From the
Tuberous sclerosis (TSC) is a human genetic syndrome
characterized by the development of benign tumors in a variety of
tissues, as well as rare malignancies. Two different genetic loci have
been implicated in TSC; one of these loci, the tuberous sclerosis-2
gene (TSC2), encodes an open reading frame with a putative
protein product of 1784 amino acids. The putative TSC2 product
(tuberin) contains a region of limited homology to the catalytic domain
of Rap1GAP. We have generated antisera against the N-terminal and
C-terminal portions of tuberin, and these antisera specifically
recognize a 180-kDa protein in immunoprecipitation and immunoblotting
analyses. A wide variety of human cell lines express the 180-kDa
tuberin protein, and subcellular fractionation revealed that most
tuberin is found in a membrane/particulate (100,000
Members of the Ras-related family of small GTP-binding proteins
are involved in many different biological
functions(1, 2) . These proteins, which bind and
hydrolyze GTP, are active when bound to GTP and inactive in the
GDP-bound state(3, 4) . Guanine nucleotide binding by
the Ras-related GTPases is regulated by cellular enzymes. Cellular
regulatory proteins include both positive regulators, the guanine
nucleotide exchange factors and negative regulators, known as GTPase
accelerating proteins (GAPs)
The Ras subfamily of small molecular weight GTPases is known to
regulate mitogenic signal transduction pathways linking plasma membrane
receptors to the nucleus. Thus Ras proteins are critical in controlling
the proliferation of many diverse cell types(4, 6) . The
critical role of GAP proteins in regulating the activity of the
Ras-like GTPases has been demonstrated by studies of the human genetic
disease, neurofibromatosis type 1 (NF1). The product of the NF1 tumor suppressor gene, neurofibromin, acts as a specific GAP for
Ras proteins (3). Loss of neurofibromin expression is associated with
the constitutive activation of Ras in cell lines derived from tumors of
patients with NF1(7, 8) .
Tuberous sclerosis (TSC)
presents many striking parallels with NF1, including autosomal dominant
inheritance, the appearance of benign tumors and other abnormalities in
multiple organ systems, and development of rare malignancies in
affected individuals(9) . Unlike NF1, TSC has been linked to two
different loci, TSC2 on chromosome 16, and an unidentified
gene on chromosome 9 (TSC1)(10, 11) . Loss of
heterozygosity at the TSC2 locus has been demonstrated in
tumors of human TSC patients, strongly suggesting that this gene
functions as a tumor suppressor(12) . This implication has been
substantiated by studies of the Eker rat strain, in which
susceptibility to bilateral renal cell carcinoma is caused by a
germline mutation in the rat TSC2 gene, and tumor development
is associated with somatic loss of heterozygosity at TSC2(13, 14) . The 5.5-kilobase transcript of TSC2 is widely expressed, consistent with the multiple organs
affected in TSC, including the brain, heart, skin, kidneys, lungs, and
others(9, 13, 15) .
The predicted TSC2 product, designated tuberin, comprises 1784 amino acids. A
58-amino acid region near the C terminus has limited homology (19
identical amino acids) with a portion of the catalytic domain of
Rap1GAP(15) . Rap1GAP acts as a negative regulator of the Rap1a
and Rap1b GTPases, which share greater than 50% amino acid identity
with Ras(16, 17) . Although originally identified as an
antagonist of Ras transformation (see ``Discussion''), Rap1
(also referred to as K-rev1 and smg p21) has been
shown to induce DNA synthesis and morphological changes when
microinjected into Swiss 3T3 fibroblast cells(18) . These
results demonstrated that like Ras, the Rap1 proteins may act as
positive mitogenic signaling molecules.
The homology between tuberin
and Rap1GAP suggests a possible molecular similarity between
neurofibromin, a regulatory GAP for Ras, and tuberin, a potential
regulator of Rap1. Investigation of this possibility has awaited the
identification and characterization of the TSC2 product. To
address these questions, we have raised antisera to bacterial fusion
proteins encoding the N-terminal and C-terminal regions of tuberin.
