From the Department of Biochemistry and Protein
Network Research Center, Yonsei University, Seoul 120-749, Korea and
the ¶ Department of Biology, Faculty of Science, Chiba
University, Chiba 263-8522, Japan
Received for publication, December 23, 2002, and in revised form, February 11, 2003
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ABSTRACT |
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The cullin-containing ubiquitin-protein
isopeptide ligases (E3s) play an important role in regulating the
abundance of key proteins involved in cellular processes such as cell
cycle and cytokine signaling. They have multisubunit modular structures in which substrate recognition and the catalysis of ubiquitination are
carried out by distinct polypeptides. In a search for proteins involved
in regulation of cullin-containing E3 ubiquitin ligases we
immunopurified CUL4B-containing complex from HeLa cells and identified
TIP120A as an associated protein by mass spectrometry. Immunoprecipitation of cullins revealed that all cullins tested specifically interacted with TIP120A. Reciprocal immunoaffinity purification of TIP120A confirmed the stable interaction of TIP120A with cullin family proteins. TIP120A formed a complex with CUL1 and
Rbx1, but interfered with the binding of Skp1 and F-box proteins to
CUL1. TIP120A greatly reduced the ubiquitination of phosphorylated I The ubiquitin-dependent proteolysis provides
a fundamental mechanism for regulating protein activity in various
processes ranging from cell cycle and developmental switches to signal
transduction (1). This process begins with the attachment of a
multiubiquitin chain to a target protein and involves several enzymatic
activities. A ubiquitin-activating enzyme
(E1)1 activates ubiquitin in
an ATP-dependent reaction by forming a thioester bond with
the C-terminal glycine of ubiquitin. The ubiquitin is then transferred
to a specific sulfhydryl group on a ubiquitin-conjugating enzyme (E2).
A ubiquitin-protein ligase (E3) transfers the activated ubiquitin from
E2 to a lysine residue of a bound substrate, forming an isopeptide
bond. Substrate specificity is determined mainly by E3s which bind both
the protein substrate and the cognate E2. Once the multiubiquitin chain
is assembled on a protein substrate by the cooperation of E1, E2, and
E3 enzymes, the target protein is recognized and degraded by the 26 S proteasome (1-3).
In mammalian cells, a wide variety of E3s are found. The cullin family
proteins play an important role in a group of multisubunit E3 ubiquitin
ligases by associating with an Rbx1 (also known as ROC1 and Hrt1)
family member of RING finger proteins to form the integral core (4).
The SCF complexes are the best characterized ones of this class (5).
They consist of CUL1, Rbx1, Skp1, and an F-box protein. Rbx1 contains
the RING-H2 finger domain, forms a catalytic core with CUL1, and
recruits the cognate E2 (6-8). Skp1 functions as an adaptor that links
an F-box protein to CUL1 (9). Substrates of the SCF complexes are bound
by F-box proteins, which contain the Skp1-binding F-box motif and a
variable protein-protein interaction domain that directly interacts
with substrates (9, 10). Since a large number of F-box proteins are
encoded by eukaryotic genomes (11-13), a variety of proteins are
expected to be substrates of the SCF complexes, assuming that most of
the F-box proteins form functional SCF E3 ubiquitin ligases. So far, a
few SCF complexes, including SCFSkp2,
SCF CUL2 and CUL5 can also assemble multisubunit E3 ubiquitin ligases that
bear a striking resemblance to SCF-type complexes. CUL2/Rbx1 and
CUL5/Rbx1 form a complex with the Elongin BC heterodimer that
functions as an adaptor analogously to Skp1 in the SCF complexes. The
Elongin BC complex binds to a large number of proteins including the von Hippel-Lindau (VHL) tumor suppressor protein (14-16) and members of the SOCS-box protein family (17, 18), each of which contains
an Elongin BC-binding site and a diverse protein-protein interaction motif (19). As a component of the VHL ubiquitin ligase
complex, the VHL protein targets the Studies on cullin/Rbx1-containing E3 ubiquitin ligases raise the
possibility that other cullin family members could function as a
component of ubiquitin ligases with distinct substrate specificities by
forming multiprotein complexes with yet unidentified adaptors and/or
substrate recognition subunits. In an effort to address this
possibility, we have been purifying cullin-containing complexes from HeLa cells and identifying specifically associated proteins. In
this report, we present purification of CUL4B-containing complexes and
demonstrate that TIP120A specifically interacts with cullin family
proteins and that it negatively regulates the activity of an SCF
ubiquitin ligase by interfering with the binding of Skp1 to CUL1.
