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PDBsum entry 2ooa
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References listed in PDB file
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Key reference
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Title
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Structural basis for ubiquitin-Mediated dimerization and activation of the ubiquitin protein ligase cbl-B.
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Authors
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P.Peschard,
G.Kozlov,
T.Lin,
I.A.Mirza,
A.M.Berghuis,
S.Lipkowitz,
M.Park,
K.Gehring.
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Ref.
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Mol Cell, 2007,
27,
474-485.
[DOI no: ]
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PubMed id
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Abstract
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Cbl proteins are E3 ubiquitin ligases that are negative regulators of many
receptor tyrosine kinases. Cbl-b and c-Cbl contain a ubiquitin-associated (UBA)
domain, which is present in a variety of proteins involved in ubiquitin-mediated
processes. Despite high sequence identity, Cbl UBA domains display remarkably
different ubiquitin-binding properties. Here, we report the crystal structure of
the UBA domain of Cbl-b in complex with ubiquitin at 1.9 A resolution. The
structure reveals an atypical mechanism of ubiquitin recognition by the first
helix of the UBA. Helices 2 and 3 of the UBA domain form a second binding
surface, which mediates UBA dimerization in the crystal and in solution.
Site-directed mutagenesis demonstrates that Cbl-b dimerization is regulated by
ubiquitin binding and required for tyrosine phosphorylation of Cbl-b and
ubiquitination of Cbl-b substrates. These studies demonstrate a role for
ubiquitin in regulating biological activity by promoting protein dimerization.
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Figure 2.
Figure 2. Ubiquitin Binding to the UBA Domain of Cbl-b Is
Required for Cbl-b Biological Activity
(A and B) Amino acid
residues Ala937 (A937), Met940 (M940), and Phe946 (F946) in the
UBA domain of Cbl-b are required for binding to ubiquitin. (A)
In vitro binding of GST-UBA[b] to ubiquitin agarose. Wild-type
and mutant proteins bound to ubiquitin (Ub-agarose) or in the
input (Input lysate, 5%) were resolved by SDS-PAGE and detected
by immunoblotting with an antibody against GST. (B) In vitro
binding to ubiquitin chains. Wild-type or mutant GST-UBA[b] was
immobilized on glutathione Sepharose beads and incubated with
either Lys48 (K48)- or Lys63 (K63)-linked ubiquitin chains.
Following elution, bound ubiquitin chains were detected with an
anti-ubiquitin antibody (IB Ub). As a control, GST-UBA[b] from
the beads was visualized by staining (Coomassie).
(C and D)
Deletion or mutation of UBA[b] decreases the phosphorylation of
Cbl-b following Met receptor activation. HeLa cells were
transfected with wild-type HA-Cbl-b or mutants unable to bind
ubiquitin (A397E, M940A, F946A, and ΔUBA). Following
stimulation with HGF for the indicated time, HA-Cbl-b proteins
were immunoprecipitated and detected with an
anti-phosphotyrosine antibody (IB pTyr) or an anti-HA antibody
(IB HA). The graphs (right) represent the quantification of
immunoblots from four (C) or two (D) independent experiments.
The degree of tyrosine phosphorylation of Cbl-b was calculated
as the ratio of anti-pTyr over anti-HA immunostaining,
normalized for the wild-type at 10 min, and averaged with
standard deviations shown.
(E) Deletion or mutation of the
Cbl-b UBA domain decreases Met receptor ubiquitination. HEK293
cells were transfected with empty vector, wild-type HA-Cbl-b,
two mutants unable to bind ubiquitin (ΔUBA and A937E), or a
mutant with no ubiquitin-ligase activity (C373A). After 24 hr,
cellular proteins were immunoprecipitated with an anti-Met
antibody (IP Met) and probed with antibodies against ubiquitin
(IB ubiquitin) or Met receptor (IB Met). The total amount of
transfected protein was determined in a whole-cell lysate (WCL)
with an anti-HA antibody (IB HA Cbl-b). The graph (right)
presents the degree of ubiquitination plotted as the normalized
ratio of anti-Ub over anti-Met immunostaining from two
independent experiments.
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Figure 5.
Figure 5. Ubiquitin Chains Promote the Dimerization of Cbl-b
(A) Model of tetraubiquitin bound to the UBA[b] dimer. The
structure of the UBA dimer (yellow) with two ubiquitin molecules
bound (purple) from the X-ray crystal structure is shown with
two bridging ubiquitin molecules (pink) added to show how the
UBA dimer could bind Lys48-linked ubiquitin chains. The
C-terminal Leu-Arg-Gly-Gly ubiquitin tails are disordered in
solution and depicted by single-letter amino acid codes.
(B) Ubiquitin chains promote the dimerization of the Cbl-b.
HEK293 cells were cotransfected with Flag-tagged Cbl-b wild-type
and HA-tagged Cbl-b wild-type or a mutant that does not bind
ubiquitin (A937E). Flag-tagged Cbl-b was immunoprecipitated and
resolved by SDS-PAGE. The presence of HA-Cbl-b proteins in the
immunoprecipitate was detected by western blotting. Where
indicated, hexaubiquitin chains were added to the lysates 30 min
prior to the immunoprecipitations. At the end of the
immunoprecipitations, aliquots of the lysates were resolved by
SDS-PAGE and immunoblotted with anti-HA and anti-ubiquitin
antibodies to detect the presence of the HA-Cbl-b proteins and
ubiquitin chains, respectively.
(C and D) Isothermal
calorimetric titrations of the UBA domain from Cbl-b with
ubiquitin (C) and Lys48-linked tetraubiquitin (D). Each panel
shows the thermogram (top) and data analysis after integration
(bottom). The experimental data (rectangles) were fit (thin
line) to a model of a single binding site, and the values of
affinity, stoichiometry, ΔH, and error estimates in the fitting
were reported.
(E and F) Dimerization of UBA[b] is required
for its association with ubiquitin chains, but not with
monoubiquitin. (E) In vitro binding to ubiquitin agarose.
Wild-type and mutant GST-UBA[b] proteins bound to ubiquitin
(Ub-agarose) or in the input (Input lysate, 5%) were detected by
immunoblotting with an antibody against GST. (F) In vitro
binding to ubiquitin chains. Wild-type or mutant GST-UBA
proteins were immobilized on glutathione Sepharose beads and
incubated with Lys48 (K48)-linked ubiquitin chains. The bound
ubiquitin chains were detected with an anti-ubiquitin antibody
(IB Ub) and GST-UBA with anti-GST antibody (IB GST).
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The above figures are
reprinted
by permission from Cell Press:
Mol Cell
(2007,
27,
474-485)
copyright 2007.
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