From the Unité d'Immunologie Virale and
Groupe des Bunyaviridés, Unité des arbovirus et
virus des fièvres hémorragiques, Institut Pasteur, 25 et
28, rue du Dr. Roux, 75724 Paris Cedex 15, France and the
¶ Institut Cochin de Génétique Moléculaire
(ICGM) INSERM CJF9703 and ** ICGM U129 INSERM, Université Paris V,
75014 Paris, France
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ABSTRACT |
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Activation of NF- NF- Cell Lines, Transfections, and Infections--
Subconfluent
cells of the 293 human embryo kidney cell line were transfected by
LipofectAMINETM Plus (Life Technologies, Inc.) with the
indicated reporter plasmids and pcDNA3 vectors expressing Antibodies and Reagents--
The 10B monoclonal antibody
directed against the amino terminus of I Electrophoretic Mobility Shift Assay--
The electrophoretic
mobility shift assay was performed with 4 µg of nuclear extract
incubated for 15 min at room temperature with a
[ To investigate the putative role of B transcription factors
requires phosphorylation and ubiquitin-proteasome-dependent
degradation of I
B proteins. We provide evidence that a human F-box
protein, h-
TrCP, a component of Skp1-Cullin-F-box protein (SCF)
complexes, a new class of E3 ubiquitin ligases, is essential for
inducible degradation of I
B
.
TrCP associates with
Ser32-Ser36 phosphorylated, but not with
unmodified I
B
or Ser32-Ser36
phosphorylation-deficient mutants. Expression of a F-box-deleted
TrCP inhibits I
B
degradation, promotes accumulation of
phosphorylated Ser32-Ser36 I
B
, and
prevents NF-
B-dependent transcription. Our findings indicate that
TrCP is the adaptor protein required for I
B
recognition by the SCF
TrCP E3 complex that ubiquitinates
I
B
and makes it a substrate for the proteasome.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
B transcription factor is regulated by I
B proteins of
which I
B
is the main and best characterized member (1-3).
Proteasome-mediated degradation of I
B
releases NF-
B and allows
its localization in the nucleus (4-9). Phosphorylation of
Ser32-Ser36 residues and subsequent
ubiquitination of I
B
are prerequisites to make the protein
susceptible to proteasome attack (10-13). While the kinase complex
accounting for I
B
phosphorylation has been recently characterized
(14-20), factors necessary for ubiquitination and targeting of
I
B
to the proteasome remain unknown. Covalent attachment of
polyubiquitin to substrate proteins involved a cascade of ubiquitin
transfer reactions with E1,1
a ubiquitin-activating enzyme, and E2 a ubiquitin-conjugating enzyme
that operates in conjunction with a specificity factor E3 (21-26). It
has been suggested that E3 functions in substrate recognition and E2
positioning. Although E2 enzymes belonging to the Ubc4/Ubc5 family can
ubiquitinate I
B
in vitro, the E3 responsible for the
signal-induced ubiquitination of I
B
remains to be identified (10,
27). A novel class of E3 is represented by the Skp1-Cullin-F-box
protein complexes (SCFs) (28-33). The core component of these newly
identified E3s is Skp1, which assembles with different F-box proteins
and has been shown in human cells to interact selectively with CUL-1,
but not with other Cullin proteins belonging to the Cdc53 family
(34-36). The role of the F-box proteins in these SCF complexes is to
recruit phosphorylated substrate proteins to trigger their
ubiquitination (28-33). Like the other members of the F-box protein
family, human
TrCP, which we recently identified (37), has a modular
organization with an F-box motif involved in proteasome targeting
through interaction with Skp1, and a seven WD repeats binding domain
for interaction with substrate proteins (see Fig. 1A). We
hypothesized that
TrCP could be the F-box adaptor protein allowing
recruitment of I
B
by a SCF E3 ubiquitin-protein ligase complex
that ubiquitinates I
B
and makes it a substrate for degradation by
the proteasome.