Immunoprecipitation and immunoblotting with these antisera have
identified tuberin as a widely expressed 180-kDa molecule, with
associated GAP activity that is specific for Rap1. These findings have
potential implications for the tumor suppressor function of TSC2.
To isolate fusion
proteins present in inclusion bodies, the bacteria were induced with 1
mM isopropyl-
Following electroblotting, the membrane
was blocked overnight with Tris-buffered saline, 0.1% Nonidet P-40, and
3% bovine serum albumin. After blocking, the membrane was incubated
with 15 µg/ml digoxigenin-labeled anti-Tub-C IgG (for Fig. 2a, 2c, 3, and 4). Immunoblotted proteins
were detected by incubation with sheep anti-digoxigenin
F
Here we have described the identification of the TSC2 gene product, tuberin, and its initial biochemical
characterization. In particular, the discovery of tuberin-associated
GAP activity toward Rap1a suggests that this activity represents a
primary biochemical function of tuberin. Full-length native tuberin,
isolated by immunoprecipitation, displayed significantly more GAP
activity than a recombinant C-terminal fragment of tuberin, suggesting
that this activity may be positively regulated by post-translational
modification of tuberin in mammalian cells. Alternatively, full GAP
activity may require a larger region of the C terminus than that which
was tested (amino acids 1387-1784). In either case, it is clear
that tuberin does interact physically with Rap1a, and the primary site
of this interaction resides within the tuberin C terminus. Additional
evidence that the C-terminal region of tuberin is critical for its
biological function comes from the study of tumor induction in the Eker
rat strain, which is caused by an insertion resulting in premature
termination of tuberin upstream from the catalytic
domain(13, 14) .
These results do not, however,
determine whether Rap1a represents the sole or even the primary target
for the GAP activity of tuberin, as more than 50 small molecular weight
GTP-binding proteins are known to be present inside mammalian
cells(3) . Rap1b, which represents the other isoform of Rap1, is
likely to be sensitive to tuberin GAP activity, as Rap1b is highly
homologous to Rap1a and no biochemical differences between the isoforms
have been demonstrated(16, 29) . The finding that Ras,
Rho, Rac, and especially Rap2 (which shares 60% homology with Rap1) are
resistant to the GAP activity of tuberin strongly suggests that Rap1 is
the primary target for this activity. The vast majority of tuberin was
found in the particulate/membrane fraction of cells, consistent with
the possibility that tuberin might co-localize with Rap1a, which is
associated with intracellular vesicles (30).
If Rap1 does serve as
the primary target for the GAP activity of tuberin, the role of this
protein in the development of tumors in TSC patients may be through one
of several different mechanisms. Evidence that Rap1 mediates a positive
growth signal has come from studies in which it was shown to induce DNA
synthesis and morphological changes, when micro-injected into Swiss 3T3
cells(18) . In another study, a gain-of-function mutant in the Drosophila melanogaster homologue of rap1 was shown
to result in a roughened eye phenotype. Revertants of this mutant
displayed significantly lower expression of the rap1 gene.
Loss-of-function mutants of rap1 were found to be lethal,
suggesting a positive role for the rap1 product in cell
proliferation or cell viability (28).
In light of these findings
suggesting a positive signaling role for Rap1, the presumed lack of
functional tuberin in tumors of patients with TSC and in renal cell
tumors of the Eker rat may reflect aberrant regulation of Rap1,
resulting in its constitutive activation. While tuberin is clearly not
the sole source of Rap1GAP activity in K-562 cells, its activity could
be critical in certain cell types, particularly in cells that are
predisposed to tumor formation in TSC patients. In this scenario,
tuberin would be functionally analogous to the NF1 product
neurofibromin, a RasGAP critical for regulation of Ras in tumor cells
of NF1 patients(7, 8) . Recent studies have demonstrated
that while neurofibromin is critical as a regulator of GTP
An
alternative model for the function of tuberin and Rap1 is suggested by
studies which have raised the possibility that Rap1 has a negative
effect on cell growth. These studies originated with the demonstration
that rap1a can antagonize ras-induced transformation. rap1a was identified as a cDNA that, when overexpressed, could
cause reversion of the transformed phenotype in NIH 3T3 bearing an
oncogenic K-ras gene(17) . In addition, expression of
constitutively active rap1 (valine 12) was shown to inhibit
the activation of MAP kinase following simulation with epidermal growth
factor and lysophosphatidic acid, although with less efficiency than a
dominant-inhibitory mutant of ras (rasN17)(33) . Further studies have demonstrated
that Rap1 is capable of binding to all of the putative mitogenic
effectors for mammalian Ras: RasGAP, Raf, and phosphatidylinositol-3`
kinase(34, 35) .
