From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
Received for publication, September 29, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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Cbl-b is implicated in setting the threshold of T
lymphocyte activation. In Cbl-b-deficient T cells, the activation of
Vav, a guanine nucleotide exchange factor, is significantly enhanced. The molecular mechanism underlying Cbl-b-regulated Vav activation was
unclear. Here it is shown that Cbl-b interacts with and induces ubiquitin conjugation to the p85 regulatory subunit of
phosphatidylinositol 3-kinase, an upstream regulator of Vav. A
functional RING finger of Cbl-b was essential for p85 ubiquitination.
However, a loss of function mutation at the well-conserved
amino-terminal variant src homology (SH) 2 domain of Cbl-b did not
affect its ligase activity. A distal carboxyl-terminal proline-rich
region in Cbl-b was mapped to contain the primary binding sequences for
the SH3 domain of p85. Deletion of either the distal proline-rich
region in Cbl-b or the SH3 domain of p85 severely reduced ubiquitin
conjugation to p85. The data suggest a molecular link for
Cbl-b-mediated negative regulation of Vav, with phosphatidylinositol
3-kinase as a direct target for Cbl-b E3 ubiquitin ligase.
Cbl-b and its close mammalian homologue, Cbl, consist of an
amino-terminal variant SH21
domain, a RING finger, and a carboxyl-terminal proline-rich domain with
potential tyrosine phosphorylation sites (1, 2). Previous studies have
shown that Cbl-b and Cbl function as adaptor proteins by interacting
with other critical signal molecules, including the variant SH2
domain-dependent interaction with cell surface receptor
tyrosine kinases or intracellular protein tyrosine kinases such as Syk
and Zap-70 and the carboxyl-terminal region-dependent interaction with Grb2, 14-3-3, phosphatidylinositol 3-kinase (PI3-K), Vav, and Crk-L (3, 4). Genetic and biochemical studies have shown that
Cbl family proteins including those from Drosophila and
Caenorhabditis elegans attenuate intracellular
signaling induced by the engagement of cell surface receptors. One
mechanism for this negative role is Cbl-mediated ubiquitination of
receptor tyrosine kinases (5-7). It is now understood that Cbl
functions as an E3 ubiquitin (Ub) ligase whose RING finger recruits a
Ub-conjugating enzyme, E2, and whose SH2 domain recognizes activated
receptor tyrosine kinases for Ub conjugation (7-10).
Ubiquitination is an important cellular process that involves ligation
of a protein substrate with Ub, thereby marking it for degradation by
the 26S proteasome (11-13), and it involves a cascade of reactions
including E1, E2, and E3 enzymes. Ub is first activated by an
activating enzyme (E1) to form a high energy thiolester bond between Ub
and E1 and is then transferred to a conjugating enzyme (E2). The E3s or
Ub protein ligases are the components responsible for specific
substrate recognition and for promoting Ub ligation to the target
protein. Therefore, the E3s can provide specificity to the Ub system.
Two recent genetic studies using Cbl-b gene-targeted mice showed that
Cbl-b deficiency can change the signaling thresholds: Cbl-b deficiency
uncouples T-cell proliferation and interleukin 2 production from
the costimulation of CD28, and the gene-targeted mice develop
spontaneous autoimmunity or become highly susceptible to exogenous
antigen-induced autoimmune diseases (14, 15). These studies suggest a
critical role of Cbl-b in the regulation of T-cell activation
thresholds and hence in the maintenance of a balance between immunity
and tolerance. In Cbl-b-deficient T cells, the tyrosine phosphorylation
and/or activation of Vav, a GDP/GTP exchange factor, are significantly
enhanced (14, 15), suggesting that Cbl-b negatively regulates T-cell
signaling by inhibiting Vav activation. However, the molecular
mechanism underlying Cbl-b-mediated negative regulation of Vav remains
to be determined.
Previous studies have shown that PI3-K, which phosphorylates PI
lipid at the D3 position of the inositol ring to form active lipid
second messengers, regulates the exchange activity of Vav through the
binding of the active lipid products to the pleckstrin homology (PH)
domain of Vav (16, 17). We have investigated whether Cbl-b acts as an
E3 Ub ligase to promote ubiquitination of PI3-K. Here we show that
Cbl-b binds p85, the regulatory subunit of PI3-K, and induces its
ubiquitination. We propose a model by which Cbl-b indirectly regulates
Vav activation by inducing ubiquitination of PI3-K.
Antibodies--
The polyclonal anti-Cbl-b and anti-Fyn
antibodies and anti-c-Myc, anti-HA, and anti-Vav monoclonal
antibodies (mAbs) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Rabbit anti-p85 antibody was from UBI (Lake Placid, NY).
Anti-Xpress mAb was from Invitrogen (Carlsbad, CA). Anti-human CD3 mAb
OKT3 was purified from ascites using a protein G-Sepharose affinity column.