EXPERIMENTAL PROCEDURES
TrCP
proteins or with no
TrCP insert. Fluorigenic substrate luciferin
served to quantify luciferase reporter gene expression in cytoplasmic
extracts obtained by lysis in phosphate buffer containing 1% Nonidet
P-40. The pcDNA3-
TrCP or -
TrCP
F constructs are described
in Ref. 37.
TrCP and
TrCP
F coding sequences were amplified by
polymerase chain reaction and inserted in fusion with the Myc/His
double tag in the pcDNA3.1 Myc/HisA vector (Invitrogen). SV5-tagged
wild type or SV5-tagged S32A/S36A phosphorylation-deficient mutant
I
B
are described in Ref. 11, 3Enh-
B-ConA and ConA luciferase
reporter plasmids are described in Ref. 38. RSV luciferase reporter
plasmid was purchased from Invitrogen.
TrCP or I
B
were
inserted in fusion with the LexA DNA binding domain and the Gal4
activation domain, respectively, as described in Ref. 37. Construction
and use of recombinant SFV was carried out as described before
(39-42). Briefly, 293 cells were infected at a multiplicity of 5 for
6 h in serum-free medium with SFV particles carrying myc-tagged
TrCP proteins. Expression of
TrCP proteins and viral nucleocapsid protein was verified by Western blot analysis, and 100% infection efficiency was confirmed by immunofluorescence (data not shown). The
same results as those shown in Fig. 2B were obtained in HeLa cells infected with SFV.
B
was described in Ref.
43; the polyclonal antibody specifically recognizing I
B
phosphorylated at serine residue 32 is from New England Biolabs
(9241S), and the monoclonal anti-myc antibody was from Santa Cruz
Biotechnology (hybridoma 9E10, SC-40). For co-immunoprecipitation
experiments (Fig. 3, A and B), 200 µg of
cytoplasmic extract were incubated with anti-myc-agarose conjugates
(SC-40 AC from Santa Cruz) for 60 min at 4 °C. Precipitated beads
were washed 10 times in phosphate-buffered saline containing 1%
Nonidet P-40 and both protease and phosphatase inhibitors. Antibody-antigen complexes were disrupted by boiling in gel loading buffer (Pierce). Precipitated proteins were fractionated by
SDS-polyacrylamide gel electrophoresis and electroblotted onto
nitrocellulose membranes.
-32P]ATP-labeled, double-stranded oligonucleotide
containing the HIV-1 long terminal repeat binding site for NF-
B
(5'-ACAAGGGACTTTCCGCTGGGACTTTCCAGGGA-3'). Samples were analyzed in
nondenaturing 6% polyacrylamide gels. Competition experiments were
performed by adding a 40-fold molar excess of homologous, unlabeled
oligonucleotide to each sample prior to addition of the radiolabeled probe.
RESULTS AND DISCUSSION
TrCP in the regulation of
NF-
B activation, we first assessed the effect of wild type (
TrCP)
and a F-box deleted
TrCP (
TrCP
F) (Fig.
1A) on the transcriptional activity of a NF-
B-dependent (3Enh-
B-ConA) promoter
driving a luciferase reporter gene (10, 11). Expression of
TrCP
resulted in a 2- to 3-fold increase in the activity of the
3Enh-
B-ConA promoter in cells of the 293 human embryo kidney cell
line stimulated by either tumor necrosis factor (TNF) or okadaic acid
(OKA), two well characterized inducers of NF-
B (Fig. 1B,
left panel). Similarly, the low level of constitutive
activation of NF-
B found in unstimulated cells (compare
3Enh-
B-ConA to ConA) is also enhanced by expression of
TrCP (Fig.
1B, left panel). In sharp contrast to
TrCP,
expression of the
TrCP
F mutant massively and consistently
inhibited NF-
B-dependent transcription by more than 90%
of the levels induced in TNF- or OKA-stimulated cells transfected with
an insertless, control plasmid (pcDNA3) (Fig. 1B,
left panel). Importantly, expression of the transdominant
negative mutant
TrCP
F fully prevented localization of NF-
B to
the nucleus upon cell activation. This finding is in keeping with an
increased stability of inhibitor I
B proteins that anchor NF-
B in
the cytoplasm in an inactive form. The failure of
TrCP
F to modify
the activity of a RSV promoter indicates that
TrCP
F does not
affect NF-
B independent mechanisms of transcription (Fig.