While Rap1 is
likely to be a primary target for the GAP activity of tuberin, we
cannot exclude the possible involvement of other small molecular weight
GTP-binding proteins. Recently, Spa-1, a protein with homology to
Rap1GAP and tuberin, was shown to have GAP activity toward both Rap1
and the nuclear-localized, Ras-related GTPase Ran(37) . Also,
given the fact that Rap1 binds to RasGAP with high affinity, it is
possible that tuberin suppresses cell growth by binding to Ras, without
stimulating its GTPase. In future studies, we will pursue the
possibility that the tumor suppressor function of tuberin is related to
its ability to regulate, or interact with, other members of the Ras
superfamily in addition to Rap1. Cell lines derived from tumors of the
Eker rat should greatly facilitate these investigations of the tumor
suppressor function of tuberin.
We thank Dr. Jean de Gunzburg, Dr. Alan Hall, and Dr.
Albert Reynolds for providing p21 proteins and/or expression plasmids,
respectively. We also thank Lisa Leonardsen and Alex Papageorge for
advice and Doug Lowy for continual support and interest.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
g)
fraction. Immunoprecipitates of native tuberin contain an activity that
specifically stimulates the intrinsic GTPase activity of Rap1a. These
results were confirmed in assays with a C-terminal fragment of tuberin,
expressed in bacteria or Sf9 cells. Tuberin did not stimulate the
GTPase activity of Rap2, Ha-Ras, Rac, or Rho. These results suggest
that the loss of tuberin leads to constitutive activation of Rap1 in
tumors of patients with tuberous sclerosis.
(
)(5) . The
GAP proteins act by stimulating the intrinsic GTPase of the GTP-binding
proteins, keeping them in the inactive, GDP-bound state(4) .
Plasmids and Cell Lines
Two cDNA fragments
encoding regions of the human TSC2 gene were obtained by polymerase
chain reaction amplification from a fetal brain cDNA library
(Clontech)(19) . One encodes amino acids Ala-2 to Ala-306 of
tuberin (Tub-N), and the other encodes amino acids Leu-1387 to Val-1784
(Tub-C)(15) . The DNA fragments were cloned into the vector
pGEX-4T2 (Pharmacia Biotech Inc.) using BamHI and SalI, for expression as glutathione S-transferase
(GST) fusion proteins. The DNA sequences of the fragments were verified
by direct DNA sequencing (U. S. Biochemical Corp.). For control
purposes, a region of the mouse CDC25mM/GRF gene, comprising the dbl-homology (DH) region (amino acids Arg-211 to Leu 512), was
also cloned into pGEX-4T2(20) . For expression as a GST fusion
protein in Sf9 cells, the fragment encoding Tub-C was subcloned into
the baculovirus transfer vector pAcG2T (Pharmingen) using BamHI and EcoRI. All cell lines were obtained from
the American Type Tissue Collection, and were cultured according to the
conditions supplied.