Plasmids--
The Cbl-b cDNAs encoding full-length Cbl-b, a
Cbl-b amino-terminal region (a.a. 1-349; Cbl-b 1-349), and Cbl-b
G298E, with a HA or Xpress epitope in an elongation factor
promoter-driven mammalian vector (pEFneo), were as described previously
(18, 19). The Cbl-b truncated constructs containing the RING finger and
the carboxyl-terminal proline-rich sequences (a.a. 341-982), the
entire carboxyl-terminal proline-rich sequences alone (a.a. 443-982),
the distal proline-rich sequences (a.a. 595-982), the amino-terminal
variant SH2 domain and RING finger (a.a. 1-481), or an addition of the
proximal proline-rich domains to Cbl-b 1-481 (a.a. 1-595) were
amplified by polymerase chain reaction, tagged with a Xpress or HA
epitope, and subcloned into the pEFneo plasmid. Point mutations,
which replaced cysteine 372 with alanine (C372A) and tryptophan
400 with alanine (W400A) in the RING finger of the full-length Cbl-b,
were made by site-directed mutagenesis (QuickChange; Stragagene,
La Jolla, CA). The Myc-tagged Ub cDNA from a HA-tagged Ub plasmid
(20) was generated by polymerase chain reaction and subcloned into
pEFneo. The p85 Cell Culture, Transfection, and Stimulation--
Jurkat T cells
were cultured in RPMI 1640 (Irvine Scientific, Santa Ana, CA)
supplemented with 10% fetal bovine serum and antibiotics. For protein
expression in Jurkat T cells, cells were transfected with the
appropriate amount of plasmids (usually 1-5 µg total) by
electroporation (240 V, 960 microFarads; Bio-Rad). After 48 h,
cells were collected, resuspended (2 × 107 cells/ml)
in 0.5 ml of medium, and treated with pervanadate, anti-CD3 (OKT3)
antibody (1 µg/ml), or MG132 (50 µM) for different time
intervals as indicated at 37 °C. Cells were then pelleted and
resuspended in 1× Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM NaPiP, 2 mM
Na3VO4, and 10 µg/ml each of aprotinin and
leupeptin). Cells were lysed for 10 min at 4 °C, and insoluble
materials were removed by centrifugation at 15,000 × g
(4 °C, 10 min). For display of ubiquitinated protein bands, 0.1%
SDS was added into lysis buffer to disrupt nonspecific protein-protein interaction.
Immunoprecipitation and Immunoblotting--
For
immunoprecipitation, lysates (~1 × 107 cells) were
mixed with antibodies (1 µg) for 2 h, followed by the addition
of 30 µl of protein G-Sepharose beads (Santa Cruz Biotechnology) for an additional 2 h at 4 °C. Immunoprecipitates were washed four times with 1× Nonidet P-40 lysis buffer and boiled in 20 µl of 2×
Laemmli's buffer. Samples were subjected to 8% or 10%
SDS-polyacrylamide gel electrophoresis analysis and electrotransferred
onto polyvinylidene difluoride membranes (Millipore). Membranes were
probed with the indicated primary antibodies (usually 1 µg/ml),
followed by horseradish peroxidase-conjugated secondary antibodies.
Membranes were then washed and visualized with an enhanced
chemiluminescence detection system (ECL; Amersham Pharmacia Biotech).
When necessary, membranes were stripped by incubation in stripping
buffer (62.5 mM Tris-HCl, pH 5.7, 100 mM
2-mercaptoethanol, and 2% SDS) for 1 h at 70 °C with constant
agitation, washed, and then reprobed with other antibodies as indicated.
Cbl-b Induces p85 Ubiquitination--
Classical PI3-K is composed
of a p85 regulatory subunit and a p110 catalytic subunit. The p85
subunit contains an amino-terminal SH3 domain and two carboxyl-terminal
SH2 domains to mediate protein-protein interactions and an interdomain
between the two SH2 domains for association with the p110 subunit (21).
Previous studies have shown that Cbl-b associates with PI3-K (14, 18).
The recent discovery that Cbl functions as a RING-type E3 ligase
for receptor tyrosine kinases prompted us to investigate whether PI3-K
could be a target protein for Cbl-b-induced ubiquitination. As the
first step, we examined whether p85 is ubiquitinated in vivo
by transient overexpression of p85 and Myc epitope-tagged Ub in Jurkat
T cells. As shown in Fig. 1A,
transient overexpression of p85 with Myc-Ub but not p85 alone resulted
in the formation of a slowly migrating species of p85 that was
recognized by anti-Myc antibody. Treatment of T cells with pervanadate
did not enhance Ub conjugation to p85. However, the ubiquitinated forms
of p85 were enhanced by further addition of cells with MG132, a
proteasome inhibitor (22).
We then examined whether Cbl-b induces Ub conjugation to p85. Cbl-b was
coexpressed with p85 together with Myc-Ub in Jurkat T cells, and the
transfected cells were immunoprecipitated with anti-p85 antibody. The
immunoprecipitates were analyzed using an anti-Myc antibody to detect
ubiquitinated p85. Coexpression with Cbl-b markedly enhanced
ubiquitinated forms of p85 (Fig. 1B), which were
further enhanced by pretreatment with MG132. To rule out the
possibility that the ubiquitinated species results from
coimmunoprecipitated proteins other than p85, we added 0.1% SDS to the
lysis buffer to reduce nonspecific protein-protein interaction. An
additional control was included by using normal rabbit serum for
immunoprecipitation, and it did not precipitate slowly migrating
species (data not shown). The data indicate that the p85 subunit of
PI3-K is polyubiquitinated and that Cbl-b can act as an E3 ligase for p85.
