From the Lineberger Comprehensive Cancer
Center,
Department of Biochemistry and Biophysics,
** Program in Molecular Biology and Biotechnology, University
of North Carolina at Chapel Hill, Chapel Hill, North Carolina
27599-7295, and the ¶ Department of Biological Chemistry,
University of Michigan Medical School, Ann Arbor, Michigan 48109
Received for publication, September 3, 2002, and in revised form, December 2, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
TSC2, or tuberin, is the product of the
tuberous sclerosis tumor suppressor gene TSC2 and acts
downstream of the phosphatidylinositol 3-kinase-Akt signaling
pathway to negatively regulate cellular growth. One mechanism
underlying its function is to assemble into a heterodimer
with the TSC1 gene product TSC1, or hamartin, resulting in
a reduction in phosphorylation, and hence activation, of the ribosomal
subunit S6 kinase (S6K). We identified a novel interaction between TSC2
and 14-3-3 Tuberous sclerosis
(TSC)1 is an inheritable
disorder in which the brain, kidneys, skin, heart, and other organs
may be affected by tumor-like growths, or hamartomas, that can result
in seizures, mental retardation, and autism (1, 2). TSC affects roughly 1 in 6,000 newborns and is caused by mutations in either one of two
genes. The TSC1 locus encodes a 130-kDa protein termed
hamartin, or TSC1, and the TSC2 locus encodes a 180-kDa
protein termed tuberin, or TSC2. Studies of hamartomas and tumors from
TSC patients, as well as those of rodent models, support the
categorization of both gene products as tumor suppressor proteins.
The proteins TSC1 and TSC2 bind each other, and this association is
compromised by many tumor-derived mutations found in either protein
(3). Studies in Drosophila melanogaster identified the TSC
genes (dTSC1 and dTSC2) as important regulators
of cell growth (4-6). Direct biochemical evidence linking the function of the TSC complex to cell growth control was recently
provided by the finding that TSC1-TSC2 inhibits mTOR effectors and that phosphorylation of TSC2 by Akt suppresses TSC1-TSC2 activity (7-10). In an effort to elucidate the function and regulation of TSC proteins, we searched for cellular protein(s) that interact with TSC2 and have
identified 14-3-3 as a novel TSC2-interacting protein. We show that
14-3-3 selectively interacts with phosphorylated TSC2 and interferes
with the function of TSC1-TSC2. Our findings identify a novel
regulation of the TSC complex and provide further insight into the
mechanisms that control TSC activity in cell signaling.
Cell Culture, Yeast, Reagents, and Plasmids--
U2OS and
HEK293T cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum, penicillin, and
streptomycin (Invitrogen). Insulin was purchased from Invitrogen and was administered at 350 nM. The HF7c strain of
Saccharomyces cerevisaiae was used in two-hybrid assays.
cDNA encoding 14-3-3 GST Fusion Protein Pull-down Assay--
GST or GST fusion
proteins were purified with glutathione-Sepharose following induction
with 0.1 mM
isopropyl-1-thio- Immunoprecipitation and Western Blotting--
Except where
indicated, cells were lysed in Nonidet P-40 lysis buffer and cleared by
centrifugation. To immunoprecipitate endogenous 14-3-3 Identification of 14-3-3 14-3-3 14-3-3 Serine 1210 of TSC2 Is Required for 14-3-3 14-3-3
Ser1210 in TSC2 is a probable site of phosphorylation by a
yet unidentified kinase and is required for binding with 14-3-3
Although mutant products of the TSC1 and TSC2
genes have long been implicated in tumorigenesis and dysregulation of
cell growth, the mechanism underlying the function of these proteins in
normal cell physiology has surfaced only recently (4-6). Genetic data indicating that the TSC1-TSC2 complex negatively regulates cell growth
have been substantiated by biochemical studies, demonstrating inhibition of mTOR by TSC1-TSC2. The activity of the TSC complex to
suppress mTOR is potent and can be inhibited by phosphorylation of TSC2
by Akt (7-10). Here we describe a distinct mechanism of regulating the
growth-suppressive activity of the TSC complex. We have found that
phosphorylated TSC2 is a ligand for 14-3-3 binding and that the
functional consequence of this ternary interaction is a decreased
ability of TSC1-TSC2 to inhibit S6K phosphorylation, both basally and
in response to stimulus. Importantly, this regulation of TSC by 14-3-3, unlike that by Akt phosphorylation, does not involve a dissociation of
TSC1-TSC2.