Plasmids--
The cDNAs encoding human TIP120A
(GenBankTM accession number BE019018), CUL2
(GenBankTM AA206544), CUL4A (GenBankTM
BC008308), and Rbx1 (GenBankTM H71993) were from
Incyte Genomics Inc. CUL3 (GenBankTM accession
number KIAA0617) and CUL4B (GenBankTM accession
number KIAA0695) cDNA clones were a kind gift from Kazusa DNA
Research Institute. Human CUL1, Skp1, and Skp2 cDNAs (kind
gifts from Y. Xiong and H. Zhang) were described previously (24, 25).
To construct plasmids for the expression of N-terminally FLAG- or
HA-tagged proteins, cDNAs were amplified by PCR with appropriate
primers and ligated into pcDNA3.1(+) vector (Invitrogen).
Stable Cell Lines, Extract Preparation, and Protein Complex
Purification--
HeLa Tet-Off (Clontech) derived
cells stably expressing EBNA-1 were transfected with an episomal
expression vector pYR-FLAG-hCUL4A or pYR-FLAG-hTIP120A that contained
the gene of interest under the tetracycline-regulated promoter, oriP
for episome replication, and the selection marker for hygromycin B. The
cells were selected and maintained in Dulbecco's modified Eagle's
medium (Invitrogen) supplemented with 10% fetal bovine serum
(Invitrogen), 100 µg/ml G418 (Sigma), 300 µg/ml hygromycin B
(Clontech), 100 units/ml penicillin, 100 µg/ml
streptomycin, 1 mM L-glutamine, and with 2 µg/ml tetracycline (Sigma). To induce the expression of FLAG-tagged proteins, cells were grown without tetracycline for 2 days. Nuclear extracts and cytosolic S100 extracts were prepared as described previously (26). Nuclear extracts were dialyzed against buffer BC (20 mM Tris-HCl (pH 7.9), 15% glycerol, 1 mM EDTA,
1 mM DTT, 0.2 mM phenylmethanesulfonyl
fluoride, 0.05% Nonidet P-40) containing 150 mM KCl
(BC150) and rotated with anti-FLAG M2-agarose (Sigma) at 4 °C for
3-6 h. After extensive washes with BC150, proteins were eluted with
0.3 mg of FLAG peptide per ml in BC150.
Protein Identification by Mass Spectrometry--
Immunopurified
protein complexes were resolved on sodium dodecyl sulfate (SDS)-4-20%
gradient polyacrylamide gels (Novex). After staining gels with
Sypro Ruby (Bio-Rad) and subsequently with colloidal Coomassie
Blue, protein bands were excised and digested with trypsin as described
previously (27). In-gel tryptic digests of proteins were analyzed by
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry using Voyager-DE STR (Applied Biosystems) and by
nanoelectrospray ionization tandem mass spectrometry on API QSTAR
Pulsar Q-TOF (Applied Biosystems). The mass spectral data were used to
search the National Center for Biotechnology Information nonredundant
and expressed sequence tag data bases.
Immunoprecipitations and Western Blotting--
Transfection was
carried out by the CaPO4-DNA precipitation method using
Hepes or BES buffer. After 36 h, cells were lysed in buffer
containing 50 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mM
phenylmethanesulfonyl fluoride, and 1.0% Nonidet P-40. Cell lysates
were adjusted to 0.1% Nonidet P-40 and incubated with anti-FLAG or
anti-HA antibody resin (Sigma) for 4 h at 4 °C. The immune
complexes were recovered by low speed centrifugation, and the resin was
washed extensively with the binding buffer with 0.1% Nonidet P-40 and
then eluted with buffer containing 20 mM Tris-HCl (pH 8.0)
and 2% SDS. Immunoprecipitated proteins were separated by SDS-PAGE and
transferred to nitrocellulose membrane (Bio-Rad) and visualized by
Western blotting with the enhanced chemiluminescence reagents (Amersham
Biosciences). For Western blotting, we used antibodies against
FLAG (Sigma), HA (Babco), TIP120A (BD Biosciences), CUL1 (Lab Vision),
Rbx1 (Lab Vision), Skp1 (Zymed Laboratories Inc.), and
Skp2 (Zymed Laboratories Inc.).