Cbl-b RING Finger but not the Variant SH2 Domain Is Essential for
Its Ligase Activity--
Recent studies including our own (7-9) have
shown that Cbl RING finger recruits a Ub-conjugating enzyme E2
and that an intact RING finger is required for E2 binding and its E3
ligase activity. Specifically, mutation of the conserved cysteine 381 to alanine (Cbl C381A) disrupts its E2 binding and ligase activity (8). We then examined whether mutation of the conserved cysteine 372 to
alanine in Cbl-b (Cbl-b C372A), which is equivalent to the Cbl C381A
mutation, affected its ligase activity toward p85 ubiquitination. Coexpression of Cbl-b C372A completely abolished its ability to promote
p85 ubiquitination (Fig. 2A).
Analysis of the cell lysates showed comparable levels of protein
expression between wild-type Cbl-b and the C381A mutant.
Previous studies have documented the importance of the Cbl
amino-terminal variant SH2 domain and RING finger in mediating ubiquitination of receptor tyrosine kinases (7, 8, 23), thus supporting
a model in which the amino-terminal variant SH2 domain specifically
targets the substrates. Consistent with this, a loss of function
mutation (G306E) at this domain, which disrupts the association of Cbl
with the tyrosine kinases, can also abrogate Cbl-induced ubiquitination
of the receptor tyrosine kinases (7, 23). Next, we examined the
functional role of the variant SH2 domain of Cbl-b in p85
ubiquitination. A loss of function mutation of glycine 298 to glutamic
acid in Cbl-b (Cbl-b G298E), which is equivalent to the Cbl G306E
mutation, was previously shown to disrupt the interaction with Zap-70
(19). However, the same mutation did not affect its ability to induce
p85 ubiquitination because both the wild-type Cbl-b and Cbl-b G298E
induced the formation of slowly migrating p85 species to a similar
degree (Fig. 2B). Analysis of the cell lysates showed
equivalent amounts of protein expression between the wild-type Cbl-b
and the G298E mutant. The result indicates that the evolutionarily
conserved variant SH2 domain in Cbl-b is not essential for p85 ubiquitination.
Cbl-b Physically Associates with p85--
E3 ligases confer
specificity to the Ub system by directly interacting with the substrate
proteins and help transfer Ub to them (24). An interaction between
Cbl-b and PI3-K has been demonstrated in primary T cells and in Jurkat
T cells (14, 18), and this interaction is not mediated by the two SH2
domains of p85 (18). To further confirm this interaction, we
cotransfected HA epitope-tagged Cbl-b with p85 in Jurkat T cells.
HA-Cbl-b was coimmunoprecipitated with p85 (Fig.
3A) and with Grb2, as
described previously (18). We then examined the effect of RING finger
mutation on p85 binding. Jurkat T cells were cotransfected with
HA-tagged p85 with Xpress-tagged Cbl-b or the Cbl-b RING finger mutant
C372A. Cbl-b C372A showed similar binding to p85 or to Grb2 as
wild-type Cbl-b (Fig. 3B), indicating that the observed loss
of Ub ligase activity toward p85 in the Cbl-b C372A mutant is not due
to the disruption of its interaction with p85. To examine whether the
interaction is activation-dependent, transfected cells were
then stimulated with anti-CD3 antibody at different intervals, and the
cell lysates were immunoprecipitated with anti-HA antibody. p85 was
coimmunoprecipitated with Cbl-b, and that did not change much upon
stimulation (Fig. 3C), suggesting that the interaction is
constitutive. Next, we analyzed whether Cbl-b-induced ubiquitination of
p85 is dependent on T-cell receptor engagement. Stimulation of Jurkat T
cells with anti-CD3 antibody for different time periods up to 1 h
did not increase Ub conjugation to p85 in either the absence or
presence of Cbl-b overexpression (Fig. 3C). The data suggest
that Cbl-b interacts with p85 and promotes its ubiquitination in an
activation-independent manner.
A Distal Proline-rich Region of the Cbl-b Carboxyl-terminal Region
Is Required for the Efficient Interaction with p85 and Its Ub
Conjugation--
The interaction region in Cbl-b was then determined
by coexpressing p85 with either a HA-tagged amino-terminal region
(Cbl-b 1-349) or a construct that contains the RING finger and the
carboxyl-terminal region (Cbl-b 341-982). Deletion of the RING finger
and the carboxyl-terminal region from Cbl-b abrogated its interaction
with p85, as shown in the Cbl-b 1-349 mutant (Fig.
4A). However, the Cbl-b
341-982 mutant still retained its ability to associate with p85 to the same degree as full-length Cbl-b (Fig. 4A).
The Cbl-b carboxyl-terminal-dependent interaction with p85
suggests that this region is responsible for the substrate targeting, at least for p85 protein, and is probably responsible for its ubiquitination. We tested this hypothesis by coexpressing wild-type Cbl-b, Cbl-b 1-349, or Cbl-b 341-982 with p85. Although Cbl-b 1-349
did not enhance Ub conjugation to p85 over the basal level, Cbl-b
341-982 and full-length Cbl-b showed similar ability to induce p85
ubiquitination (Fig. 4B). The data collectively suggest that
the RING and the carboxyl-terminal region are sufficient for p85
binding and ubiquitination.
We tried further to determine the binding region of p85 in Cbl-b. To
this end, we generated two Cbl-b constructs containing Cbl-b
amino-terminal variant SH2 domain plus the RING finger (Cbl-b 1-481)
or Cbl-b 1-481 plus the proximal proline-rich domain (Cbl-b 1-595),
as depicted in Fig. 5A.