Since Akt inactivates the TSC1-TSC2 complex via TSC2 phosphorylation,
we were surprised to find that phosphorylation of TSC2 by Akt does not
appear to provide the primary binding site for 14-3-3. We found that 14-3-3
does not interfere with
TSC1-TSC2 binding and can form a ternary complex with these two
proteins. Association between 14-3-3
and TSC2 requires
phosphorylation of TSC2 at a unique residue that is not a known Akt
phosphorylation site. The overexpression of 14-3-3
compromises the
ability of the TSC1-TSC2 complex to reduce S6K phosphorylation. The
antagonistic activity of 14-3-3
toward TSC is dependent on the
14-3-3
-TSC2 interaction, since a mutant of TSC2 that is not
recognized by 14-3-3
is refractory to 14-3-3
. We suggest that
14-3-3 proteins interact with the TSC1-TSC2 complex and negatively
regulate the function of the TSC proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
was PCR amplified from the HeLa cDNA
library and subcloned in-frame with either Myc-, HA-, or
GST-epitope tags. Site-directed mutagenesis was performed by standard
PCR techniques using the QuikChange kit (Stratagene) and verified by
DNA sequencing.
-D-galactopyranoside over 4 h in exponentially growing Escherichia coli, BL21(DE3), cultured at 30 °C. For HEK293T cell lysate, transfected cells were
lysed in a Nonidet P-40 lysis buffer (15 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% Nonidet P-40, 1 mM
PMSF, 1 mM dithiothreitol, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 10 µg/ml tripsin inhibitor, and 150 µg/ml
benzamidine) and 1 mg incubated at 30 °C for 1 h with or
without 10 units of CIP and terminated with 10 µM
Na2VO3. Equal amounts of GST or GST fusion
proteins (~20 µg) were used to precipitate binding proteins from
250 µg of treated lysate.
-TSC2
complexes, 1.5 mg of total protein lysate was used; for all other
immunoprecipitations, 300-500 µg of cell lysate was used. Western
blotting was performed with 50-100 µg of protein extract separated
by SDS-PAGE and transferred to nitrocellulose membrane. Polyclonal
antibody to TSC2 was raised using purified fusion protein consisting of
GST and a fragment of human TSC2 (residues 1283-1807) as an immunogen.
Affinity-purified antibodies to Myc (clone 9E10, NeoMarkers), HA
(clone 12CA5, Roche Molecular Biochemicals), TSC2 (C-20, Santa Cruz),
14-3-3
(K19, Santa Cruz), and phosphorylated Thr389 of
S6K (#9205, Cell Signaling Technologies) were purchased commercially. Quantitative analyses were performed with Scion Image software (www.scioncorp.com/).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
as a TSC2-interacting Protein--
In
an attempt to uncover novel TSC2-interacting proteins we screened
3 × 106 independent clones derived from a HeLa cell
cDNA library using full-length rat TSC2 as bait in a yeast
two-hybrid assay. Among the 20 total positive colonies isolated in the
screen were 11 independent clones of 14-3-3
and three of 14-3-3
.
A directed yeast two-hybrid assay confirmed the binding of 14-3-3
to
TSC2 but did not indicate an interaction between 14-3-3
and TSC1
(Fig. 1A). When TSC2 was
ectopically expressed in HEK293T cells we were able to detect 14-3-3
in the anti-TSC2 immunoprecipitate (Fig. 1B). Reciprocally,
TSC2 could also be detected in the 14-3-3
immunocomplex (Fig.
1B). An in vivo binding between endogenous TSC2
and 14-3-3 was detected in HEK293 cells (Fig. 1C). These results suggest that multiple isoforms of 14-3-3 can physically interact with the TSC2 gene product, tuberin.
View larger version (48K):
[in a new window]
Fig. 1.
