From the Departments of Surgery and Pathology, University of Washington, Seattle, Washington 98195
Received for publication, March 20, 2001
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ABSTRACT |
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Hamartin and tuberin are products of the
tumor suppressor genes, TSC1 and TSC2, respectively. When mutated, a
characteristic spectrum of tumor-like growths develop resulting in the
syndrome of tuberous sclerosis complex. The phenotypes
associated with TSC1 and TSC2 mutations are largely indistinguishable
suggesting a common biochemical pathway. Indeed, hamartin and tuberin
have been shown to interact stably in vitro and in
vivo. Factors that regulate their interaction are likely critical
to the understanding of disease pathogenesis. In this study, we showed
that tuberin is phosphorylated at serine and tyrosine residues in
response to serum and other factors, and it undergoes serial
phosphorylation that can be detected by differences in electrophoretic
mobilities. A disease-related TSC2 mutation (Y1571H) nearly abolished
tuberin phosphorylation when stimulated with pervanadate. Expression of this mutant tuberin caused a marked reduction in TSC1-TSC2 interaction compared with wild-type protein and significantly curtailed the growth
inhibitory effects of tuberin when overexpressed in COS1 cells,
consistent with a loss of function mutation. Examination of a second
pathologic mutation, P1675L, revealed a similar relationship between
limited phosphorylation and reduced interaction with hamartin. Our data
show for the first time that 1) tuberin is phosphorylated at tyrosine
and serine residues, 2) TSC1-TSC2 interaction is regulated by tuberin
phosphorylation, and 3) defective phosphorylation of tuberin is
associated with loss of its tumor suppressor activity. These findings
suggest that phosphorylation may be a key regulatory mechanism
controlling TSC1-TSC2 function.
The autosomal dominant syndrome of tuberous sclerosis complex
(TSC)1 is a disorder
classified as a phakomatosis and is characterized by the development of
hamartomatous lesions that are patchy in distribution in multiple organ
systems (1). The common clinical manifestations stem from growths in
the brain (cortical tubers), kidney (angiomyolipoma and cysts), heart
(rhabdomyomas), and skin (2). As such, it commonly results in epilepsy,
mental retardation, renal complications, and premature deaths. Although
pathologically diverse, the lesions in TSC share common features of
excessive proliferation, abnormal differentiation, and aberrant
migration. Genetic studies of families have successfully identified two
genetic loci that are responsible for this disease (3, 4). Within the
last few years, the two genes, TSC1 and TSC2, have been positionally cloned, allowing investigations that will lead to the understanding of
the molecular basis of TSC.
The function of these genes has been partially elucidated to date.
There is evidence to suggest that the TSC2 product, tuberin, suppresses
tumorigenicity (5, 6), controls cell cycle (7-9), affects normal brain
development (10), exhibits in vitro Rap1 GTPase-activating
protein activity (11), modulates transcription in vitro
(12), interacts with the TSC1 protein, hamartin (13, 14), and
participates in vesicular trafficking (15). In addition, hamartin was
shown to interact with ezrin-radixin-moesin and regulate cell
adhesion (16). However, there is no unifying concept of how these two
genes may influence these processes. Further, there has not been any
study addressing the regulation of hamartin and tuberin in the context
of their physiologic role as tumor suppressor genes.
Among the various properties of these two proteins, their ability to
interact and to form a stable complex has been the most consistent
finding (13, 14). Their interaction fits well with the concept that the
gene products function along a common biologic pathway. Thus, mutation
of either gene can give rise to a similar clinical phenotype. This led
to the hypothesis that hamartin and tuberin may function as a complex,
and factors regulating their interaction will be important in
understanding their physiologic roles. In this study, we discovered
that tuberin undergoes reversible phosphorylation at serine and
tyrosine residues that can be detected by mobility shifts on SDS-PAGE
gels in response to serum and other stimuli. Two disease-related
missense mutations of TSC2 gave rise to proteins that were defective in
their ability to be phosphorylated. As such, they were unable to
interact efficiently with hamartin, thus abolishing the tumor
suppressor activity of the complex. These findings provide novel
insights into the mechanisms of TSC1-TSC2 gene functional regulation.