Consistent with our data that carboxyl-terminal region is required for
p85 ubiquitination (Fig. 3C), deletion of the entire
carboxyl-terminal region (Cbl-b 1-481) completely abrogated its
interaction with p85, and deletion of the distal proline-rich sequences
severely reduced its interaction with p85 (Fig. 5B).
We further mapped the p85 binding region in the Cbl-b carboxyl-terminal
portion. There are more than 10 proline-rich stretches spanning the
Cbl-b carboxyl-terminal region (2). Two cDNA fragments, which
encode proteins encompassing the entire proline-rich sequences from
a.a. 443 to a.a. 982 or the distal proline-rich region from a.a. 595 to
a.a. 982, were generated and tagged with a HA epitope. The interaction
of these two proline-rich domains with p85 was analyzed by
coimmunoprecipitation. Both fragment proteins bound to p85 to a similar
degree (Fig. 5C) under both resting and
pervanadate-stimulated conditions, suggesting that the proximal
proline-rich sequences are less important for p85 interaction.
To further confirm that the distal proline-rich sequences are the
primary binding region for p85, we compared the p85 interaction of the
full-length Cbl-b with that of the 595-982 (a.a. 595-982) mutant. The
595-982 mutant and wild-type Cbl-b showed comparable binding to p85
(Fig. 5D). Reprobe of the same membrane showed equivalent
amounts of proteins for wild-type Cbl-b and the 595-982 mutant. The
data are consistent with the idea that the distal proline-rich
region contains the primary binding sequences for p85.
A functional role for the carboxyl-terminal proline-rich region of
Cbl-b was assessed in p85 ubiquitination. Removal of the distal
proline-rich sequence in Cbl-b (Cbl-b 1-595), which reduced its
interaction with p85 (Fig. 5B), severely hampered its
ability to promote p85 ubiquitination (Fig.
6A). The slight augmentation of p85 ubiquitination could be due to the residual binding of the
1-595 mutant to p85, as shown in Fig. 5B. Analysis of the cell lysates showed equivalent amounts of protein expression for wild-type Cbl-b and the 1-595 mutant (Fig. 6B). We conclude
that the Cbl-b distal proline-rich sequences are required for both efficient p85 binding and its ubiquitination.
The SH3 Domain of p85 Binds to Cbl-b and Is Required for p85
Ubiquitination--
The p85 subunit of PI3-K contains an SH3 domain at
the amino terminus. The constitutive interaction between p85 and the
distal proline-rich region of Cbl-b suggests that the SH3 domain of p85 mediates the interaction with Cbl-b. We then generated a p85 construct with the SH3 domain deleted (p85
The p85 SH3 domain-dependent interaction with the distal
proline-rich sequences of Cbl-b and the defect of p85 ubiquitination by
the Cbl-b mutant lacking the distal proline-rich domain suggest that
the SH3 domain of p85 is required for its ubiquitination. To test this
hypothesis, we examined the Cbl-b-promoted ubiquitination of p85 or
p85 The results in this report demonstrated that Cbl-b acts as an E3
Ub ligase by interacting with the p85 regulatory subunit of PI3-K and
promoting its ubiquitination. A functional RING finger is essential for
the Ub ligase activity toward p85, whereas the well-conserved
amino-terminal variant SH2 domain is dispensable for this event. We
further demonstrated that the distal proline-rich sequences in Cbl-b
and the SH3 domain of p85 are required for the efficient interaction
and subsequent Ub conjugation to p85. Our data suggest a novel role for
Cbl-b in regulating T-cell activation by inducing p85 ubiquitination.
Recent genetic studies on Cbl-b-deficient T cells showed that the
activation of Vav is increased in these cells, which was proposed to be
responsible for costimulation-independent cytokine production and cell
proliferation (14, 15). This finding is supported by a study showing
that coexpression of Cbl-b and Vav inhibits the nucleotide exchange
activity of Vav (25). The recent findings, including our own, showing
that Cbl functions as an E3 ubiquitin ligase (7-9) prompted us to
investigate a potential role of Cbl-b on Vav ubiquitination. Our
efforts failed to prove that Vav is a direct target for Cbl-b E3 Ub
ligase activity, even after repeated experiments (data not shown). A
recent study showed that Vav could be ubiquitinated by the suppressor
of cytokine signaling 1, a component of elongin Ub ligase complex.
Suppressor of cytokine signaling 1 interacts with Vav and induces its
ubiquitination and degradation (26). Although it cannot be ruled out
that Vav is a target for Cbl-b E3 ligase activity, we favor a model in which suppressor of cytokine signaling 1 is the primary E3 ligase component for Vav, and Cbl-b may indirectly regulate Vav activation.
We then investigated the possibility that Cbl-b regulates the upstream
regulators for Vav activation. One of the well-established regulators
is PI3-K, whose lipid product can bind to the pleckstrin homology
domain of Vav and activates Vav nucleotide exchange activity (16, 17).
We demonstrated that Cbl-b induces Ub conjugation to p85, the
regulatory subunit of PI3-K, which is dependent on its RING finger
domain and distal proline-rich sequences, suggesting that Cbl-b acts as
an E3 Ub ligase for p85. Besides PI3-K, Src kinases can also regulate
the tyrosine phosphorylation of Vav in synergy with PI3-K (16).
Although we did not address a potential role for Cbl-b on Src kinases
in this study, our data support a model in which Cbl-b indirectly
regulates Vav activation by promoting Ub conjugation to p85.