14-3-3 binds
TSC2. A, a directed yeast two-hybrid assay was
performed by co-transforming yeast with bait construct (pGBT8) listed
first and prey construct (pGAD) listed second, each with or without
cDNA insert. Transformed cells were replica-plated to -Leu,
-Trp plates and -Leu, -Trp, -His plates supplemented with
3-aminotrizole. B, HEK293T cells were transfected with
plasmid encoding indicated proteins, and lysates were either
immunoprecipitated (IP) first (upper two panels)
or examined directly (lower two panels) by Western blotting
with the indicated antibody (WB). C, lysate from
exponentially growing HEK293T cells was immunoprecipitated with
anti-Myc (control) or anti-TSC2 monoclonal antibodies and
resolved by SDS-PAGE. Proteins were detected by immunoblotting with
antibodies to TSC2, TSC1, and 14-3-3, and the Western blot shown is
from a single exposure. D, experiment was performed as
outlined in B.
Forms a Ternary Complex with TSC1 and TSC2
Proteins--
In contrast to tuberin, TSC1 did not interact with
14-3-3
by yeast two-hybrid analysis (Fig. 1A). Since
there is no obvious homologue to TSC1 in the genome of the
host yeast, S. cerevisiae, these results indicate that
TSC2 directly interacts with 14-3-3 and that this interaction is not
dependent on the formation of a TSC1-TSC2 complex. Akt negatively
regulates the TSC1-TSC2 complex by phosphorylating TSC2 and causing its
disassembly from TSC1 (8, 9). We questioned whether binding of
14-3-3
to TSC2 might similarly affect the TSC1-TSC2 interaction. We
co-transfected plasmid encoding a GST-14-3-3
fusion protein into
cells expressing Myc-TSC1 and HA-TSC2, either individually or in
combination. GST-14-3-3
was precipitated from the lysate by addition
of gluthathione-agarose beads, and binding of TSC1 and TSC2 was
examined by immunoblotting. Fig. 10 shows that GST-14-3-3
efficiently precipitated TSC2 (lane 4), but not TSC1
(lane 3). When overexpressed alone, the TSC1 protein
presents at a very low level in the Nonidet P-40 lysate (lane
3), presumably due to its association into an insoluble fraction
(11) and its rapid degradation when not complexed with TSC2.2 When expressed with
both TSC1 and TSC2, however, 14-3-3
was able to complex with TSC1
(Fig. 1D). Prolonged film exposure increased the signal of
TSC1 (lane 3), but did not reveal any association of TSC1
with GST-14-3-3
(data not shown). These data indicate that binding
of 14-3-3
with TSC2 does not impair TSC1-TSC2 association and that
14-3-3
can form a ternary complex with both proteins.
Binds a Phosphorylated Form of Tuberin--
Members of
the 14-3-3 family of proteins typically interact with phosphoproteins,
recognizing the consensus binding sequence RSXpS/TXP where pS/T represents a
phoshorylated serine or threonine residue (12). Structural analyses
have pinpointed a cluster of positively charged amino acids that are
highly conserved among 14-3-3 proteins and that lay within an
amphipathic groove on the surface of the protein (13, 14). These
residues coordinate the phosphoamino acid located on the 14-3-3 binding
partner, and their mutation completely disrupts the binding of 14-3-3 proteins to their specific interacting polypeptide (15-17). To
determine whether these residues are also critical to the binding of
TSC2 by 14-3-3
, we replaced lysines 58 and 62 with alanine
(14-3-3
K58A,K62A) and asked whether this mutant
14-3-3
was capable of associating with TSC2. No interaction between
14-3-3
K58A,K62A and TSC2 was detected (Fig.
2A), indicating that lysines
58 and 62 in 14-3-3
are essential for binding TSC2 and therefore
that TSC2 may be recognized by 14-3-3
as a phosphoprotein. To
further test this notion, we purified GST fusion proteins with both the wild-type 14-3-3
and the 14-3-3
K58A,K62A mutant (Fig.
2B). Equal amounts of these proteins were added to lysate
from HEK293T cells expressing HA-TSC2 either left untreated or treated
with CIP. Treatment with CIP caused the collapse of multiple otherwise
broad TSC2 bands to a tight-packed species (Fig. 2C,
lanes 1 and 2), confirming phosphorylation of
TSC2. GST-14-3-3
was able to selectively precipitate slowly
migrating phosphorylated forms of TSC2, but not the CIP-treated TSC2
(Fig. 2C, lanes 5 and 6). Neither GST
alone nor GST-14-3-3
K58A,K62A was able to pull down TSC2
from the lysates. These findings demonstrate a requirement of TSC2
phosphorylation for binding to 14-3-3
and are in support of a direct
interaction between the two polypeptides.