Expression and Purification of Recombinant
Proteins--
His6-tagged yeast Uba1, human Ubc3,
and UbcH5A (Ref. 28, a kind gift from J. W. Conaway) were
expressed in Escherichia coli strain BL21(DE3) and purified
by Ni2+-agarose beads (Qiagen). Ub containing a
His6 tag and a protein kinase C recognition site (a kind
gift from Z.-Q. Pan) was purified as described previously (29). Rat
TIP120A containing N-terminal FLAG and His6 tags was
expressed in Sf21 cells and purified as described previously
(30). Generation of recombinant baculoviruses encoding human CUL1 with
a His6 tag, human I Purification of CUL4B-containing Complex--
To facilitate the
purification of CUL4B-containing complex, we first established a
HeLa-derived cell line that conditionally expressed FLAG-tagged CUL4B
using an episomal expression vector. The tetracycline-regulated
expression of FLAG-CUL4B in stable transfectants was tested by
immunoblotting of cell extracts with anti-FLAG antibody. FLAG-CUL4B was
induced upon removal of tetracycline, while it was not detected in
cells grown in the presence of tetracycline (Fig.
1A). FLAG-CUL4B and its
associated proteins were purified from extracts of induced and
uninduced cells by a single-step immunoaffinity purification procedure,
which had proved to be a gentle and efficient method (33). Several
cellular proteins specifically co-purified with FLAG-CUL4B under the
experimental conditions employed, as judged by SDS-polyacryamide gel
electrophoresis of purified proteins from induced and uninduced cells
(Fig. 1B). Bands corresponding to specifically interacting
proteins were excised and analyzed by matrix-assisted laser
desorption/ionization mass spectrometry and nanoelectrospray tandem
mass spectrometry. The p14 band was identified as Rbx1 (data not
shown), which forms a stable complex with all cullin family members
(4). Another protein p50 turned out to be a novel protein of unknown
function. However, since reciprocal immunoprecipitation experiments did not confirm the specificity of the interaction between p50 and CUL4B,
p50 was not further studied. As shown in Fig.
2, mass spectrometric analyses of tryptic
peptides derived from p120 revealed it as human TIP120A, which was
shown to be a transcription factor that enhanced transcription by RNA
polymerases I, II, and III (34, 35). The fact that TIP120A was found in
CUL4B-containing complex raised the possibility that in addition to the
reported function of TIP120A in transcription, it might be involved in
regulation of E3 ubiquitin ligase and/or ubiquitin- dependent
proteolysis.
TIP120A Interacts with Cullin Family Proteins--
To address the
specificity of CUL4B-TIP120A interaction, we tested other cullin family
members for their binding to TIP120A. HeLa cells were transfected with
expression constructs of FLAG-tagged CUL1, CUL2, CUL3, CUL4A, or CUL4B,
and anti-FLAG immunoprecipitation was carried out on the cell lysates.
Western blotting of immunoprecipitates with anti-TIP120A antibodies
indicated that TIP120A associated with all cullins tested (Fig.
3A). Immunoprecipitates of an
unrelated protein FLAG-Chk2kd did not contain TIP120A (lane
1), indicating that the interaction between TIP120A and cullins
was specific. To confirm the association of TIP120A with cullins,
FLAG-TIP120A was immunopurified using anti-FLAG antibodies from cells
that conditionally expressed the protein. SDS-polyacryamide gel
electrophoresis of purified proteins revealed several protein bands
with their molecular masses of 80-90 kDa that were present only in the
preparation derived from induced cells (Fig. 3B). Mass
spectrometric analyses identified these proteins as CUL1, CUL2, CUL3,
CUL4A, and CUL4B (data not shown), confirming that TIP120A specifically
interacted with cullins. A protein band with the molecular mass of 14 kDa was identified as Rbx1, suggesting that TIP120A formed a trimeric complex with a cullin and Rbx1. Since TIP120A interacted with most, if
not all, cullins, it might function as a global regulator of
cullin-containing ubiquitin ligases.