Previous studies on Cbl have focused on the functional role of its
amino-terminal variant SH2 domain in targeting the substrate proteins
such as cell surface receptor tyrosine kinases (5-9, 23). A loss of
function mutation (G306E) at this domain of Cbl, which disrupts its
interaction with receptor tyrosine kinases, can also abrogate its Ub
ligase activity toward these kinases (7, 23). However, a corresponding
mutation at Cbl-b (G298E) did not affect its activity to promote p85
ubiquitination, suggesting that the amino-terminal variant SH2 domain
of Cbl-b is dispensable for Ub conjugation to p85. Rather, we showed
that the carboxyl-terminal proline-rich region and, more specifically,
the distal proline-rich sequences of Cbl-b are required for efficient
p85 ubiquitination. Thus, our results suggest a novel mechanism by
which the Cbl-b carboxyl-terminal region can also recruit target
proteins and, with the help of the RING finger, induce the transfer of
Ub to them.
Our finding that the Cbl-b carboxyl-terminal region can recruit protein
substrates is consistent with the recent crystal structural study on
Cbl RING finger and an E2, Ub-conjugating enzyme H7 (10). In this
complex, the active cysteine of Ub-conjugating enzyme H7, which forms a
thiol-ester bond with Ub, is on the opposite side of the complex
relative to the binding site for tyrosine kinase binding domain
(amino-terminal domain). Therefore, a substrate that associates with
the carboxyl-terminal region would presumably allow an easy transfer of
Ub to the substrate. To further support our model, we recently found
that the Cbl carboxyl-terminal region could also recruit protein
substrates such as Stat5 and that the RING finger and the
carboxyl-terminal proline-rich region are sufficient for promoting
Stat5 ubiquitination.2 In
fact, the Cbl family proteins including those from C. elegans or Drosophila are well conserved in the
amino-terminal variant SH2 domain and the RING finger domain (3),
whereas the carboxyl-terminal regions between Cbl and Cbl-b are more
divergent, and some Cbl family members (Sli-1, D-Cbl, and Cbl-3) lack
or contain only a short form of the carboxyl-terminal proline-rich
region. Because both Cbl and Cbl-b carboxyl-terminal regions contain
several protein interaction motifs, it is conceivable that differential
recruitment of protein substrates by the carboxyl-terminal region would
provide diversity and specificity to Cbl- and/or Cbl-b-mediated protein ubiquitination.
The SH3 domain of p85 recognizes a consensus proline-rich motif:
RXXRPLPPLPP (28), which is also present in p85 itself. Inspection of the Cbl-b carboxyl-terminal region did not find a perfect
match for this consensus sequence. We attempted but failed to map the
exact binding motif in the distal proline-rich sequences, suggesting
that more than one proline-rich motif is required for the interaction
with the SH3 domain of p85. In support of this notion, it was
previously shown that the proximal proline-rich stretches in Cbl,
rather than one proline-rich motif, mediate the interaction with Grb2
(29).
The SH3 domain of p85 has been shown to mediate interaction with the
proline-rich sequence of another p85 molecule to form a p85 heterodimer
(30) or interaction with the proline-rich sequences of other molecules
(31, 32). We have shown here that the p85 SH3 domain can also interact
with Cbl-b proline-rich sequences, which results in p85 ubiquitination
promoted by Cbl-b E3 Ub ligase. Thus, our study suggests a novel role
for the SH3 domain of p85 in mediating its ubiquitination.
Polyubiquitination of protein substrates by the Ub system targets them
for degradation by the 26S proteasome (11, 12). However, modification
of biological functions through ubiquitination of protein substrates
without proteolysis has been reported recently in several systems,
including regulation of transcription by transcription factor (33) or
ubiquitination-dependent processing of precursor proteins
(27). In consideration of the fact that the protein levels of
Cbl-b-binding proteins do not change in Cbl-b-deficient T cells such as
Zap-70, Lck, or even Vav (14, 15), it can be postulated that Cbl-b, as
an E3 Ub ligase, may play a general role in functional regulation of
its target proteins through ubiquitination in a protein
degradation-independent manner. The present work provides us with a
molecular basis for our ongoing study on how Cbl-b-promoted
ubiquitination of p85 could affect its biological function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
plasmid with or without a HA tag, the p85
SH3
domain with a HA tag, and the HA-tagged p110
plasmid of PI3-K in
pEFneo were provided by T. Mustelin (La Jolla Institute for
Allergy and Immunology, San Diego, CA). An SH3 domain-deficient p85
(p85
SH3) was constructed by subcloning the BglII and
XbaI fragment of p85 into pEFneo with a HA tag. The Vav
plasmid in pEFneo was from A. Altman (La Jolla Institute for
Allergy and Immunology, San Diego, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cbl-b induces ubiquitination of p85.
A, Jurkat T cells were transfected with 3 µg of plasmid
containing HA-tagged p85 cDNA in the absence or presence of
Myc-tagged Ub plasmid (1 µg). Transfected cells were either left
untreated or treated with PV or PV plus MG132 for 30 min. Cell lysates
were immunoprecipitated with anti-HA antibody. The immunoprecipitates
were subjected to immunoblotting with anti-Myc mAb (top
panel). The positions of polyubiquitinated p85 protein
((Ub)n-p85) are indicated. The membrane was reprobed with
anti-HA (bottom panel). The position of p85 is indicated by
an arrow. B, Jurkat T cells were transfected with
plasmids containing HA-p85, Xpress-tagged Cbl-b, and Myc-Ub.