View larger version (56K):
[in a new window]
Fig. 2.
Binding of TSC2 by 14-3-3
requires phosphorylation of TSC2. A, U2OS cells
were transfected to express proteins as indicated and lysates were
either immunoprecipitated (IP) with anti-HA antibody
(upper two panels) or examined directly (lower two
panels) by Western blotting with the indicated antibody
(WB). * indicates a nonspecific band. B, purified
GST and GST fusion proteins were separated by SDS-PAGE followed by
staining with Coomassie Brilliant Blue. Molecular mass standards
are given to the left. C, lysate from HEK293T
cells transfected with HA-TSC2 expression plasmid was incubated either
with or without CIP as indicated and mixed with the GST or the GST
fusion protein shown. Precipitated material was detected by Western
blotting against the HA epitope. Equal amounts of each lysate (20%
input) were included in lanes 1 and 2 to
demonstrate even loading and CIP activity.
Binding--
The
binding of phosphoproteins to 14-3-3s is often regulated by the
serine/threonine protein kinases A, B/Akt, or C, since their
phosphorylation consensus sites are similar to that recognized by
14-3-3s (18). Akt has been shown to directly phosphorylate TSC2 at
multiple sites and thereby negatively regulate its activity (7-9). We
questioned whether phosphorylation of TSC2 by Akt enabled its
interaction with 14-3-3
. Mapping of Akt target sites in TSC2 by
two-dimensional gel electrophoresis identified four residues phosphorylated by Akt: serine 939, serine 1086, serine 1088, and threonine 1422 (8). We tested mutants of TSC2 in which each, or all
(4×A), of these residues was substituted by alanine to assess the role
of Akt in regulating the TSC2-14-3-3
interaction. Strikingly, no
single mutation had any effect on the ability of TSC2 to
co-immunoprecipitate 14-3-3
(Fig.
3A). However, simultaneous mutation of all four Akt phosphorylation sites in TSC2 reduced its
binding with 14-3-3
(4×A mutant, lane 9). This suggested that 14-3-3 proteins bind TSC2 at a phosphoamino acid(s) distinct from
those phosphorylated by Akt and that Akt-mediated phosphorylation, although not required, may influence the interaction of 14-3-3 with
TSC2. With the aid of SCANSITE (scansite.mit.edu) we identified three putative 14-3-3 binding motifs within TSC2: serine 540, serine
1210, and serine 1355 (Fig. 3B). Binding analysis performed with the three individual TSC2 mutants revealed a requirement that
Ser1210 alone must remain intact in order for 14-3-3
to
bind (Fig. 3C). Because Ser1210 has not been
identified as an Akt phosphorylation site (7-9), these results suggest
that binding of 14-3-3
to TSC2 is dependent on a kinase(s) other
than Akt.
View larger version (39K):
[in a new window]
Fig. 3.
Serine 1210 of TSC2 is necessary for
14-3-3 docking. A, HEK293T
cells were transfected with pcDNA3-Myc-14-3-3
and plasmid
encoding the indicated mutant or wild-type form of HA-TSC2. Cells were
lysed, immunoprecipitated (IP) with anti-Myc
antibody, and immunoprecipitates examined by Western blot
(WB) (upper two panels), and total cell lysate
was analyzed in parallel (lower two panels). B, schematic
diagram using single amino acid letter code to show consensus binding
site for 14-3-3 proteins and putative 14-3-3 binding sites in TSC2.
Phosphorylated residues are indicated in bold type.
C, experiment was performed as outlined in
A.
Antagonizes TSC Function--
The interaction of 14-3-3 proteins with various binding partners is known to have diverse
biochemical consequences leading to various physiological effects (18).
We were unable to detect altered subcellular localization of TSC2
following overexpression of 14-3-3
(data not shown). We were
likewise unable to detect disruption of the TSC1-TSC2 protein complex
nor an alteration in their stability by overexpression of 14-3-3
(data not shown). Several recent reports have indicated that TSC1-TSC2
activity can be demonstrated by a reduction in S6K phosphorylation on
threonine residue 389 (7-9). To determine whether there might be a
functional consequence resulting from the interaction of 14-3-3
with
TSC2, we assayed for the ability of TSC2 to inhibit phosphorylation, and hence activation, of S6K. Under normal growth conditions in U2OS
cells S6K is phosphorylated on Thr389, and we detected an
evident reduction in phosphorylation when TSC1 and TSC2 were
co-expressed (Fig. 4A).