Bacterial Protein Synthesis and Partial
Purification
HB101 Escherichia coli cells bearing the
pGEX-4T2 plasmids encoding GST-Tub-N, GST-Tub-C, or GST-DH were grown
to an optical density of 1.0 (600 nm) and induced with 50
µM isopropyl--D-thiogalactopyranoside for 2
h at 28 °C. The cells were pelleted, resuspended in 1/50 volume of
phosphate-buffered saline (PBS), 0.1% Nonidet P-40, and 1 mM dithiothreitol (DTT), and lysed by sonication. Sonicated lysates
were centrifuged at 15,000
g to pellet insoluble
material. Aliquots of the supernatants were used immediately for
experiments measuring GAP activity. To estimate the concentration of
the GST fusion proteins in the lysates, glutathione-coupled Sepharose
beads (Pharmacia) were added to an aliquot of the supernatant,
incubated for 2 h at 4 °C, washed four times with PBS, and
subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) together
with purified carbonic anhydrase (Sigma) as a standard. The gels were
stained with Coomassie Brilliant Blue-G (BDH).
-D-thiogalactopyranoside for 3 h at
37 °C. Bacteria were lysed by sonication in PBS, 1% Nonidet P-40,
and 1 mM DTT. Unlysed cells were removed by low-speed
centrifugation, and the inclusion bodies were pelleted by 15,000
g for 15 min, washed twice with PBS, 1% Nonidet P-40,
1 mM DTT, and 2 M urea, and repelleted. Concentration
and purity were estimated by SDS-PAGE. The purity of GST-Tub-N and
GST-Tub-C in these preparations was 80-90%. Acetone powder
preparations of HB101 cells expressing GST-Tub-N, GST-Tub-C, and GST-DH
were prepared as described(21) .
Antisera
300-400 µg of purified
GST-Tub-N and GST-Tub-C (in the form of inclusion bodies) were used to
immunize New Zealand White rabbits with Freund's adjuvant.
Booster injections were carried out 3, 5, and 9 weeks later. For use in
Western blotting, immunoglobulin G (IgG) was isolated from the sera of
rabbits immunized with Tub-C by binding to protein A, and conjugated
with digoxigenin-3-O-methylcarbonyl--aminocaproic
acid-N-hydroxysuccinimidine ester according to the
manufacturer's instructions (Boehringer Mannheim).
Immunoprecipitation and Immunoblotting of
Tuberin
For analysis by combined immunoprecipitation/Western
blotting, 10 cells per sample lane (in a volume of 300
µl) were used. Cell lysis and immunoprecipitation were performed as
described (22), using 10 µl of antiserum (a 1:30 dilution). The
samples were subjected to SDS-PAGE on 6% gels, and proteins were
electroblotted onto an Immobilon-P membrane (Millipore). For blocking
experiments, antisera were incubated with acetone powder suspensions
(0.1% w/v) of bacterial proteins for 2 h at 4 °C, then clarified by
centrifugation before use.
-alkaline phosphatase conjugate, and chemiluminescence
substrate Lumi-Phos530 (Boehringer Mannheim). The immunoblot shown in Fig. 2b employed different nonconjugated antisera at a
dilution of 1:2,000, as indicated. This blot was developed with an
anti-rabbit peroxidase-based enhanced chemiluminescence detection kit
(Kirkegaard and Perry). To estimate the molecular weight of tuberin,
both prestained molecular weight standards (BRL) and unstained
standards (Pharmacia) were employed.
Figure 2:
Identification of tuberin by
immunoprecipitation/Western blot analysis. Protein extracts were
prepared from cultured cell lines and subjected to immunoprecipitation
with the indicated antiserum. Immunoprecipitates were washed, separated
by SDS-PAGE, and transferred to polyvinylidene difluoride membranes,
and immunoblotting was carried out with the indicated antibody. a, lysates of K-562 cells were subjected to
immunoprecipitation (IP) with preimmune, unrelated, or
anti-Tub-N serum, and probed with anti-Tub-C serum. b, lysates
of G-401 cells were analyzed with anti-Tub-N or anti-Tub-C serum, and
the immunoblot was incubated with preimmune, unrelated, or anti-Tub-C
serum. A specific band of 180 kDa corresponding to tuberin was observed
in both cell lines (arrowhead). c, prior to
immunoprecipitation of K-562 cell lysates, the anti-Tub-N serum was
incubated with acetone powder preparations of bacteria expressing
GST-Tub-C, GST-DH, GST-Tub-N, or with buffer alone. Specific blocking
of the anti-Tub-N serum by the GST-Tub-N preparation was observed,
following immunoblotting with anti-Tub-C
serum.
