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Figure 1.
Figure 1 The  -catenin-binding
sites of APC. (A) Schematic of the APC primary structure. The
conserved axin binding (SAMP1-3), oligomerization (olig.),
armadillo repeat (arm.), basic and discs large interaction (dlg)
regions are indicated. The 15 amino acid -catenin-binding
repeats are labeled A, B and C (white boxes). The 20 amino acid
-catenin-binding
repeats are labeled 1 -7 (black boxes). Truncations in the
midpoint cluster region (MCR), which eliminate all of the
axin-binding and most of the -catenin-binding
repeats, account for >60% of oncogenic mutations in APC (Miyoshi
et al., 1992). The APC constructs used in binding experiments
and crystallization are shown, with the beginning and end
residue numbers in human APC indicated. (B) Alignment of the APC
15 and 20 amino acid repeats with E-cadherin and XTcf3. The
alignment of the 15mers with E-cadherin and XTcf3 was performed
based on the homologous regions of the E-cadherin - -catenin,
XTcf3 - -catenin
and APC-rA - -catenin
structures (boxed). The 20mers were aligned with the 15mers
based on alignment of the core homology regions. For an
alternative alignment using the SLSSL sequences of E-cadherin
and the 20mers, see Figure 4A, bottom panel. The residues that
constitute the 15 and 20 amino acid repeat sequences are in
bold. The homologous residues of the 15 and 20mer 'core homology
region' are shaded gray; those conserved only in the 15mers are
blue. The phosphorylation-specific binding motif of E-cadherin
and the homologous APC 20mer sequences are highlighted in
yellow. Beginning residue numbers based on the full-length
proteins are indicated before the alignment. Residues from
APC-rA that form contacts with -catenin
are indicated by asterisks (contacts by side chain only or main
chain and side chain atoms) or plus signs (contacts by mainchain
atoms only) above the alignment. hAPC-A, hAPC-B, hAPC-C: human
APC 15mer repeats A, B and C. hAPC-D: hypothesized fourth human
15mer. dAPC-A, dAPC-B: Drosophila APC 15mers. eAPC-A, eAPC-B:
Drosophila APC2 15mers. hAPC-1, hAPC-2, etc.: human APC 20mers.
(C) Competition experiments to test the relative affinities of
several -catenin-binding
peptides. GST-pulldown assays were performed using GST - -catenin
(full length) in the presence of a 5-fold excess of APC-fA.
Increasing quantities of the APC-rA, APC-rAL, Tcf-ext or Cad-ext
peptides were tested for their ability to compete with APC-fA
for binding to limiting -catenin.
Fold molar excess of peptide (as compared with APC-fA) is
plotted on the x-axis, as a pseudo log base-4 plot. APC-fA band
intensities were quantified using the NIH Image program and are
shown on the y-axis as percent of binding relative to that with
no peptide competitor. Each point is plotted as mean  SD
of three experiments, except for the 256-fold excess of APC-rA,
for which only two data points were obtained. APC-fA did not
bind to GST alone (data not shown). See Materials and methods
for details.
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