Materials--
HeLa, COS1, and NIH3T3 cells were obtained from
the American Type Culture Collection (Manassas, VA). EEF126-4, EEF
126-8, and LEF8 cells were derived as described previously (5, 15). All cell culture reagents, LipofectAMINE, and Plus reagent were purchased from Life Technologies, Inc. Thin layer cellulose
plates were obtained from EM Science (Gibbstown, NJ).
[32P]Orthophosphate was purchased from PerkinElmer
Life Sciences. Recombinant LAR (catalytic domain), Site-directed Mutagenesis--
Polymerase chain reaction-based
mutagenesis was performed with the QuikChangeTM
site-directed mutagenesis kit (Stratagene, La Jolla, CA). The previously described rat wild-type TSC2 in pBS plasmid was used as the
template (5). The oligonucleotide primers designed to introduce the
mutations were as follows (the base change is underlined): forward primer Y1571H,
5'- GCATGGCTCTTATAGGCACACAGAGTTTCTG-3'; reverse primer
Y1571H, 5'-CAGAAACTCTGTGTGCCTATAAGAGCCATGC-3'; forward
primer P1675L, 5'-CCATGTGATCATCACACTGCTGGACTATAAATGC-3'; reverse primer P1675L,
5'-GCATTTATAGTCCAGCAGTGTGATGATCACATGG-3'. After
sequencing, Y1571H and P1675L were subcloned into wild-type TSC2 by
digesting with AocI and HindIII. The entire TSC2
sequence was then subcloned into the NotI and
XhoI sites of pcDNA3 (Invitrogen, Carlsbad, CA).
Cell Culture and Treatments--
EEF126-4 and EEF126-8 cells
were cultured as described previously (5, 15). HeLa, NIH3T3, and COS1
cells were cultured according to ATCC protocol. The TSC1 and TSC2
plasmids were transfected into 60-80% confluent COS1 cells using
LipofectAMINE as described by the manufacturer. 48 h
post-transfection cells were treated as shown in figures. A stock
solution of pervanadate (PV) was prepared fresh each time by mixing
equal volumes of 0.1 M H2O2 and 0.1 M Na3VO4. The stimulation reactions
were stopped by washing culture dishes with ice-cold phosphate-buffered
saline. Cells were immediately lysed in harvest buffer (50 mM Tris-HCl, 150 mM NaCl, 2.5 mM
EGTA, 1 mM sodium vanadate, 1% Nonidet P-40 plus 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.25 mg/ml AEBSF). Cells
were broken by passing through a syringe with a 22-gauge needle, and
the extract was cleared by centrifugation at 15,000 × g for 10 min. The resulting supernatant was used for experiments.
Immunoprecipitation and Immunoblotting--
Aliquots containing
equivalent amounts of cellular protein were incubated for 2 h at
4 °C with 4 µl of either IgG-purified L3-2 (TSC2) or 4050 (TSC1)
antibodies. 20 microliters of 1:1 (v/v) of protein A-Sepharose in
harvest buffer was added to each tube and incubated overnight.
Immunoprecipitates were washed three times with harvest buffer and
resuspended in 30 µl of 1.5× sample buffer. For phosphatase
treatment, immunoprecipitates were washed with harvest buffer
containing no EGTA or sodium vanadate, incubated at 30 °C for 30 min
under conditions suggested by the manufacturer, and stopped by adding
4× sample buffer. Cell extracts or immunoprecipitates were
electrophoresed on a 7% low (30:0.4) bisacrylamide SDS-PAGE gel and
transferred to PVDF membrane for Western blotting. Tuberin was
visualized using polyclonal IgG-purified anti-tuberin (L3-2) (15) at a
dilution of 1:500. The hamartin antibody was used at a dilution of
1:100, and both were followed by horseradish peroxidase-conjugated
anti-rabbit secondary antibodies at a dilution of 1:5000. Antibody
complexes were visualized by chemiluminescent detection using ECL reagent.
32P Labeling and Phosphoamino Acid
Analysis--
COS1 cells in 60-mm dishes were transfected as above.