Although increased expression of 14-3-3
did not alter the levels of
either TSC1 or TSC2, the phosphorylation of Thr389 on S6K
was partially restored in the presence of 14-3-3
(Fig. 4A). In all experiments, however, we were unable to achieve
a complete inhibition by 14-3-3
of the TSC1-TSC2 activity against S6K regardless of the cell line used, plasmid transfection ratios, or
protein expression levels (data not shown). Nevertheless, in five
independent experiments, co-expression of 14-3-3
was able to
increase phosphorylation of Thr389 in S6K an average of
2.3-fold over the levels observed in the presence of exogenous
TSC1-TSC2 alone (Fig. 4A). This represents, on average, a
63% recovery of the S6K phosphothreonine 389 levels detectable in the
absence of ectopically expressed TSC1-TSC2. The partial effect observed
is consistent with the possibility that a portion of ectopically
expressed TSC2 is refractory to 14-3-3
binding and, therefore,
inhibition by 14-3-3
.
View larger version (43K):
[in a new window]
Fig. 4.
14-3-3 decreases the
ability of the TSC1-TSC2 complex to reduce S6K phosphorylation.
A, U2OS cells were transfected as indicated, and whole cell
lysates were separated by SDS-PAGE followed by Western blotting. A
phosphospecific antibody recognizing phosphothreonine 389 of S6K is
shown as pT389. Five independent experiments were carried out as
described, and S6K phosphorylation at Thr389 as detected by
Western blotting was quantified using Scion Image software. The lowest
amounts of expressed 14-3-3
(lane 4) were used to
calculate the third bar. B, HEK293T cells were
transfected and serum-starved for 24 h prior to stimulation with
350 nM insulin for 30 min. Samples were analyzed as
outlined in A, and quantification was performed on two
independent experiments.
(Fig. 3C). To ensure that the ability of 14-3-3
to
compromise the inhibition by TSC2 of S6K phosphorylation was due to the
binding of 14-3-3
to TSC2 and not an indirect effect, we tested
whether TSC2S1210A was also sensitive to regulation by
14-3-3
. The addition of insulin to serum-starved HEK293T cells
caused an increase in phosphorylation of S6K at Thr389
(Fig. 4B). Expression of TSC1 and TSC2 not only prevented
the insulin-induced phosphorylation of S6K but caused the
phosphothreonine 389 level to drop below that of serum-starved
conditions (Fig. 4B, lanes 1-3). Although
TSC2S1210A behaved like wild-type TSC2 to the extent that
it could bind TSC1 (data not shown) and cause reduced phosphorylation
of Thr389 in S6K, the inhibitory activity of this mutant
toward S6K was not affected by elevated amounts of 14-3-3
, whereas
activity of the wild-type TSC2 was (Fig. 4B). These data are
consistent with a regulatory scheme in which the binding of 14-3-3
to phosphoserine 1210 of TSC2 results in a diminished ability of the
TSC1-TSC2 complex to negatively regulate the phosphorylation of S6K on
Thr389.
. Rather,
phosphorylation at serine 1210 of TSC2 by an unknown kinase is required
for efficient binding of 14-3-3
. This implies that in addition to
Akt, another kinase, and possibly a distinct cell growth-inducing
signaling pathway, may converge at the inactivation of the TSC complex.
We speculate that phosphorylation on serine 1210 of TSC2 is
rate-limiting under our experimental conditions, since the inhibitory
effect of increased 14-3-3
expression on TSC2 activity is saturable
(Fig. 4A). With over 70 reported binding proteins to date,
the cellular activities influenced by the 14-3-3 protein family is
broad and diverse (18). Our results suggest that in mammalian cells
14-3-3 proteins may cooperate with mTOR to enhance cellular growth at
least in part through the negative regulation of TSC1-TSC2. We provide
evidence in support of a model in which direct, regulated binding by
14-3-3 to phosphorylated TSC2 does not disrupt the TSC1-TSC2 complex
yet impairs its ability to function as a negative regulator of cell
growth. The identification of the serine 1210 kinase and a
determination of how 14-3-3 proteins restrain TSC activity by forming a
ternary complex with TSC1-TSC2 will undoubtedly provide insight into
the regulation and function of TSC in cell growth control.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Kun-Liang Guan for generously providing TSC2 expression constructs and for the exchange of unpublished data. We thank Sima Zacharek for discussion and reading of the manuscript, Yizhou He for technical assistance, and other members of the Xiong laboratory for helpful criticisms.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant CA65572 (to Y. X.).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.
