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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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