Cells were grown in Dulbecco's modified Eagle's medium with 0.1% FBS overnight, washed with phosphate-free Dulbecco's modified Eagle's medium, and then incubated with 0.5 mCi of orthophosphate in 2 ml of
phosphate-free Dulbecco's modified Eagle's medium with 0.1% FBS for
6 h. 10% FBS and pervanadate (0.1 mM final
concentration) were added to the cells 30 min prior to the end of
labeling. The cells were harvested, and ~50 µg of total protein was
immunoprecipitated as above. The immunoprecipitates were resolved by
SDS-PAGE and transferred to PVDF membrane, and the
32P-labeled tuberin was visualized by autoradiography.
Phosphoamino acid analysis was performed as described previously (17,
18). Briefly, the individual 32P-labeled tuberin bands were
excised and subjected to acid hydrolysis in 6 N HCl at 110 °C for
1 h. The hydrolyzed protein was transferred to a new
microcentrifuge tube, lyophilized, and dissolved in 12 µl of
H2O. Two-dimensional TLC was performed by spotting 1/12 of
the hydrolyzed protein plus 1 µl of a 1 mg/ml mix of phosphoserine, phosphothreonine, and phosphotyrosine in the corner of a TLC plate. The
first-dimension separation was by chromatography in ethanol/glacial acetic acid/water (1:1:1; v/v/v) for 90 min at room temperature. Separation in the second dimension was performed by chromatography in
isobutyl alcohol/formic acid/water (8:3:4, v/v/v) for 40 min at room
temperature. Phosphoamino acids were visualized by spraying with a
solution of 0.5% ninhydrin in 0.5% acetone and heating at 80 °C
for 5 min. Labeled amino acids were visualized by autoradiography.
Cell Proliferation Assay--
COS1 cells were transfected as
above. 24 h post-transfection, cells were trypsinized and counted
using a hemocytometer. The cells were then spun down, washed with
phosphate-buffered saline, and lysed by addition of harvest buffer as
above. Protein assays were performed using the Pierce BCA protein assay kit.
Tuberin Undergoes Gel Mobility Shifts According to Phosphorylation
State--
It has been observed previously that polyclonal
anti-tuberin antibodies can detect multiple bands at the expected
molecular mass range of 180-195 kDa on Western blotting (11).
This has been attributed to the existence of known TSC2 splice
variants. In this study, we examined whether the variation in tuberin
mobility is related to a change in phosphorylation states. Endogenous
tuberin was examined in cell lines derived from the Eker rat, which
carries a germline mutation of TSC2 (Fig.
1A). The 126-4 rat embryo
fibroblasts contain wild-type tuberin that migrated as a broad band, as
compared with a faster migrating TSC2 protein from the LEF8 cells
derived from a renal tumor. The TSC2 gene in the latter cells does not exhibit abnormal splicing of exons 25 or 31, and the remaining TSC2
coding regions did not show any deletion (data not shown). This led us
to believe that other modifications such as phosphorylation could be
responsible.
To determine whether the mobility difference was because of
phosphorylation, cells were treated with 100 µM PV, a
protein-tyrosine phosphatase inhibitor and kinase activator. Following
30 min of treatment, wild-type tuberin in 126-4 cells showed a shift
toward the slower migrating forms, whereas the tuberin from LEF8 cells showed minimal mobility change (Fig. 1A). The observed
mobility shift of the wild-type tuberin was also noted in other cell
lines with the exception of COS1 cells, where the majority of the
endogenous tuberin migrates slower constitutively (Fig. 1B).
Wild-type tuberin overexpressed in COS1 cells, however, exhibited a
band shift in response to pervanadate (Fig. 1C). This, in
part, was associated with tyrosine phosphorylation as shown by specific
immunoreactivity to the anti-phosphotyrosine antibody, PY99. Further,
the slower migration of the TSC2 band caused by pervanadate can be
reversed by treatment with various phosphatases (Fig. 1C).