TIP120A Interferes with Binding of Skp1 and F-box Proteins to
CUL1--
Since CUL1 was the best studied member of the cullin family,
we chose CUL1 to further characterize its interaction with TIP120A. To
examine whether the interaction of TIP120A with CUL1 affects the SCF
complex formation of CUL1, HeLa cells were transfected with expression
constructs of epitope-tagged TIP120A, CUL1, Rbx1, Skp1, and an F-box
protein Skp2 or TIP120A Inhibits Ubiquitination of I
TIP120A was initially identified as a TBP-interacting protein using
in vitro affinity purification procedures (34) and
subsequently shown to function as a transcriptional activator (35). The
results presented here demonstrate a novel function of TIP120A, namely, negative regulation of SCF E3 ubiquitin ligases by inhibiting Skp1
binding to CUL1. Since Skp1 is an adapter subunit of SCF complexes that
links the F-box protein to CUL1 (9, 25, 37), inhibition of Skp1 binding
by TIP120A results in inhibition of association of F-box proteins and
reduction of functional SCF complexes. It has been reported that
TIP120A expression was up-regulated during the differentiation process
of certain cells. For instance, retinoic acid treatment of P19 mouse
embryonal carcinoma cells, which induces differentiation and withdrawal
from the cell cycle, elevated expression of TIP120A (38). In addition,
it has been shown that overexpression of TIP120A in P19 cells arrested
cell growth (38). Since SCF E3 ligases play a key role in progression of cell division cycle, it is tempting to speculate that induction of
TIP120A down-regulates SCF E3 ligases which may, in turn, help cells
exit from cell cycle.
The function of TIP120A is not likely to be restricted to negative
regulation of SCF complexes. Recent evidence suggests that SCF
ubiquitin ligases may directly deliver substrate proteins to the
proteasome for degradation (39, 40). For instance, it has been shown
that Skp1 interacts with an Snf1-related protein kinase (SnRK) and B
by SCF
-TrCP ubiquitin ligase. These
results suggest that TIP120A functions as a negative regulator of SCF
E3 ubiquitin ligases and may modulate other cullin ligases in a similar fashion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPRERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-TrCP, SCFCdc4, and SCFGrr1,
have been demonstrated to have E3 activities for specific substrates.
subunits of the hypoxia-inducible transcription factors HIF1 and HIF2 for
ubiquitination (20, 21). In the case of SOCS-1, Vav and JAK2 are known
to be specific substrates of the E3 complex (22, 23). Most SOCS-box proteins may function as a substrate-binding subunit of an E3 ubiquitin
ligase complex.
EXPRERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPRERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-TrCP with a FLAG tag, and human Skp1
with a His6 tag (a generous gift from M. Pagano) were
described previously (31). Human Rbx1 linked with an N-terminal FLAG
tag was subcloned into a baculovirus expression vector, pFastBac (Invitrogen), and expressed in Sf21 cells. Sf21 cells
were cultured at 27 °C in TNM-FH (Sigma) with 5% fetal calf
serum, penicillin (100 units/ml), and streptomycin (100 µg/ml).
Sf21 cells were co-infected with the recombinant baculoviruses
indicated in Fig. 5. Sixty hours after infection, cells were collected
and lysed in ice-cold buffer containing 40 mM Hepes-NaOH
(pH 7.9), 150 mM NaCl, 1 mM DTT, 0.5% (v/v)
Triton X-100, 10% (v/v) glycerol, 5 µg/ml leupeptin, 5 µg/ml
antipain, 5 µg/ml pepstatin A, and 5 µg/ml aprotinin. Lysates were
clarified by centrifugation at 10,000 × g for 20 min
at 4 °C. FLAG-
-TrCP/His-Skp1 complex was purified by applying the
supernatant onto an M2-agarose column (Sigma) equilibrated with the
lysis buffer. After extensive washing of the column with the lysis
buffer, the bound proteins were eluted with the lysis buffer containing
0.3 mg/ml FLAG peptide (Sigma). His-CUL1/FLAG-Rbx1 was purified using
Co2+-agarose beads (Clontech) according
to the manufacturer's instructions.
