View larger version (58K):
[in a new window]
|
Fig. 1. Sequence and constructs for Xenopus Cullin-1. (A)
Protein alignment of the mouse, human and X. tropicalis Cullin-1. The
full-length protein is 99.6% identical at the amino acid level. The Rbx-1- and
Skp1-binding domains are boxed in red and blue, respectively. (B)
Diagrammatic representation of Cullin-1 constructs. Two C-terminally
truncated versions of Cullin-1 were used. Cul1-C75 and Cul1-C477 are missing
75 and 477 amino acids from the C terminus respectively (C-terminal
truncations indicated by purple bars in A). As a consequence,
Cul1-C477 contains none and Cul1-C75 only contains a subset
of the Rbx-1-binding sites. Moreover, lysine 720, the neddylation site (green
star in A) is missing in both constructs. These constructs should lose or have
reduced binding ability to Rbx-1, one of the components of the SCF complex,
and are likely to interfere with the correct formation of the endogenous SCF.
A fourth construct (N148-Cul1-C477), missing the 148 N-terminal and the 477
C-terminal amino acids, was also created. This deletes both the
Skp-1- (yellow) and the Rbx-1- (red) binding domains and
should not interfere with the endogenous SCF complex. (C) A wild-type
SCF complex (with its substrate ß-catenin) versus a complex based on the
truncated Cullin proteins (Cul1-C75). In the wild-type scenario, Cullin
proteins provide the bridge between the F-box protein that binds
ß-catenin and the E2 conjugating enzyme that supplies ubiquitin
molecules. The truncation is likely to inhibit the interaction between the
Cul1-C75 and Rbx-1/E2, hence preventing SCF function. Polyubiquitination of
ß-catenin is shown by a series of grey circles, which are missing in the
Cul1-C75-based SCF complex.
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
-tubulin as a loading control. (B) The same was carried
out for full-length Cullin-1 RNA. Embryos were analysed at stage 8
and surprisingly an upregulation of ß-catenin levels was also observed.
(C) ß-Catenin levels were also analysed at stage 19 (neurula
stage), a time during which neural crest tissue becomes specified. As at stage
8 and 10, injections of 1.6 and 2.5 ng of either the Cul1-C75 and the
full-length Cullin-1 lead to an increase in ß-catenin levels.
This was also found for stage 24 (data not shown). To determine whether both
constructs are translated correctly, western blots were stripped and
subsequently analysed for Cullin-1 protein levels (with an antibody raised
towards Cullin-1's N-terminus). The full-length Cullin-1 RNA (lane 4
and 5) results in higher levels of proteins of the correct size when compared
with uninjected embryos (lane 1). A clear size difference between Cul1-C75 and
Cullin-1 is also visible. (D) A wide range of Cullin-1 RNA
doses were injected (10 pg-3 ng into each blastomere at the two-cell stage)
and embryos were analysed at stage 10. A decrease in ß-catenin was never
observed; indeed the increase of ß-catenin correlates with the increased
dose of Cullin-1 injection.
10% of
the lysate was removed previous to the IP and analysed for total levels of
ß-catenin by anti-HA staining. Co-transfection of Cullin-1 led
to increased ß-catenin levels in the lysate control in 4/6 experiments
(not shown here). An increase in total ß-catenin is not always detected
in these experiments because of the high levels of exogenous ß-catenin
and because the ubiquitinated form is only a fraction of the total
ß-catenin.