Exposure of wild-type tuberin to
To determine whether tuberin phosphorylation is part of the physiologic
response to serum and other stimuli, electrophoretic migration of
tuberin was examined following exposure to various factors. Among the
treatments, FBS (10%) and anisomycin (25 µg/ml) produced a band
shift, whereas platelet-derived growth factor-BB (20 ng/ml) and
forskolin (10 µM) did not have a detectable effect on the
migration of tuberin from TSC2-transfected COS1 cells (Fig. 2A). Endogenous tuberin also
responded in a similar fashion. Upon serum starvation, tuberin in
NIH3T3 cells shifted toward the dephosphorylated state whereas
serum-fed cells maintained a level of phosphorylation in between the
starved and pervanadate-treated states (Fig. 2B). In
contrast, tuberin in the tumor-derived LEF8 cells did not respond to
the treatments and remained in a mobility state that was faster than
that seen with wild-type tuberin during serum starvation. Taken
together, the data showed that tuberin undergoes reversible phosphorylation in response to multiple extracellular signals, and its
phosphorylation state can be detected by electrophoretic migration.
Also, one might infer that the tuberin from LEF8 cells could be
defective in its ability to be phosphorylated. Interestingly, hamartin
did not show a significant change in band migration under the same
conditions (Fig. 2B, lower panel); future studies
will address the role of phosphorylation for hamartin.
Tuberin Is Phosphorylated at Tyrosine and Serine Residues--
To
determine which amino acids are phosphorylated, tuberin overexpressed
in COS1 cells (starved or stimulated with FBS/PV) was labeled in
vivo with [32P]orthophosphate followed by
immunoprecipitation with anti-tuberin antibodies. The
immunoprecipitates were resolved by SDS-PAGE, transferred onto PVDF
membranes, and examined by autoradiography. The bands corresponding to
phosphorylated tuberin were excised from the membrane, digested, and
subjected to phosphoamino acid analysis and Cerenkov counting. As shown
in Fig. 3A, LAR phosphatase reduced total phosphorylation to 82% of the PV-treated level while maintaining much of the tyrosine phosphorylation despite the loss of
PY99 signal on Western blotting. Both PP1 and Disease-causing Mutations of TSC2 Affect Its
Phosphorylation--
To evaluate the biologic significance of TSC2
phosphorylation, two naturally occurring TSC2 mutants were examined.
The first mutant was found in the LEF8 cells, an Eker-derived renal
tumor cell line that has not undergone loss of heterozygosity at the TSC2 locus. Sequence analysis of the TSC2 coding regions confirmed a
single base pair substitution (T4800C, rat) (data not shown). This
results in a non-conservative amino acid change, from tyrosine to
histidine (Y1571H, human; Y1573H, rat). This tyrosine lies within the
putative Rap1 GTPase-activating protein homology domain of TSC2 and is
highly conserved in evolution.
To examine the phosphorylation properties of the Y1571H mutant, an
expression construct containing this change was made by site-directed
mutagenesis and was overexpressed in COS1 cells. Western blotting
confirmed the increased mobility of the expressed mutant protein and
the minimal shift in response to PV treatment, similar to that seen in
the parental LEF8 cells (data not shown). Based on the amount of
[32P]orthophosphate incorporation, the Y1571H mutant
labeled very minimally after FCS/PV treatment, compared with wild-type
tuberin (Fig. 3B). Further, TLC analysis showed that both
tyrosine and serine phosphorylation were significantly reduced. This is
consistent with a model in which tyrosine 1571 is a phosphorylation
site that may be required for serial phosphorylation of both serine and
tyrosine residues.
A second TSC2 missense mutation that has been recurrently identified in
patients with tuberous sclerosis was also examined. The P1675L mutation
was isolated from 10 unrelated TSC individuals, and it lies within the
rabaptin5-binding domain (21). The expression construct containing this
amino acid change was generated by site-directed mutagenesis and
confirmed by direct sequencing. Overexpression of the P1675L construct
produced a stable protein that migrated faster than the wild-type
tuberin, similar to that seen with the Y1571H mutant (Fig.
4B). The P1675L mutant tuberin
did not respond to pervanadate treatment, and phosphoamino acid
analysis confirmed minimal incorporation of
[32P]orthophosphate affecting both the serine and
tyrosine residues (Fig. 3B). Our data suggest that tuberin
phosphorylation is affected by disease-causing mutations.