B
Ubiquitination Assays--
SCF
-TrCP was
reconstituted with recombinant His-CUL1/FLAG-Rbx1 and
FLAG-
-TrCP/His-Skp1 and its activity of I
B
ubiquitination assayed using the procedure previously described (29, 32) with minor
modifications. The Ub ligation reaction mixture (30 µl) contained 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM NaF, 10 nM okadaic acid, 2 mM
ATP, 0.6 mM DTT, 0.8 µg of Ub, 50 ng of E1, 200 ng of E2,
500 ng of His-CUL1/FLAG-Rbx1, 300 ng of FLAG-
-TrCP/His-Skp1, and 1.2 µg of phosphorylated glutathione S-transferase
(GST)-I
B
1-54. Reaction mixtures were incubated at
37 °C for 20 min, terminated by adding 30 µl of 2× Laemmli
loading buffer, and resolved by SDS-PAGE followed by autoradiography to
visualize the ubiquitinated I
B
. For substrate preparation, 18 µg of purified GST-I
B
1-54 was phosphorylated with
HA-tagged, constitutively active IKK
S177E/S181E kinase
purified from 293 cells following transfection. The reaction was
carried out in the presence of 10 µCi of [
-32P]ATP
at 37 °C for 20 min in a total volume of 30 µl of kinase buffer
containing 50 mM Tris-HCl (pH 7.4), 5 mM
MgCl2, 5 µM ATP, 2 mM NaF, 10 nM okadaic acid, and 0.6 mM DTT.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPRERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Immunoaffinity purification of FLAG-tagged
CUL4B. A, conditional expression of FLAG-CUL4B. Cells
expressing FLAG-CUL4B in a tetracycline-controlled manner were
established as described under "Experimental Procedures." Cells
were grown in the presence (lane 1) or the absence
(lanes 2-5) of tetracycline for the indicated time.
FLAG-CUL4B expression was examined by immunoblotting of the cell
lysates with anti-FLAG antibody. B, SDS-PAGE analysis of
purified FLAG-CUL4B. Ten µl each of purified FLAG-CUL4B samples from
uninduced and induced cells was separated by a 4-20%
SDS-polyacrylamide gel and visualized by Sypro Ruby-Coomassie
double staining. Protein size markers (in kilodaltons) are indicated on
the left.
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Fig. 2.
Identification of TIP120A as a
CUL4B-interacting protein. A, an MS/MS spectrum of a
peptide derived from p120. MS/MS analyses were performed as described
under "Experimental Procedures." B, sequences determined
by MS/MS analyses of tryptic peptides obtained from p120. All peptides
were identical to sequences in human TIP120A.
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Fig. 3.
TIP120A interacts with members of the cullin
family. A, immunoprecipitations of cullins. HeLa cells
were transiently transfected with expression constructs for FLAG-tagged
CUL1, CUL2, CUL3, CUL4A, and CUL4B (lanes 2-6). As a
negative control, an unrelated protein FLAG-tagged Chk2kd was expressed
(lane 1). Cell lysates were immunoprecipitated with
anti-FLAG antibody-coupled beads. The immunoprecipitates were subjected
to SDS-polyacryamide gel electrophoresis and analyzed by Western
blotting using anti-FLAG antibody to detect FLAG-tagged proteins
(lower panel) and anti-TIP120 antibody to detect
co-immunoprecipitated TIP120 (upper panel). B,
immunoaffinity purification of FLAG-tagged TIP120A. Ten µl each of
purified FLAG-TIP120A samples from uninduced and induced cells was
separated by a 4-20% SDS-polyacrylamide gel and visualized by Sypro
Ruby-Coomassie double staining.
-TrCP. HA-CUL1 and FLAG-TIP120A were then
immunoprecipitated with anti-HA and anti-FLAG antibodies, respectively,
and co-purified proteins were probed by immunoblotting with suitable
antibodies (Fig. 4A).
FLAG-tagged TIP120A, Rbx1, Skp1, and
-TrCP were specifically
detected in HA-CUL1 immunoprecipitates (lanes 1 and
2). In contrast, FLAG-TIP120A immunoprecipitates contained
HA-CUL1 and HA-Rbx1, but not Skp1 and Skp2 (lanes 3 and
4), indicating that FLAG-TIP120A did not form a complex with Skp1 and Skp2. To confirm that F-box proteins Skp2 and
-TrCP do not
interact with TIP120A, immunoprecipitation of HA-Skp2 or HA-
-TrCP
was performed and associated proteins analyzed by immunoblotting (Fig.