1B, left panel).
View larger version (42K):
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Fig. 1.
TrCP is required for cell
activation-induced NF-
B transcription.
A, physical map of
TrCP showing the F-box responsible for
proteasome targeting through Skp1 binding and the seven WD40 repeats.
The F-box deletion in the mutant
TrCP
F is indicated.
B, regulation of NF-
B-dependent transcription
by
TrCP or
TrCP
F. Human 293 embryo kidney cells (2 × 106 cells) were transiently co-transfected with
NF-
B-dependent (3Enh-
B-ConA luc) or -independent
(ConA luc, RSV luc) DNA luciferase expression vectors (1 µg) and
CMV-IE-promoter-driven (pcDNA3) vectors (3 µg) expressing either
wild type (
TrCP) or mutant (
TrCP
F) forms of
TrCP (10, 11).
Equal amounts of the pcDNA3 plasmid without
TrCP cDNA insert
were transfected as a control. The amount of wild type and mutant
TrCP proteins was assessed by Western blot analysis of cytoplasmic
extracts to ensure that wild type and mutant
TrCP proteins were
expressed at comparable levels (not shown). After transfection, cells
were left untreated (NS) or were stimulated with either 5 ng/ml TNF or 75 ng/ml OKA for 6 h. Experiments were repeated three
times in 293 cells and confirmed in HeLa cells. Results of a
representative experiment are shown. Luciferase activity is expressed
as relative luciferase units (RLU) per µg of protein and
results from subtracting the background signal from the values obtained
for each sample. Comparative electrophoretic mobility shift assay of
nuclear extracts from HeLa cells expressing either
TrCP or the
TrCP
F from SFV. A radiolabeled oligonucleotide encoding the
NF-
B consensus was used as a probe to bind transcription factors. A
SFV replicon without insert was used as a control. The
asterisk indicates where competitor cold oligonucleotide was
added to demonstrate the specificity of NF-
B/DNA interaction.
Thus, on the one hand, the capacity of TrCP to enhance both basal or
signal-induced activation of NF-
B and, on the other hand, the
specific inhibition of NF-
B dependent transcription promoted by
TrCP
F, strongly suggest that
TrCP is an essential component of
the NF-
B activation pathway. In support of this assumption, we found
that, concomitant with the enhancement of NF-
B-dependent
transcription, overexpression of
TrCP in 293 cells stimulated by TNF
induced an accelerated degradation of I
B
(Fig.
2A, middle panel).
In contrast, expression of the
TrCP
F mutant either from a
eukaryotic vector (Fig. 2A, right panel) or a SFV
replicon (Fig. 2B, right panel), stabilized
I
B
and delayed the kinetics of I
B
degradation. Moreover,
the presence of
TrCP
F promoted the accumulation of slow migrating
forms of I
B
characteristic of the phosphorylation of residues
Ser32 and Ser36 required for subsequent
ubiquitination and degradation of I
B
by the proteasome (Fig. 2,
A and B, right panel). Thus, these findings exclude an inhibitory effect of
TrCP
F in the
transduction pathway leading to phosphorylation of I
B
and suggest
that stabilization of I
B
is due to the blockade of a
post-phosphorylation event in the metabolism of the inhibitor.
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Slow migrating forms of IB
predominated following induction with
TNF but could even be detected in unstimulated cells (Fig. 2,
A and B, right panel, time 0). This
latter phenomenon likely reflects the inhibitory effect of
TrCP
F
on basal breakdown of I
B
and is in keeping with the low and
constitutive NF-
B-dependent transcription observed in
293 cells (Fig. 1B, left panel).
Characterization of the slow migrating band of IB
(Fig.
2A, right top panel), as a phosphorylated form of
I
B
accumulated in
TrCP
F expressing cells, was accomplished
(Fig. 2A, middle row of panels) using a
polyclonal antibody that does not recognize the unphosphorylated form
of I
B
but specifically reacts with Ser32-Ser36-phosphorylated I
B
which
accumulates in the presence of the proteasome inhibitor
Z-LLL-H following induction with TNF (Fig. 2C) (44).
