|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
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.
|