4B). CUL1 and Skp1 were present in HA-Skp2 and HA-
-TrCP immunoprecipitates, but TIP120A was not pulled down in the precipitates (lanes 5 and 6). Since CUL1 directly associates
with Skp1 (9, 36) and TIP120A (data not shown), interactions among
CUL1, Skp1, and TIP120A were examined by coupled immunoprecipitation
and immunoblotting (Fig. 4C). Whereas CUL1 was
immunoprecipitated with both TIP120A and Skp1, the immunoprecipitates
of FLAG-TIP120A contained only CUL1, but not Skp1, and those of
FLAG-Skp1 contained only CUL1, but not TIP120A (lanes 4-6).
Collectively, these data demonstrated that in cells existed at least
two distinct CUL1-containing complexes either with TIP120A or with Skp1
and that the binding of TIP120A and Skp1 to CUL1 was mutually
exclusive. To further test whether TIP120A and Skp1 compete for the
binding to CUL1, HeLa cells were transfected with expression constructs
of FLAG-CUL1, HA-TIP120A, HA-Skp1, and HA-Skp2. FLAG-CUL1 was
immunoprecipitated with anti-FLAG antibodies, and HA-tagged TIP120A,
Skp1, and Skp2 were probed by immunoblotting with anti-HA and
anti-TIP120A antibodies (Fig. 4D). Overexpression of TIP120A
reduced co-precipitation of Skp1 to 4-18% in triplicate experiments
and Skp2 to 30-40% (lanes 5 and 6). In a
similar competition experiment with TIP120A and
-TrCP, TIP120A
inhibited the association of
-TrCP with CUL1 in a
dose-dependent manner (lanes 12-14). These data
showed that TIP120A interfered with binding of Skp1 and F-box proteins
Skp2 and
-TrCP. It was noted that inhibition of Skp1 binding to CUL1
by TIP120A was greater than that of Skp2 or
-TrCP binding. Recent
structural studies on SCFSkp2 showed that the F-box motif
of Skp2 directly interacts with CUL1 as well as Skp1 (36). This
Skp2-CUL1 interaction might not be affected by TIP120A binding and
might contribute to less inhibition of Skp2 binding by TIP120A. Further
investigation is required to clarify this point.
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Fig. 4.
TIP120A interferes with binding of Skp1 and
F-box proteins to CUL1. A, immunoprecipitations of CUL1
and TIP120A. HeLa cells were transiently transfected with plasmid
vectors expressing epitope-tagged or untagged TIP120A, CUL1, Rbx1,
Skp1, Skp2, and -TrCP proteins as indicated. Lysates were prepared 36 h
after transfection and immunoprecipitated with
-HA (lanes
1 and 2) or
-FLAG antibodies (lane 4).
The precipitates were separated by SDS-PAGE and immunoblotted with
indicated antibodies. B, immunoprecipitations of Skp2 and
-TrCP. HeLa cells were transiently transfected with plasmid vectors
expressing FLAG- or HA-tagged TIP120A, CUL1, Skp1, Skp2, and
-TrCP
proteins as indicated. Immunoprecipitations with
-HA antibodies
(lanes 4-6) and immunoblotting experiments were performed
as described in the legend to A. C, association
of CUL1 either with Skp1 or with TIP120A. HeLa cells were transiently
transfected with plasmid vectors expressing epitope-tagged or untagged
TIP120A, CUL1, and Skp1 proteins as indicated. Immunoprecipitations and
immunoblotting experiments were performed as described in the legend to
A. D, competition of TIP120A and Skp1 for the
binding to CUL1. HeLa cells were transiently transfected with plasmid
vectors expressing HA-TIP120A, FLAG-CUL1, HA-Skp1, HA-Skp2, and
HA-
-TrCP proteins in combinations as indicated. Immunoprecipitations
with
-FLAG antibodies and immunoblotting experiments were performed
as described in the legend to A.
B
by
SCF
-TrCP--
To determine the functional consequence
of association of TIP120A with CUL1, we examined the effect of TIP120A
on the substrate-specific ubiquitin ligase activity of
SCF
-TrCP using I
B
as a substrate. To devise an
in vitro reconstituted system for the ubiquitination of
I
B
, we produced all components used in the ubiquitination
reaction as recombinant proteins by Escherichia coli or the
baculovirus system. Fig. 5A
shows dye staining patterns of the purified subunits of
SCF
-TrCP after separation by SDS-PAGE.