Sf9 Cell Expression
Sf9 cells were co-transfected
with the baculovirus transfer vector pAcG2T-GST-Tub-C and BaculoGold
baculovirus DNA (Pharmingen), and recombinant baculoviruses encoding
the GST-Tub-C protein were isolated and enriched according to the
manufacturer's protocol. Sf9 cells (3 10
)
were infected with virus (encoding either GST-Tub-C, or bovine
papilloma virus L1 protein as a control), and were harvested after 50
h. Cells were washed with GAP assay buffer, and lysed by sonication in
1 ml of GAP assay buffer. Lysates were clarified by centrifugation, and
the concentration of GST-Tub-C was estimated as for bacterial lysates.
Cell Fractionation
1.4 10
K-562 cells were pelleted, washed with PBS, and split in two. One
portion of the cells was lysed with 2 ml of lysis buffer, while the
other aliquot was used to prepare the 100,000
g supernatant (S100) and particulate/membrane (P100) fractions, as
described previously(22) . Equal portions of the S100, P100, and
the lysis-buffer lysates were subjected to immunoprecipitation and
immunoblotting as described above.
GAP Assays
For each GAP assay reaction with immune
complexes of tuberin, the anti-Tub-C serum was used to
immunoprecipitate tuberin from lysates of 4 10
K-562, prepared in 120 µl of lysis buffer. Immunoprecipitates
were bound to protein A-Sepharose beads, and the immune complexes were
washed three times with lysis buffer, then three times with GAP assay
buffer (20 mM Tris
HCl, pH 7.5, 10 mM MgCl
, 100 mM NaCl, 1 mM DTT, and 40
µg/ml bovine serum albumin). Then the beads were resuspended in GAP
assay buffer in one-fourth of the original volume. GAP assays were
carried out essentially as described(23) . Briefly, 0.5
µM GTP-binding proteins were loaded with 5 µM [
-
P]GTP in loading buffer (0.1 M sodium phosphate, 5 mM MgCl
, 0.5 mM EDTA, 0.5 mM DTT, 0.5 mg/ml bovine serum albumin, and 50
µg/ml sodium deoxycholate), in a volume of 10 µl, then diluted
into 600 µl of cold GAP assay buffer with 10 µM unlabeled GTP. 50 µl of this mixture was then added to the
different tuberin or GST-Tub-C preparations. Assays included 30 µl
of native tuberin bound to protein A-Sepharose beads (isolated from 4
10
K-562 cells), 10 µl of crude (200 nM GST-Tub-C) bacterial lysate, or (1 µM GST-Tub-C) Sf9
cell lysate. Reactions were carried out in duplicate at 32 °C, and
10-µl portions of each reaction were removed at the start or after
the indicated times. Remaining p21-bound
[
-
P]GTP was determined by nitrocellulose
filter binding.
GTP-binding Proteins
H6-tagged Ha-Ras and Rap1a,
purified from Sf9 cells, were provided by Dr. Albert Reynolds;
bacterial Rho and Rac were provided by Dr. Alan Hall. Rap2 was
expressed as described in HB101 cells containing a rap2 expression plasmid, provided by Dr. Jean de Gunzburg(24) .
Identification of Tuberin
To allow for the
specific detection of tuberin, antisera were raised against an
N-terminal and a C-terminal region of the predicted protein product
(designated anti-Tub-N and anti-Tub-C, respectively) (Fig. 1).
Extracts of the K-562 erythroleukemia cell line were subjected to
immunoprecipitation with anti-Tub-N, unrelated or preimmune sera. After
SDS-PAGE analysis, immunoprecipitates were transferred to a filter, and
the filter was incubated with anti-Tub-C serum (Fig. 2a). We observed the presence of a specific band
of 180 kDa in the anti-Tub-N immunoprecipitates. Next, extracts of the
G-401 Wilms tumor cell line were subjected to immunoprecipitation with
either anti-Tub-C or anti-Tub-N sera, and the immunoprecipitates were
blotted. The blot was cut into strips, which were incubated with
preimmune, anti-Tub-C, or unrelated serum (Fig. 2b).