To ascertain whether the regulatory effect of TrCP on I
B
metabolism requires association with I
B
, carboxyl-terminal
c-myc-tagged variants of either
TrCP or
TrCP
F were expressed
in HeLa cells from SFV (Fig.
3A) or in 293 cells from
eukaryotic expression vectors (Fig. 3B), and their
co-precipitation with I
B
was investigated. Cytoplasmic extracts
of cells treated or not with TNF and the proteasome inhibitor
Z-LLL-H, were incubated with an anti-myc tag
antibody bound to protein A-coated agarose beads. Proteins precipitated
by the anti-myc antibody were probed with anti-I
B
antibodies
(Fig. 3A, middle and bottom panels).
In cells expressing either the wild type or the mutated counterpart of
TrCP, a single band of co-precipitated I
B
was recognized by a
monoclonal antibody that detects both native and phosphorylated forms
of I
B
(Fig. 3A, bottom panel, lanes
4-6). This band migrates with a pattern characteristic of the
typical upshift induced by phosphorylation of I
B
Ser32 and Ser36. Western blot analysis using
the antibody specifically recognizing the phosphoserine
Ser32-Ser36 I
B
confirmed that the
proteins detected are phosphorylated at the critical residues that
permit subsequent ubiquitination of I
B
(Fig. 3A,
middle panel, lanes 4-6). No unphosphorylated form of I
B
was detected in immunoprecipitates (Fig.
3A, lower panel). Detection of phosphorylated
I
B
did not require TNF induction when the mutant
TrCP
F was
expressed (Fig. 3A, middle panel, lane
5). Moreover, larger amounts of endogenous phosphorylated I
B
co-precipitated with
TrCP
F, compared with wild type
TrCP (Fig.
3A, compare lanes 6 with lane
4), confirming that the
TrCP
F mutant acts as a transdominant
negative regulator of both constitutive and TNF-induced proteolysis of
I
B
.
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To confirm the selective association of phosphorylated IB
and
TrCP, we performed experiments using a S32A/S36A mutant of I
B
lacking the capacity to be phosphorylated by cell activation signals
promoting NF-
B activation (3). The presence of a 15-amino acid SV5
carboxyl terminus tag allows distinction of endogenous from transiently
expressed wild type or S32A/S36A I
B
. Despite expression of
similar amounts of wild type or S32A/S36A-SV5 tagged proteins (data not
shown), only the endogenous and wild type SV5-tagged I
B
(Fig.
3B, lanes 4-6), but not the SV5-S32A/S36A
phosphorylation-deficient mutant (Fig. 3B, lanes
7-10) were able to associate with either
TrCP or
TrCP
F.
Expression of either wild type or phosphorylation-deficient tagged
I
B
proteins from SFV was consistently detected in more than 90%
of cells (data not shown). The high infection efficiency of this system
allows us to conclude that co-precipitation of the endogenous, but not
the S32A/S36A-SV5 I
B
(Fig. 3B, lanes 7-10), reflects the incapacity of the phosphorylation-deficient mutant to compete for binding to I
B
.
Further evidence of IB
interaction with
TrCP was provided by
the yeast two-hybrid system (carried out as described previously (37)).
We observed that
TrCP fused to the LexA DNA binding domain
(LexA-
TrCP) associates specifically with I
B
fused to the Gal4
activation domain (Gal4AD-I
B
), as detected by histidine auxotrophy or
-galactosidase expression (Fig. 3C). The
interaction between I
B
and
TrCP is likely accounted for by the
existence in yeast of phosphorylated I
B
as shown by the
recognition of the Gal4AD-I
B
hybrid by the antibody that
specifically reacts with Ser32-Ser36
phosphorylated I
B
(Fig. 3D). This hypothesis is
reinforced by the fact that the Gal4AD-I
B
S32A/S36A mutant, which
is not recognized by the anti-phosphoserine I
B
antibody (Fig.