Transfected cells were left untreated or treated with MG132, and the
cell lysates were incubated with anti-HA. The immunoprecipitates were
subjected to immunoblotting with anti-Myc (top panel). The
positions of polyubiquitinated p85 protein ((Ub)n-p85) are
indicated. The same membrane was reprobed with anti-HA mAb
(middle panel). An aliquot of the cell lysates was
immunoblotted with anti-Xpress antibody to detect Cbl-b expression
(bottom panel).
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Fig. 2.
Cbl-b RING domain is required for its E3
ligase activity. A, Jurkat T cells were transfected
with the HA-p85 plasmid plus Xpress-Cbl-b or its C372A mutant, together
with Myc-Ub plasmid. Transfected cells were left untreated or treated
with MG132, and the cell lysates were incubated with anti-HA antibody.
The immunoprecipitates were subjected to immunoblotting with anti-Myc
antibody (top panel). The membrane was reprobed with anti-HA
(middle panel). Aliquots of the cell lysates were
analyzed with anti-Xpress antibody (bottom panel).
B, Jurkat T cells were transfected with HA-p85 plus
Xpress-Cbl-b or its loss of function mutant, G298E, at the
amino-terminal variant SH2 domain of Cbl-b. The transfected cells were
analyzed as described in A.
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Fig. 3.
Cbl-b associates with p85 and promotes its
ubiquitination in a constitutive manner. A, Jurkat T
cells were transfected with p85 plasmid without an epitope tag in the
absence or presence of HA-tagged Cbl-b plasmid. Cell lysates were
immunoprecipitated with anti-HA, and the immunoprecipitates were
subjected to immunoblotting with anti-p85 antibody. The same membrane
was then reprobed with anti-HA or anti-Grb2. An aliquot of the cell
lysates was immunoblotted with anti-p85. B, Jurkat T cells
were transfected with HA-tagged p85 plasmid with Xpress-tagged Cbl-b or
Cbl-b C372A mutant. Cell lysates were incubated with anti-Xpress, and
the immunoprecipitates were blotted with anti-HA. The same membrane was
reprobed with anti-Xpress or anti-Grb2 antibody. An aliquot of the
lysates was immunoblotted with anti-HA. C, Jurkat T cells
were transfected with p85 together with HA-tagged Cbl-b. Transfected
cells were stimulated with OKT3, an anti-CD3 antibody, at different
time points as indicated (in minutes) or with PV for 10 min. Cell
lysates were immunoprecipitated with anti-HA, and the
immunoprecipitates were blotted with anti-p85 antibody (top
panel). The same membrane was reprobed with either anti-HA
(middle panel) or anti-Grb2 (bottom panel).
D, Jurkat T cells were transfected with HA-p85 plasmid plus
Xpress-Cbl-b and Myc-Ub plasmids. Cells were treated as described in
C, and the cell lysates were immunoprecipitated with
anti-HA. The cell lysates were blotted with anti-Myc antibody
(top panel). The positions of polyubiquitinated p85
((Ub)n-p85) are indicated. The same membrane was reprobed
with anti-HA to detect the p85 protein (bottom panel).
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Fig. 4.
Cbl-b carboxyl-terminal region binds to p85
and mediates its ubiquitination. A, Jurkat T cells were
transfected with plasmids containing p85 plus HA-Cbl-b, an
amino-terminal fragment (a.a. 1-349), or a construct containing the
RING finger and the entire carboxyl-terminal region (a.a. 341-982).
Transfected cells were subjected to immunoprecipitation with anti-HA,
and the immunoprecipitates were blotted with anti-p85 (top
panel). The same membrane was reprobed with anti-HA (middle
panel). The positions of Cbl-b or its truncated mutants are
indicated by arrows. An aliquot of cell lysates was
immunoblotted with anti-p85 (bottom panel). B,
Jurkat T cells were transfected as described in A, with the
further addition of Myc-Ub plasmid. The cell lysates were
immunoprecipitated with anti-p85, and the immunoprecipitates were
analyzed with anti-Myc (top panel). The same membrane was
reprobed with anti-p85 (bottom panel).
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[in a new window]
Fig. 5.
A distal carboxyl-terminal proline-rich
region of Cbl-b contains the primary binding sequences for p85.
A, schematic representation of Cbl-b mutants.
SH2, the variant SH2 domain; RING, the RING
finger domain; Pro, the carboxyl-terminal proline-rich
domain. The numbers indicate the amino acid numbers in
Cbl-b. B, HA-tagged p85 plasmid together with Xpress-Cbl-b
or its Xpress-tagged mutants, as indicated, were transfected into
Jurkat T cells. Transfected T cells were left untreated or stimulated
with PV for 10 min. The lysates were incubated with anti-Xpress, and
the immunoprecipitates were blotted with anti-HA (top
panel). The same membrane was reprobed with anti-Xpress
(middle panel). Positions of Xpress-tagged Cbl-b or its
mutants are indicated by arrows. An aliquot of cell lysates
was immunoblotted with anti-HA to detect p85 expression (bottom
panel). C, Jurkat T cells were transfected with
epitope-untagged p85 plasmid plus HA-tagged Cbl-b fragments, HA-Cbl-b
443-982 (443-982) or HA-Cbl-b 595-982
(595-982). The cells were left untreated or treated with
PV, and the lysates were incubated with anti-HA. The immunoprecipitates
were blotted with anti-p85 (top panel). The same membrane
was reprobed with anti-HA (middle panel). The positions of
HA-tagged Cbl-b fragments are indicated by arrows. An
aliquot of the lysates was immunoblotted with anti-p85 (bottom
panel). D, Jurkat T cells were transfected with p85
plasmid plus HA-Cbl-b or HA-Cbl-b 595-982 plasmids. The cells were
analyzed as described in C.