§ Supported by the Postodoctoral Training Program, Lineberger Comprehensive Cancer Center.
Supported in part by United States Department of Defense Career
Development Award DAMD17-99-1-9574. To whom correspondence should be
addressed: CB# 7295, Lineberger Comprehensive Cancer Center, The
University of North Carolina at Chapel Hill, Chapel Hill, NC
27599-7295. Tel.: 919-962-2142; Fax: 919-966-8799; E-mail: yxiong@email.unc.edu.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.C200499200
2 S. Zacharek and Y. Xiong, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TSC, tuberous sclerosis complex; mTOR, mammalian target of rapamcyin; GST, glutathione S-transferase; HA, hemagglutinin; CIP, calf intestinal phosphatase; S6K, subunit S6 kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gomez, M., Sampson, J., and Whittemore, V. H. (1999) Tuberous Sclerosis Complex , 3rd Ed. , Oxford University Press, New York |
2. | Young, J., and Povey, S. (1998) Mol. Med. Today 4, 313-319[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Hodges, A. K., Li, S.,
Maynard, J.,
Parry, L.,
Braverman, R.,
Cheadle, J. P.,
DeClue, J. E.,
and Sampson, J. R.
(2001)
Hum. Mol. Genet.
10,
2899-2905 |
4. |
Gao, X.,
and Pan, D.
(2001)
Genes Dev.
15,
1383-1392 |
5. | Tapon, N., Ito, N., Dickson, B. J., Treisman, J. E., and Hariharan, I. K. (2001) Cell 105, 345-355[Medline] [Order article via Infotrieve] |
6. | Potter, C. J., Huang, H., and Xu, T. (2001) Cell 105, 357-368[Medline] [Order article via Infotrieve] |
7. | Manning, B. D., Tee, A. R., Logsdon, M. N., Blenis, J., and Cantley, L. C. (2002) Mol. Cell 10, 151-162[Medline] [Order article via Infotrieve] |
8. | Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. (2002) Nat. Cell Biol. 4, 648-657[CrossRef][Medline] [Order article via Infotrieve] |
9. | Potter, C. J., Pedraza, L. G., and Xu, T. (2002) Nat. Cell Biol. 4, 658-665[CrossRef][Medline] [Order article via Infotrieve] |
10. | Gao, X., Zhang, Y., Arrazola, P., Hino, O., Kobayashi, T., Yeung, R. S., Ru, B., and Pan, D. (2002) Nat. Cell Biol. 4, 699-704[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Nellist, M.,
van Slegtenhorst, M. A.,
Goedbloed, M.,
van den Ouweland, A. M.,
Halley, D. J.,
and van der Sluijs, P.
(1999)
J. Biol. Chem.
274,
35647-35652 |
12. | Fu, H., Subramanian, R. R., and Masters, S. C. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 617-647[CrossRef][Medline] [Order article via Infotrieve] |
13. | Liu, D., Bienkowska, J., Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191-194[CrossRef][Medline] [Order article via Infotrieve] |
14. | Xiao, B., Smerdon, S. J., Jones, D. H., Dodson, G. G., Soneji, Y., Aitken, A., and Gamblin, S. J. (1995) Nature 376, 188-191[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Zhang, L.,
Wang, H.,
Liu, D.,
Liddington, R.,
and Fu, H.
(1997)
J. Biol. Chem.
272,
13717-13724 |
16. |
Wang, H.,
Zhang, L.,
Liddington, R.,
and Fu, H.
(1998)
J. Biol. Chem.
273,
16297-16304 |
17. |
Thorson, J. A., Yu, L. W.,
Hsu, A. L.,
Shih, N. Y.,
Graves, P. R.,
Tanner, J. W.,
Allen, P. M.,
Piwnica-Worms, H.,
and Shaw, A. S.
(1998)
Mol. Cell. Biol.
18,
5229-5238 |
18. |
Tzivion, G.,
and Avruch, J.
(2002)
J. Biol. Chem.
277,
3061-3064 |