Effects of Phosphorylation on Hamartin-Tuberin
Interaction--
Substantial evidence indicates that tuberin and
hamartin form a stable complex in vivo (13, 14). We
hypothesized that phosphorylation of tuberin may play a role in
regulating its interaction with hamartin. Co-immunoprecipitation
experiments were performed on endogenous and overexpressed, exogenous
TSC1 and TSC2 proteins. As expected, the wild-type hamartin and tuberin
in EEF126-4 and NIH3T3 fibroblasts formed a stable complex as they
were able to be immunoprecipitated by both anti-tuberin and
anti-hamartin antibodies (Fig. 4A). However, the mutant form
of tuberin found in tumor cells, LEF8, did not interact with hamartin.
The lack of co-immunoprecipitation of the endogenous proteins was not
because of the absence of hamartin. In addition, when the mutant Y1571H
and wild-type TSC1 were transiently overexpressed in COS1 cells, only a
fraction of the two proteins were recovered as a complex, compared with
the wild-type TSC2 (Fig. 4B). Under conditions of protein
overexpression (>20-fold increase), some degree of interaction was
retained by the mutant product. When the expression constructs were
transiently transfected into tuberin-deficient rat embryonic
fibroblasts, EEF126-8 cells, transgene expression was modest
(~2-fold), and only the wild-type tuberin, not the Y1571H mutant, was
shown to interact with hamartin (data not shown). We also tested the
ability of the P1675L mutant to interact with hamartin when expressed
in COS1 cells. Again, the amount of complex formation relative to the
expression levels of the individual proteins was significantly less for
the mutant, compared with the wild-type tuberin (Fig.
4B).
Effects of Mutant Tuberin on Proliferation--
Previous
experiments have shown that wild-type tuberin exerts growth inhibitory
effects when overexpressed in cells, transiently or stably (5). We
tested the biologic activity of the Y1571H mutant in transient
transfection of COS1 cells. 24 h after transfection, the cell
number and protein concentration were measured as an index of cell
proliferation (Fig. 5). In accordance
with previous studies, cells overexpressing wild-type tuberin showed a
reduction of 49% in cell number and a drop of 50% in protein
concentration, compared with the vector control. In contrast, the
Y1571H mutant induced only a modest reduction of 20% in cell number
and 27% in protein concentration. In both cases, the exogenous tuberin was expressed to nearly equal levels of at least 20-fold over the
endogenous levels. The same result in cell number reduction was
observed when the experiment was repeated in NIH3T3 cells (data not
shown).
Tuberous sclerosis is an example of the "two genes, one
disease" paradigm where mutation of either one of its two responsible loci, TSC1 and TSC2, will result in the expression of a similar phenotype. Evidence from genetic and biochemical analyses is congruent with the concept of functional cooperativity of the two gene
products, hamartin and tuberin. However, the biologic
significance of their interaction and its regulation have not been
reported. In this study, we demonstrated that tuberin is phosphorylated
at serine and tyrosine residues, and this process is altered by the two missense mutations examined. One consequence of the hypophosphorylated mutant forms of tuberin is the reduction in their interaction with
hamartin and correspondingly, the loss of the growth inhibitory function of tuberin. Our data support the notion that regulation of
TSC1-TSC2 interaction through tuberin phosphorylation is critical to
their biologic function. Mutations that disrupt the integrity of the
tuberin-hamartin functional complex can lead to the initiation of
disease. We also showed that various phosphorylation states of tuberin
can be easily detected by their different electrophoretic mobilities.
The regulation of tuberin phosphorylation is likely complex and has not
been fully elucidated in this study. The wild-type TSC2 product
undergoes reversible phosphorylation in response to physiologic
stimuli, such as serum. Based on phosphoamino acid analysis and
electrophoretic mobility shifts, there exists multiple sites involving
serine and tyrosine residues. Assuming that each mobility shift
represents a different phosphorylation state, we can deduce a minimum
of four levels of TSC2 phosphorylation beyond the basal state, but
mapping of specific phosphorylation sites awaits further studies.
Of the two mutants described, the effect of Y1571H on tuberin
phosphorylation strongly suggests that the Y1571 can itself be
phosphorylated. Review of other pathologic missense TSC2 mutations has
identified four additional tyrosine or serine residues that may be
relevant to its phosphorylation (21). Based on 32P
labeling, the effect of the P1675L mutant on reducing tuberin phosphorylation can be interpreted as more complete than the Y1571H mutation. The disruption of the proline residue is predicted to have
profound effects on the structural conformation of the protein and
consequently, is likely to abolish interaction with the appropriate kinase(s). This site resides in the rabaptin5-binding domain (15) and
represents a common amino acid altered by naturally occurring mutations; it was reported in 10 independent cases of tuberous sclerosis (21). Our findings lead us to believe that tuberin phosphorylation is targeted by natural mutations and is important to
its function.