View larger version (34K):
[in a new window]
Fig. 6.
The distal proline-rich region of Cbl-b is
required for p85 ubiquitination. A, Jurkat T cells were
transfected with the HA-p85 plasmid together with Xpress-Cbl-b or a
truncated mutant with a carboxyl-terminal distal proline-rich region
deleted in Cbl-b (Xpress-Cbl-b 1-595 (1-595)) plus Myc-Ub
plasmid. Transfected cells were left untreated or treated with MG132,
and the cell lysates were incubated with anti-HA. The
immunoprecipitates were blotted with anti-Myc (top panel).
The positions of polyubiquitinated p85 ((Ub)n-p85) are
indicated. The same membrane was reprobed with anti-HA (bottom
panel). B, the cell lysates from A were
immunoblotted with anti-Xpress. The positions of the wild-type Cbl-b
and the 1-595 mutant are indicated by arrows.
SH3) and analyzed whether deletion of the SH3 domain of p85 could affect its interaction with Cbl-b. HA-p85 or HA-p85
SH3 was coexpressed with Xpress-tagged Cbl-b. The
protein-protein interaction was analyzed by coimmunoprecipitation with
anti-Xpress antibody. Removal of the SH3 domain from p85 completely
abolished its interaction with Cbl-b, although Cbl-b associated with
the full-length p85 and Grb2 under the same conditions (Fig.
7A).
View larger version (38K):
[in a new window]
Fig. 7.
The SH3 domain of p85 mediates the
interaction with Cbl-b and is required for p85 ubiquitination.
A, Jurkat T cells were transfected with HA-p85 plasmid or
with its SH3 domain-deleted mutant, HA-p85 SH3, plus Xpress-Cbl-b
plasmid. Cell lysates were incubated with anti-Xpress antibody, and the
immunoprecipitates were blotted with anti-HA. The same membrane was
reprobed with anti-Xpress or anti-Grb2. An aliquot of the cell lysates
was immunoblotted with anti-HA. The positions of p85, Cbl-b, Grb2, or
p85
SH3 are indicated by arrows. B, cells were
transfected as described in A, with the addition of Myc-Ub
plasmid. Transfected cells were left untreated or treated with MG132,
and the cell lysates were incubated with anti-HA. The
immunoprecipitates were blotted with anti-Myc (top panel).
H, the immunoglobulin heavy chain. The same membrane was
reprobed with anti-HA (bottom panel). The positions of p85
and p85
SH3 are indicated.
SH3 in Jurkat T cells. Under a condition that coexpression of Cbl-b with the full-length p85 induced Ub conjugation to p85, deletion of the SH3 domain in p85 severely reduced its ubiquitination induced by Cbl-b, even with MG132 treatment (Fig. 7B). Taken
together, the results indicate that the p85 SH3 domain is required for
Cbl-b interaction and for Cbl-b-promoted ubiquitination of p85.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1DK56558 (to Y.-C. L.).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: Division of Cell
Biology, La Jolla Institute for Allergy and Immunology, 10355 Science
Center Dr., San Diego, CA 92121. E-mail: yuncail@liai.org.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M008901200
2 H.-Y. Wang, D. Fang, L. Qiu, Y. Altman, C. Elly, and Y.-C. Liu, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Ub, ubiquitin; PI3-K, phosphatidylinositol 3-kinase; SH, src homology; mAb, monoclonal antibody; HA, hemagglutinin; a.a., amino acid(s); PV, pervanadate.
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REFERENCES |
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1. | Langdon, W. Y., Hartley, J. W., Klinken, S. P., Ruscetti, S. K., and Morse, H. C. d. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1168-1172[Abstract] |
2. | Keane, M. M., Rivero-Lezcano, O. M., Mitchell, J. A., Robbins, K. C., and Lipkowitz, S. (1995) Oncogene 10, 2367-2377[Medline] [Order article via Infotrieve] |
3. | Lupher, M. L., Jr., Rao, N., Eck, M. J., and Band, H. (1999) Immunol. Today 20, 375-382[CrossRef][Medline] [Order article via Infotrieve] |
4. | van Leeuwen, J. E., and Samelson, L. E. (1999) Curr. Opin. Immunol. 11, 242-248[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Miyake, S.,
Lupher, M. L., Jr.,
Druker, B.,
and Band, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7927-7932 |
6. |
Lee, P. S.,
Wang, Y.,
Dominguez, M. G.,
Yeung, Y. G.,
Murphy, M. A.,
Bowtell, D. D.,
and Stanley, E. R.
(1999)
EMBO J.
18,
3616-3628 |
7. | Levkowitz, G., Waterman, H., Ettenberg, S. A., Katz, M., Tsygankov, A. Y., Alroy, I., Lavi, S., Iwai, K., Reiss, Y., Ciechanover, A., Lipkowitz, S., and Yarden, Y. (1999) Mol. Cell 4, 1029-1040[Medline] [Order article via Infotrieve] |
8. |
Joazeiro, C. A.,
Wing, S. S.,
Huang, H.,
Leverson, J. D.,
Hunter, T.,
and Liu, Y. C.