GST-I
B
1-54 was phosphorylated with
IKK
S177E/S181E in the presence of
[
-32P]ATP and incubated in the Ub ligation reaction
with recombinant CUL1/Rbx1 and
-TrCP/Skp1 (Fig. 5B). A
high molecular weight 32P-labeled protein ladder was
formed, indicating that phosphorylated GST-I
B
1-54
was polyubiquitinated. The ubiquitination of
GST-I
B
1-54 was dependent not only on E1 and E2 (data
not shown) but on CUL1/Rbx1 and
-TrCP/Skp1 (lanes 1-4),
showing that intact SCF
-TrCP complex was required for
I
B
ubiquitination. Addition of increasing amounts of recombinant
TIP120A greatly reduced the ubiquitination of I
B
by CUL1 complex
in a dose-dependent manner (lanes 4-7). To test
whether the reduction of I
B
ubiquitination by TIP120A is
correlated with the decrease of Skp1 binding to CUL1, the CUL1/Rbx1 complex in the ubiquitination reactions was immunoprecipitated with
anti-Rbx1 antibodies and bound TIP120A and Skp1 monitored by Western
blotting. As the association of TIP120A with CUL1 increased, the
binding of Skp1 to CUL1 decreased (Fig. 5B, lower
panel). These results indicated that TIP120A inhibited ubiquitin
ligase activity of CUL1 by interfering with the binding of Skp1 and
-TrCP to CUL1.
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Fig. 5.
TIP120A inhibits ubiquitination of
I B
by
SCF
-TrCP. A,
SDS-PAGE analysis of purified FLAG-TIP120A, His-CUL1/FLAG-Rbx1, and
FLAG-
-TrCP/His-Skp1. FLAG-TIP120A, His-CUL1/FLAG-Rbx1 and
FLAG-
-TrCP/His-Skp1 were expressed in Sf21 cells and purified
as described under "Experimental Procedures." Five µl each of
purified proteins was separated by a 4-20% SDS-polyacrylamide gel and
visualized by Coomassie Blue staining. Protein size markers (in
kilodaltons) are indicated on the left. B,
inhibition of I
B
ubiquitination by TIP120A. Purified
GST-I
B
1-54 was phosphorylated with
IKK
S177E/S181E in the presence of
[
-32P]ATP and incubated with recombinant
His-CUL1/FLAG-Rbx1(500 ng), FLAG-
-TrCP/His-Skp1 (300 ng), and
FLAG-TIP120A (0.5, 1, and 2 µg in lanes 5, 6,
and 7, respectively) as indicated. Reactions were performed
at 37 °C for 20 min, terminated by adding Laemmli loading buffer,
and resolved by SDS-PAGE followed by autoradiography to visualize the
ubiquitinated I
B
ladders. For the immunoblotting experiments
shown in the lower panel, reactions were performed without
the substrate and ATP, and the CUL1/Rbx1 complex was immunoprecipitated
with anti- Rbx1 antibodies.
4
subunit of the 20 S proteasome in Arabidopsis (39),
suggesting that SnRK and/or
4 subunit may function as a docking site
for the SCF complexes on the proteasome. Thus, competitive binding of
TIP120A to CUL1 against Skp1 may help release of SCF ligases from a
proteasomal docking site and unloading of the substrate. Our finding
that TIP120A interacts not only with CUL1 but with other cullins
suggests that TIP120A has a general role common to most, if not all,
cullin-containing E3 ligases. Further structural and functional
dissection of TIP120A-cullin complexes is expected to further clarify
the role of TIP120A in regulation of ubiquitination and proteolysis.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. M. Pagano, J. W. Conaway, Z.-Q. Pan, Y. Xiong, and H. Zhang for generous gifts of reagents.
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FOOTNOTES |
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* This work was supported in part by grants from the Korea Science and Engineering Foundation through Protein Network Research Center at Yonsei University and from the Korean Ministry of Science and Technology through 21C Frontier Project.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.
§ These two authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
82-2-2123-2704; Fax: 82-2-392-3488; E-mail:
yoonj@yonsei.ac.kr.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M213070200
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ABBREVIATIONS |
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The abbreviations used are: E1 or Uba, ubiquitin-activating enzyme; E2 or Ubc, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; Ub, ubiquitin; GST, glutathione S-transferase; HA, hemagglutinin; SCF, Skp1-Cdc53/cullin-F box; SOCS, suppressor of cytokine signaling; VHL, von Hippel-Lindau; DTT, dithiothreitol; BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; MS/MS, tandem mass mass spectrometry.
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