3D), did not associate with LexA-
TrCP (Fig.
3C).
We have previously documented that TrCP is a component of a SCF
complex involved in HIV-1 Vpu-mediated CD4 degradation. The WD domain
of
TrCP is responsible for the interaction with Vpu (37). Although
TrCP is involved in both Vpu-mediated CD4 and I
B
proteolysis,
it should be stressed that important differences between the two
degradation pathways exist. Indeed, if both Vpu and I
B
can be
phosphorylated at serine in DSGXXS motifs, phosphorylation of Vpu occurs constitutively (45-46) while that of I
B
requires activation of cell signaling. Furthermore, and in contrast to I
B
,
Vpu has not yet been characterized as a substrate for ubiquitination or
degradation by the proteasome. No human protein recognized as
ubiquitination substrate by an SCF complex and undergoing degradation by the proteasome has been documented so far. I
B
phosphorylated at critical serine residues represents the first example of this kind
of substrate (8).
In the SCF complexes with E3-ubiquitin-protein ligase activity, the
F-box component allows specific recognition of substrates (28-33). The
existence of a large number of F-box-containing proteins revealed by
genome sequencing and the combinatorial interactions of SCF components
that belong to different protein families (Cullin, E2 ubiquitin-protein
conjugating enzymes) suggest that the growing family of E3 ligases is
composed of a large number of different SCF complexes. This diversity
has likely hampered the identification of the E3 ligase responsible for
ubiquitin conjugation of IB
required for I
B
degradation and
NF-
B activation. While precise characterization of the ubiquitin
conjugating activity associated with SCF
TrCP is still
missing, our findings provide evidence that
TrCP is ultimately
responsible for recognition of phosphorylated I
B
by the SCF complex.
As shown for other F-box proteins in yeast, TrCP could target
different substrates as well as I
B
to the proteasome. This hypothesis is sustained by the recent discovery that, Slimb, the Drosophila homolog of
TrCP, may be involved in an as yet
unidentified step of regulation of the wingless and hedgehog pathways
(47). However, it cannot be assumed from this that
TrCP is a broad, universal adaptor for ubiquitinated substrates. Indeed, in the SCF
complex, F-box proteins determine and restrict substrate recognition by
the proteasome. Thus, the yeast F-box protein Cdc4 is able to
selectively bind phosphorylated Sic1, but not phosphorylated Cln1 or
Cln2, whereas Grr1, another yeast F-box protein, shows selective
association with the latter substrates but not with Sic1-(30-32).
In conclusion, our findings characterize TrCP as a F-box protein,
which selectively associates with Ser32-Ser36
phosphorylated, but not unmodified, I
B
.
TrCP represents the SCF adaptor which ultimately accounts for recognition of phosphorylated I
B
by the ubiquitination machinery and allows targeting of the ubiquitinated inhibitor to the proteasome (see model in Fig.
4). While this manuscript was completed
and sent for review, a report by Yaron et al. (48) was
published that supports the involvement of
TrCP in I
B
degradation and NF-
B activation. Thus,
TrCP can be considered as
a new target for pharmacological intervention in the physiopathological
processes regulated by NF-
B.
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ACKNOWLEDGEMENTS |
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We thank Michèle Bouloy and Agnès Billecocq of the Bunyaviridés research group at Institut Pasteur for their generous support to this project. S. Michelson is acknowledged for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by the European Union Concerted Action BIOMED II (ROCIO II project), Agence Nationale de Recherche sur le SIDA, SIDACTION, and Association pour la Recherche sur le Cancer (France).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 a fellowship of the German Academic Exchange Service (Program HSP III).
To whom correspondence should be addressed. Tel.:
33-1-44-41-25-65; E-mail: benarous{at}cochin.inserm.fr.
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ABBREVIATIONS |
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The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; SCF, Skp1-Cullin-F-box protein; ConA, concanavalin A; RSV, Rous sarcoma virus; SFV, Semliki forest virus; TNF, tumor necrosis factor; OKA, okadaic acid.
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REFERENCES |
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