells or HEK293 cells that overexpress RAFT1 (Fig. 1B). Similarly, antibodies
-782-RAFT1 and
-C-term-RAFT1 recognize a protein of the same apparent molecular
mass (data not shown), which also corresponds to that of the RAFT1
protein purified using a rapamycin-FKBP12 affinity
column(11, 12, 13, 15) .
Figure 1:
Characterization of
anti-RAFT1 peptide antibodies. A, shown is a diagram of the
location of the RAFT1 sequences that were used in the generation of
peptide antibodies (Ab), the minimal FKBP-rapamycin-binding
domain (FRB)(47) , the serine (Ser-2035) that when
mutated to arginine inhibits FKBP-rapamycin binding(47) , and
the putative lipid kinase domain. B, on Western blots, the
-64-RAFT1 antibody recognizes a 220-kDa protein in membranes from
rat brain (60 µg), MG63 cells (150 µg), and HEK293 cells (150
µg) and in detergent lysates (10 µg) of 293 cells transfected
with a cytomegalovirus vector containing the cDNA for RAFT1 (RAFT1), but not with the vector alone (vector). The
ECL exposure times were as follows: brain, 1 min; MG63 and 293 cells, 2
min; and transfected cells, 30 s. C, the
-64-RAFT1
antibody immunoprecipitates a 220-kDa protein and recognizes proteins
of the same molecular mass in immunoprecipitates made with two other
-RAFT1 peptide antibodies. Shown is a Western blot probed with the
-64-RAFT1 antibody of immunoprecipitates (I.P.) prepared
from rat brain with the
-64-RAFT1,
-782-RAFT1, and
-C-term-RAFT1 antibodies(-) or with the antibodies
preincubated with the peptides used in their generation (+). The
ECL exposure time was 30 s.
We
conducted immunoprecipitation experiments in combination with Western
blotting to ascertain that the epitopes recognized by the three
different antibodies are all part of the same molecule. When
-64-RAFT1 is used to probe Western blots of immunoprecipitates
prepared with each of the three anti-RAFT1 antibodies, a band of M
= 220,000 is always detected, which is
eliminated by preincubation of the immunoprecipitating antibody with
the peptide against which it was raised (Fig. 1C).
Immunoreactive bands of lower molecular weight that are also eliminated
by antigen preincubation probably represent degradation products of the
large RAFT1 protein.
Figure 2:
RAFT1 immunoprecipitates contain PI kinase
activity that is activated by nonionic detergent and not inhibited by
adenosine or wortmannin. A, PI kinase assays using PI and PS
as substrates in the presence of 0.2% Nonidet P-40 were performed on
RAFT1 immunoprecipitates (I.P.) from brain or MG63 or HEK293
cells prepared as described for Fig. 1B. Phosphorylated
lipids were chloroform-extracted, separated by TLC, and detected by
autoradiography. The migration position of the PIP standard is
indicated. The TLC plate was exposed to film for 10.5 h (brain), 36 h
(MG63), and 24 h (HEK293). B, RAFT1 was immunoprecipitated
from brain with the -64-RAFT1 antibody and assayed for PI kinase
activity under the conditions indicated. Shown is an autoradiograph of
the TLC plate (toppanel). The activity of each
reaction was quantitated by densitometry of the autoradiograph and is
presented as percentage of total activity in the presence of 0.2%
Nonidet P-40 (bottompanel). This experiment was
repeated three times with similar results.
To characterize the
type of PI kinase involved, we took advantage of the known properties
of certain PI kinases(30) . Type II PI 4-kinase is activated by
nonionic detergents and inhibited by adenosine, while type III PI
4-kinase activity is much less sensitive to either treatment. The PI
kinase activity is stimulated over 20-fold by the detergent Nonidet
P-40, but is decreased only 20% with a high concentration of
adenosine (Fig. 2B), indicating that it does not
represent either a conventional type II or III PI 4-kinase.
Wortmannin is a potent in vitro inhibitor of PI 3-kinase that is used extensively to implicate this enzyme in biological processes(31) . The observation that both wortmannin and rapamycin inhibit p70 S6 kinase activity (32) raises the possibility that these drugs target the same molecule, i.e. RAFT1. We detect no inhibition of PI kinase activity with 5 nM wortmannin, a concentration sufficient to inhibit the known mammalian PI 3-kinase(31) , and only a slight reduction in PI kinase activity at 1000 nM wortmannin (Fig. 2B), a concentration that inhibits reported PI 3-kinase activities(33, 34) , type III PI 4-kinase activity(34) , and a novel wortmannin-sensitive PI 4-kinase activity(35) . The inability of wortmannin to inhibit RAFT1 PI kinase activity indicates that RAFT1 is not likely to be a target of wortmannin in vivo and that the similar effects of wortmannin and rapamycin upon p70 S6 kinase activity are mediated through different molecules. Recent studies that identify the domains of the p70 S6 kinase molecule required for its mitogen-stimulated activation and rapamycin and wortmannin inhibition support this conclusion(36) .
To identify the position on the inositol
ring of PI that is phosphorylated by RAFT1 immunoprecipitates, we
conducted HPLC analysis on the PIP produced after its deacylation (Fig. 3). The P-labeled product of the
phosphorylation activity migrates identically to the
[
H]PI-4-P standard. In this HPLC system, PI-3-P
reproducibly elutes from the column at an earlier time
point(28) .