Again, a specific band of 180 kDa was detected in the anti-Tub-N and
anti-Tub-C immunoprecipitates, but only when the blot was probed with
anti-Tub-C serum (the anti-Tub-N serum did not bind to tuberin in
immunoblot analyses). The observed migration of this band is close to
the predicted molecular mass of tuberin (198 kDa)(15) .
Furthermore, the combined immunoprecipitation/immunoblot analysis
provides a high degree of certainty that the observed protein is in
fact tuberin, since antisera against different regions of tuberin were
employed for immunoprecipitation and for immunoblotting. As additional
proof of specificity, a significant reduction in the intensity of the
180-kDa band was observed following preincubation of the anti-Tub-N
serum with an acetone powder extract prepared from E. coli expressing GST-Tub-N, but not by preincubation with acetone powder
preparations of E. coli expressing GST-Tub-C or GST-DH (Fig. 2c).
Figure 1:
Structure of
tuberin and bacterial fusion proteins. Tuberin contains a potential
leucine zipper domain (LZ), and a region of homology to the
catalytic domain of Rap1GAP, in which 19 of 58 amino acid residues are
identical (15). The recombinant GST fusion proteins (GST-Tub-N and
GST-Tub-C) encode the N- and C-termini of tuberin. GST-Tub-C encodes
the putative catalytic (GAP) domain of tuberin, based on sequence
alignment with Rap1GAP.
Immunoprecipitation of extracts from
[S]methionine-labeled K-562 and G-401 cells with
these antisera again revealed a 180-kDa band, which could be
specifically blocked by preadsorption with acetone powder preparations
from E. coli expressing the cognate fusion protein, but not by
preadsorption with control acetone powder preparations (data not
shown). Immunoblot analyses performed without prior immunoprecipitation
also revealed a 180-kDa protein in the cell lysates. However, the
background when either procedure was employed singly was higher than in
the combined immunoprecipitation/immunoblot analyses, which led us to
use the latter procedure in most of the subsequent experiments.
Subcellular Localization of Tuberin
To determine
the subcellular localization of tuberin, K-562 cells were lysed by
Dounce homogenization in the absence of detergent. The postnuclear
supernatant was centrifuged at 100,000 g to yield
membrane/particulate (P100) and nonparticulate (S100) fractions.
Control lysates from an equal amount of cells were prepared with
detergent, and each of the fractions was analyzed by
immunoprecipitation (with anti-Tub-N, preimmune, or anti-Tub-C)
followed by Western blotting (with anti-Tub-C). A substantial majority
of the tuberin was recovered in the P100 fraction (Fig. 3),
raising the possibility that tuberin may associate with the cell plasma
membrane, or with intracellular membranes or organelles.
Figure 3:
Intracellular localization of tuberin.
Biochemical subcellular fractionation was performed with K-562 cells.
Cells were lysed by Dounce homogenization as described, and the
post-nuclear supernatant was centrifuged at 100,000 g to yield the supernatant (S100) and the pellet (P100) fractions.
Equal proportions of these fractions and of a whole cell detergent
extract (LYSATE) were subjected to immunoprecipitation with
preimmune, anti-Tub-N, or anti-Tub-C sera, and immunoblotted with
anti-Tub-C serum. Arrowhead denotes the position of the
180-kDa tuberin band.
Expression of Tuberin in Different Cell Lines
The
wide variety of organ systems affected in TSC patients suggests that
the 180-kDa tuberin protein is expressed in many different cell types.
Indeed, expression of the TSC2 mRNA in diverse cell types has
been demonstrated by Northern blot analysis. We tested a panel of 13
different human cell lines for tuberin expression (Fig. 4). These
lines represent a variety of cell types (see legend to Fig. 4).
Cells were lysed, and portions of each lysate containing an equal
amount of protein were subjected to combined
immunoprecipitation/immunoblot analysis. With the exception of HL-60
and U-937 cells, tuberin was expressed in all of the cell lines tested.