(1999)
Science
286,
309-312 |
9. |
Yokouchi, M.,
Kondo, T.,
Houghton, A.,
Bartkiewicz, M.,
Horne, W. C.,
Zhang, H.,
Yoshimura, A.,
and Baron, R.
(1999)
J. Biol. Chem.
274,
31707-31712 |
10. | Zheng, N., Wang, P., Jeffrey, P. D., and Pavletich, N. P. (2000) Cell 102, 533-539[Medline] [Order article via Infotrieve] |
11. | Hochstrasser, M. (1996) Annu. Rev. Genet. 30, 405-439[CrossRef][Medline] [Order article via Infotrieve] |
12. | Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425-479[CrossRef][Medline] [Order article via Infotrieve] |
13. | Hicke, L. (1999) Trends Cell Biol. 9, 107-112[CrossRef][Medline] [Order article via Infotrieve] |
14. | Bachmaier, K., Krawczyk, C., Kozieradzki, I., Kong, Y. Y., Sasaki, T., Oliveira dos Santos, A., Mariathasan, S., Bouchard, D., Wakeham, A., Itie, A., Le, J., Ohashi, P. S., Sarosi, I., Nishina, H., Lipkowitz, S., and Penninger, J. M. (2000) Nature 403, 211-216[CrossRef][Medline] [Order article via Infotrieve] |
15. | Chiang, Y. J., Kole, H. K., Brown, K., Naramura, M., Fukuhara, S., Hu, R. J., Jang, I. K., Gutkind, J. S., Shevach, E., and Gu, H. (2000) Nature 403, 216-220[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Han, J.,
Luby-Phelps, K.,
Das, B.,
Shu, X.,
Xia, Y.,
Mosteller, R. D.,
Krishna, U. M.,
Falck, J. R.,
White, M. A.,
and Broek, D.
(1998)
Science
279,
558-560 |
17. |
Ma, A. D.,
Metjian, A.,
Bagrodia, S.,
Taylor, S.,
and Abrams, C. S.
(1998)
Mol. Cell. Biol.
18,
4744-4751 |
18. | Elly, C., Witte, S., Zhang, Z., Rosnet, O., Lipokowitz, S., Altman, A., and Liu, Y.-C. (1999) Oncogene 18, 1153-1162 |
19. | Zhang, Z., Elly, C., Qiu, L., Altman, A., and Liu, Y.-C. (1999) Curr. Biol. 9, 203-206[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Qiu, L.,
Joazeiro, C.,
Fang, N.,
Wang, H. Y.,
Elly, C.,
Altman, Y.,
Fang, D.,
Hunter, T.,
and Liu, Y. C.
(2000)
J. Biol. Chem.
275,
35734-35737 |
21. |
Rameh, L. E.,
and Cantley, L. C.
(1999)
J. Biol. Chem.
274,
8347-8350 |
22. |
Kim, T. K.,
and Maniatis, T.
(1996)
Science
273,
1717-1719 |
23. |
Lill, N. L.,
Douillard, P.,
Awwad, R. A.,
Ota, S.,
Lupher, M. L., Jr.,
Miyake, S.,
Meissner-Lula, N.,
Hsu, V. W.,
and Band, H.
(2000)
J. Biol. Chem.
275,
367-377 |
24. | Koepp, D. M., Harper, J. W., and Elledge, S. J. (1999) Cell 97, 431-434[Medline] [Order article via Infotrieve] |
25. | Bustelo, X. R., Crespo, P., Lopez-Barahona, M., Gutkind, J. S., and Barbacid, M. (1997) Oncogene 15, 2511-2520[CrossRef][Medline] [Order article via Infotrieve] |
26. |
De Sepulveda, P.,
Ilangumaran, S.,
and Rottapel, R.
(2000)
J. Biol. Chem.
275,
14005-14008 |
27. | Hoppe, T., Matuschewski, K., Rape, M., Schlenker, S., Ulrich, H. D., and Jentsch, S. (2000) Cell 102, 577-586[Medline] [Order article via Infotrieve] |
28. | Rickles, R. J., Botfield, M. C., Weng, Z., Taylor, J. A., Green, O. M., Brugge, J. S., and Zoller, M. J. (1994) EMBO J. 13, 5598-5604[Abstract] |
29. |
Donovan, J. A.,
Ota, Y.,
Langdon, W. Y.,
and Samelson, L. E.
(1996)
J. Biol. Chem.
271,
26369-26374 |
30. |
Harpur, A. G.,
Layton, M. J.,
Das, P.,
Bottomley, M. J.,
Panayotou, G.,
Driscoll, P. C.,
and Waterfield, M. D.
(1999)
J. Biol. Chem.
274,
12323-12332 |
31. |
Li, E.,
Stupack, D. G.,
Brown, S. L.,
Klemke, R.,
Schlaepfer, D. D.,
and Nemerow, G. R.
(2000)
J. Biol. Chem.
275,
14729-14735 |
32. |
Yudowski, G. A.,
Efendiev, R.,
Pedemonte, C. H.,
Katz, A. I.,
Berggren, P. O.,
and Bertorello, A. M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6556-6561 |
33. | Kaiser, P., Flick, K., Wittenberg, C., and Reed, S. I. (2000) Cell 102, 303-314[Medline] [Order article via Infotrieve] |