Although direct interaction between TSC1 and TSC2 proteins has been
confirmed by yeast two-hybrid analyses and co-immunoprecipitation studies (13, 14), the binding domains have not been completely elucidated. Reports have suggested that the N terminus of tuberin is
essential for hamartin interaction (13, 22). Here, we showed that
phosphorylation sites outside the putative interaction domains are also
critical to hamartin-tuberin interaction. Because there exists a high
degree of concordance between interaction and phosphorylation in the
mutants studied in this report, it is difficult to isolate the effects
of interaction on phosphorylation. The fact that wild-type tuberin
overexpressed alone can undergo phosphorylation in response to
pervanadate treatment to the same degree as when it is co-expressed with hamartin would strongly suggest that tuberin
phosphorylation is not dependent on its interaction with
hamartin.2
The concept of phosphorylation-regulated protein interaction has been
noted in diverse signaling pathways. Our characterization of tuberin
phosphorylation and its effects on hamartin interaction provides new
insights into the regulation of its tumor suppressor function. Analyses
of the missense mutations support the biologic significance of TSC2
phosphorylation. Further understanding of the pathways that modify its
phosphorylation state will provide opportunities to manipulate its
function exogenously as therapy for patients with tuberous sclerosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and PP1
(catalytic subunit) phosphatases were purchased from New England
Biolabs (Beverly, MA). AEBSF and anisomycin were obtained from
Calbiochem. Horseradish peroxidase-conjugated donkey anti-rabbit and
anti-mouse antibodies and ECL reagent were purchased from Amersham
Pharmacia Biotech. Anti-phosphotyrosine (PY99) was obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). The tuberin (15) and hamartin
antibodies were purified using the EconoPac serum IgG purification
columns from Bio-Rad. Polyclonal anti-hamartin was raised in rabbits
against a 15-amino acid peptide, KDHELDPRRWKRLET (238), conjugated
to bovine serum albumin. The antiserum was affinity-purified using the
Pierce Aminolink Plus immobilization kit. Immobilon-P PVDF membranes
were purchased from Millipore (Bedford, MA). Other chemicals were
purchased from Sigma. Wild-type TSC1 in pcDNA3 (Invitrogen, Carlsbad, CA) was a kind gift from Elizabeth Henske (Fox Chase Cancer
Center, Philadelphia, PA) (14).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Electrophoretic mobility of tuberin varies
according to cell type and treatments with pervanadate and
phosphatases. A, Western blot analysis of tuberin in
cells treated with or without PV for 30 min. Eker rat-derived embryonic
fibroblasts, 126-4 (TSC2+/+) and 126-8
(TSC2 /
) cells served as positive and negative controls,
respectively. Renal tumor-derived LEF8 cells expressed a more mobile
form of tuberin. B, electrophoretic mobility of tuberin in
response to PV in COS1, HeLa, and NIH3T3 cells. C, COS1
cells were transiently transfected with wild-type TSC2 vector, and
after 48 h they were exposed to pervanadate for 30 min. Tuberin
was immunoprecipitated, treated with phosphatases (Phos) as
shown, and blotted with anti-tuberin antibodies and
anti-phosphotyrosine antibodies (PY99). Molecular weight
markers are shown on the left.
phosphatase dramatically enhanced
its mobility and eliminated immunoreactivity to PY99. Recombinant PP1
(catalytic subunit), which possesses serine/threonine and some tyrosine
phosphatase activities (19), caused a similar shift in mobility, as
well as loss of PY99 immunoreactivity. Treatment with a tyrosine
phosphatase, LAR (catalytic domain), eliminated the PY99 signal but did
not affect mobility of the tuberin band. These observations are
consistent with multiple phosphorylation events affecting tuberin
mobility. Although other modifications such as ubiquitination can
affect electrophoretic mobility, such changes are not reversible by
phosphatase treatment (20). We conclude that the frequently observed
"doublets" and band shifts of tuberin on Western blotting can be
accounted for by various states of protein phosphorylation.