Figure 3:
The PIP generated by RAFT1
immunoprecipitates is PI-4-P. The P-labeled PIP generated
as described for Fig. 2A was excised from the TLC
plate, deacylated(29) , and subjected to HPLC
analysis(28) . The positions of the standards
[
H]PI-4-P and
[
H]PI-4,5-P
are shown (thinline). The [
P]PIP (thickline with diamonds) comigrates with the PI-4-P
standard. The positions of ADP and ATP, internal standards used to
compare separate HPLC analyses, are indicated with arrows.
This experiment was repeated three times with similar results. In
addition, HPLC analyses demonstrate that the PIP generated by the RAFT1
immunoprecipitates in the absence of detergent is also PI-4-P (data not
shown).
Figure 4:
Rapamycin-FKBP12 does not inhibit the
RAFT1 PI 4-kinase activity, but is able to bind to RAFT1
immunoprecipitates. Top panel, RAFT1 immunoprecipitates were
preincubated with 100 nM FKBP12, 100 nM FK506-FKBP12,
or 100 nM rapamycin-FKBP12, and PI kinase assays were
performed. All samples contain equivalent amounts of ethanol, the
vehicle for the drugs. The experiment was repeated five times with
similar results. Bottom panel, RAFT1 immunoprecipitates were
treated as described above, except that 10,000 cpm of P-labeled FKBP12 (11) were substituted for
unlabeled FKBP12. After a 45-min incubation, the immunoprecipitates
were washed twice and resolved by 18% SDS-polyacrylamide gel
electrophoresis, and the dried gel was exposed to film. The experiment
was repeated twice with similar results.
The failure of
rapamycin-FKBP12 to inhibit the PI 4-kinase activity in the previous
experiments could result from an inability of the drug-receptor complex
to interact with RAFT1 after it has been immunoprecipitated. We showed,
however, that under the experimental conditions used in the lipid
kinase assay, P-labeled FKBP12 does bind to the
immunoprecipitated RAFT1 in a rapamycin-dependent fashion (Fig. 4, bottompanel). Furthermore, the
addition of rapamycin to brain homogenates prior to immunoprecipitation
in order to elicit the binding of rapamycin-FKBP12 to RAFT1 also does
not result in an inhibition of the PI 4-kinase activity of the
immunoprecipitates (data not shown).
In summary, we have
demonstrated that immunoprecipitates of RAFT1, which has a lipid kinase
domain with amino acid homology to both PI 4- and 3-kinases, contain PI
4-kinase activity. We have only tested PI, PI-4-P, and PI-4,5-P as in vitro substrates, and it is possible that RAFT1
could act upon other phosphatidylinositols that already possess more
than one phosphate. For instance, RAFT1 might phosphorylate PI-3-P on
the 4-position, and it may be the true in vivo substrate.
Furthermore, our results do not rule out that RAFT1 may phosphorylate
specific protein substrates. The mammalian PI 3-kinase and the yeast PI
3-kinase VPS34 display protein kinase
activity(37, 38, 39) , but this activity has
not been reported for PI 4-kinases.
The inability of rapamycin-FKBP12 to inhibit the PI 4-kinase activity of RAFT1 raises questions about the mechanism of rapamycin's pharmacological actions. Although our results do not address the effects of rapamycin-FKBP12 on the PI 4-kinase activity of RAFT1 in vivo, it is likely that the drug does not directly inhibit the kinase activity of the protein. The drug could still effectively inhibit the lipid kinase activity by altering the intracellular localization of the enzyme and preventing its access to lipid substrates located on membranes. In addition, rapamycin-FKBP12 may prevent RAFT1 from interacting with substrates for a putative protein kinase activity or inhibit post-translational modifications of RAFT1, such as phosphorylation, that may be necessary for activity. Alternatively, RAFT1 may associate with a PI 4-kinase that coimmunoprecipitates with RAFT1 but is unaffected by rapamycin-FKBP12.
The finding that RAFT1
immunoprecipitates contain PI 4kinase activity is also of importance
outside of the field of immunophilin research. Multiply phosphorylated
forms of PI are involved in numerous aspects of signal transduction.
Most notably, PI-4-P is the precursor for PI-4,5-P, which
is hydrolyzed by phospholipase C into inositol 1,4,5-trisphosphate and
diacylglycerol, important mediators of intracellular calcium release
and protein kinase C activity, respectively(40) . PI-4-P
synthesis can be activated by ligand binding to membrane receptors for
neurotransmitters, hormones, and growth
factors(41, 42, 43) . Therefore, one might
expect that the lipid kinase activity of RAFT1 will be tightly
regulated.
PI-4-P and PI-4,5-P are emerging as important
regulators of the cytoskeleton and of vesicle fusion during membrane
trafficking (44) . Furthermore, the finding that the gene
product mutated in ataxia telangiectasia (45) is a RAFT1
homolog indicates that derangements of phosphoinositide synthesis may
cause human disease. The cDNA for only one PI 4-kinase has been
isolated from mammalian tissues(23) . The identification of
these PI 4-kinases, as well as the recent cloning of the cDNA for a
PI-4-P 5-kinase(46) , may clarify the regulation of
phosphoinositide biosynthesis and their role in controlling cell
growth.