Interestingly, each of the tuberin-negative cell lines represents a
different maturation level of the myelocytic/monocytic lineage;
abnormalities in this lineage have not been linked to TSC
patients(9, 25) . Small differences in the migration
rate of tuberin were observed in some of the cell lines (e.g. SW-13 and HT-29 cells). These differences may reflect
post-translational modification of tuberin, or the products of
alternatively-spliced variants of the TSC2 mRNA. Splicing
variants have been demonstrated for the TSC2 homolog in
rats.(
)
Figure 4:
Expression of tuberin in different human
cell lines. Cultures of 13 human cell lines were lysed as described,
and 450 µg of each lysate was subjected to immunoprecipitation with
anti-Tub-N or preimmune serum, followed by Western blot analysis with
anti-Tub-C serum. Cell lines analyzed were 293 (embryonic kidney
cells), WI-38 (lung fibroblasts), U-937 (histiocytic lymphoma), 769-P
(primary renal cell carcinoma), G-402 (renal leiomyoblastoma), SW-13
(adenocarcinoma of the adrenal cortex), HT-29 (colon adenocarcinoma),
G-401 (Wilm's tumor), K-562 (erythroleukemia), HL-60
(promyelocytic leukemia), HeLa (cervical carcinoma), SW 1088
(astrocytoma), and MOLT-3 (T-cell lymphoblastic leukemia). Arrowhead denotes the position of the 180-kDa tuberin
band.
GAP Activity in Tuberin
Immunoprecipitates
The region of tuberin bearing homology to
Rap1GAP (58 amino acids) is substantially shorter than the Rap1GAP
catalytic domain (331 residues), and is also shorter than the homology
seen between different Ras-specific GAP proteins (about 350
residues)(3, 26) . However, if tuberin does indeed
function as a GAP for Rap1, then immunoprecipitates of tuberin should
contain this type of activity. Therefore, native tuberin was isolated
by immunoprecipitation from K-562 cell lysates, and the
immunoprecipitates were incubated with
[-
P]GTP-bound Rap1a to assay potential GAP
activity (Fig. 5a). We observed significant levels of
GAP activity toward Rap1a in the tuberin immunoprecipitates, but not in
immunoprecipitates in which a control antiserum (anti-papillomavirus
structural protein L2) was used. As a positive control, we analyzed the
GAP activity toward Rap1a present in non-immunoprecipitated lysates of
K-562 cells. Three µl of K-562 lysate contained more GAP activity
toward Rap1a than tuberin immunocomplexes isolated from 120 µl of
K-562 lysate. These differences may be due to incomplete isolation of
tuberin from the lysates, although the reaction kinetics of Sepharose
bead-bound tuberin are undoubtedly different from those of soluble
proteins present in the K-562 lysate. In addition, the K-562 cells may
express other sources of Rap1-specific GAP activity, such as one of the
previously characterized Rap1GAP activities(23, 27) .
Figure 5:
Tuberin
possesses GAP activity toward Rap1a. GTP remaining bound to Rap1a was
determined following binding of [-
P]GTP to
Rap1a, using a nitrocellulose filter assay. The plot represents
Log
(Rap1a
GTP{t}/Rap1a
GTP{t
}
(as %)) versus time. a, GAP assay using native
tuberin. Immunoprecipitation of K-562 lysates (120 µl per GAP
reaction) was performed using either anti-Tub-C or control antibody.
Immune complexes were collected using protein A-Sepharose and washed,
then incubated with [
-
P]GTP
Rap1a. An
equal amount of protein A-Sepharose beads with:
, anti-Tub-C;
, control antibody, or 3 µl of K-562 lysate (⊞) was
used. As K-562 lysates exhibit more GAP activity than immune complexes
of tuberin, tuberin may be only one of the sources of Rap1GAP activity. b, GAP assay using bacterially expressed GST-Tub-C; different
amounts of E. coli lysates expressing GST-Tub-C (
, 40
nM;
, 8 nM; &cjs2125;, 1.6 nM) or
GST-DH (
, 10 nM) were used for this assay. GST-Tub-C
exhibited a weak dose dependent GAP activity (⊞, 3 µl of NIH
3T3 lysate). c, GAP assay using Sf9 cell lysates expressing
GST-Tub-C (
, 200 nM), control protein (
), or buffer
control (
). Sf9 cells possess endogenous Rap1GAP activity (28).