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Fig. 2.
Exogenous and endogenous tuberin responded to
various stimuli. A, wild-type TSC2-transfected COS1
cells were starved and then treated with the indicated stimuli for
defined time periods. Tuberin was detected by Western blotting.
Anis, anisomycin; PDGF, platelet-derived growth
factor; Fk, forskolin. B, Western blot analysis
of endogenous tuberin (top) and hamartin (bottom)
in LEF8 and NIH3T3 cells, under starved (S), serum-fed
(C), and pervanadate-treated (PV)
conditions.
phosphatases decreased phosphorylation of tuberin to 14 and 11% of the
pervanadate-treated control, respectively. Overall, the amount of
[32P]orthophosphate incorporation correlated well with
tuberin mobility. When the samples were analyzed by two-dimensional
TLC, it was clearly shown that tuberin was phosphorylated on serine and
tyrosine but not threonine residues (Fig. 3A).
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Fig. 3.
Phosphoamino acid analysis showed that
tuberin is phosphorylated on distinct residues. A,
Wild-type TSC2 overexpressed in COS1 cells were labeled with
[32P]orthophosphate, treated with pervanadate,
immunoprecipitated with anti-tuberin antibodies, and then treated with
, LAR, and PP1 phosphatases (Phos), or control
(C). Shown are the results of SDS-PAGE (top) and
two-dimensional TLC (bottom). Dotted circles
representing phosphorylated serine (p-S), threonine
(p-T), and tyrosine (p-Y) as detected by
ninhydrin are shown. B, mutant TSC2 constructs were
transfected into COS1 cells, proteins were labeled with
[32P]orthophosphate under starved and
serum/pervanadate-treated conditions, and tuberin was
immunoprecipitated. wt, wild-type.
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Fig. 4.
Effects of TSC2 mutants on the interaction of
tuberin with hamartin. A, endogenous proteins were
immunoprecipitated (IP) with either anti-tuberin
(top) or anti-hamartin (bottom) antibodies. The
LEF8 cells contain the Y1571H mutant allele and express both tuberin
and hamartin. Negative control, 126-8 (TSC2 /
);
positive controls, 126-4 (TSC2+/+) and NIH3T3
(TSC2+/+). C, control; S,
starved. B, extracts of COS1 cells overexpressing TSC2
mutants, Y1571H (Y/H), P1675L (P/L), or wild-type
(wt) TSC2 were subjected to immunoprecipitation
(IP) with anti-tuberin (left) or anti-hamartin
(right) antibodies. Relative amount of co-precipitated
proteins is shown by immunoblotting with anti-tuberin (top)
and anti-hamartin (bottom) antibodies.
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Fig. 5.
Effects of the TSC2 (Y1571H) mutation on cell
proliferation. Transient transfections of vector control, Y1571H
mutant, and wild-type (WT) TSC2 in COS1 cells were
quantified for cell number and protein concentration. The experiment
was repeated three times, and the results were standardized to vector
control. Compared with wild-type TSC2, the mutant transfectants had a
significantly higher cell number and protein concentration (*,
p = 0.01 and 0.04, respectively, using unpaired
t tests).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Guenter Daum, Yuji Yamamoto, Baldwin Mak, Katie Jones, Trevor Dundon, and Denise Spring for helpful suggestions and Hong Gu and Ana Kobayashi for technical assistance. We are grateful to Elizabeth Henske for providing the TSC1 construct.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (CA77882) and the Tuberous Sclerosis Alliance.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 Surgery,
University of Washington, 1959 NE Pacific St., Box 356410, Seattle, WA
98195. Tel.: 206-616-6405; Fax: 206-616-6406; E-mail: ryeung@u.washington.edu.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.C100136200
2 Unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: TSC, tuberous sclerosis complex; PV, pervanadate; LAR, leukocyte antigen-related protein-tyrosine phosphatase; PP1, protein phosphatase 1; FBS, fetal bovine serum; PVDF, polyvinylidene fluoride; PAGE, polyacrylamide gel electrophoresis; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride, HCl.
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