Lysates of Sf9 cells expressing GST-Tub-C exhibited increased GAP
activity.
GAP Activity of Recombinant Tuberin Fragment
The
results described above strongly suggest that tuberin itself possesses
GAP activity for Rap1, although we could not exclude the possibility
that this activity is due to a coimmunoprecipitating Rap1GAP. To
distinguish between these possibilities, we tested the ability of the
GST-Tub-C fusion protein, which contains the last 398 amino acids (and
includes the putative catalytic domain) of tuberin, to stimulate the
GTPase activity of Rap1a. The GST-Tub-C protein, expressed in both E. coli and Sf9 cells, was tested for GAP activity toward
Rap1a. When lysates of bacteria expressing GST-Tub-C were tested, a
dose dependent, albeit weak, GAP activity was observed, while E.
coli lysates expressing a control GST-fusion protein lacked
detectable GAP activity (Fig. 5b). In addition, lysates
of Sf9 cells expressing GST-Tub-C contained enhanced GAP activity
toward Rap1a, when compared with lysates of Sf9 cells expressing a
control protein (Fig. 5c). Insect cells express
endogenous Rap1GAP, which probably accounts for the activity observed
in the control cell lysates(28) .
Specificity of Tuberin GAP Activity
To determine
the specificity of tuberin GAP activity, we incubated
immunoprecipitates of native tuberin with
[-
P]GTP-bound recombinant Rap1a, Rap2, Ras,
or Rho proteins. The GAP activity of tuberin toward all four of the
GTPase proteins was measured in the same experiment. Consistent with
the results of Fig. 5a, immunoprecipitates with
anti-tuberin antisera contained significant levels of GAP activity
toward Rap1a, compared to control immunoprecipitates (Fig. 6a). By contrast, the intrinsic GTPase activity of
Ras, Rap2, and Rho was not stimulated by incubation with anti-tuberin
immunoprecipitates (Fig. 6, b, c, and d). In
additional experiments, the GTPase activity of Rac was not enhanced by
tuberin (data not shown).
Figure 6:
GAP
activity of native tuberin is specific for Rap1a. Immunoprecipitates of
K-562 cells obtained by anti-Tub-C () or control antibody (
)
were used to test sensitivity of different Ras-like protein toward GAP
activity of native tuberin. Assays were conducted as described for Fig.
5. a, Rap1a; b, Rap2; c, Ha-Ras; d,
Rho. Different slopes reflect the different intrinsic GTPase activities
of these proteins. Two or three independent experiments were performed
for each protein, and the data shown are representative of the results
obtained in each trial.
Ras in
Schwann cells, it is not required as a regulator in other cell types (e.g. NIH 3T3)(31, 32) . In spite of this fact,
overexpression of NF1 does inhibit the growth of NIH
3T3(31) . One explanation for these findings is that
neurofibromin binds to Ras, preventing Ras from interacting with its
mitogenic effectors. Such a model could also apply to tuberin and
Rap1a: loss of tuberin expression in cells would free up Rap1, thereby
allowing it to transmit a positive signal through its effectors.
(
)These results
have been interpreted as supporting a model whereby Rap1 antagonizes
Ras through competitive binding of Rap1 to Ras effector molecules. An
alternative (although not mutually exclusive) possibility is that Rap1
proteins participate in the transduction of growth-inhibitory signals,
thereby overcoming the mitogenic signals originating from
Ras(36) . In accordance with this scenario, tuberin might
function as an effector protein (as well as a GAP) for Rap1, and the
loss of tuberin expression would prevent transmission of the
growth-inhibitory signals originating from Rap1.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.