Expression of β-Catenin Mutant Proteins in MDCK Cells
Mutant β-catenin proteins lacking NH2
-terminal 90 (ΔN90), 131 (ΔN131), 151 (ΔN151) amino acids, or COOH-terminal 86 amino acids (ΔC), and full-length β-catenin (β-catenin*) were expressed in MDCK cells (Fig. ). The sequence for the SV-40 large T antigen epitope recognized by the mAb KT3 (MacArthur and Walter, 1984
) was added to the 3′ termini of all cDNA constructs to distinguish protein products from endogenous β-catenin. All constructs were expressed under the control of the Tet-repressible transactivator; in the presence of either Tet or Dox, expression of exogenous protein is completely repressed (Gossen and Bujard, 1992
MDCK clones cultured for 4 d without or with doxycycline (−/+ Dox) were analyzed for expression of mutant β-catenins (Fig. ). Mutant β-catenins (Fig. , a–c; marked with stars in β-cat.C, β-cat.N, and KT3 blots) were detected in protein extracts from clones cultured without Dox, but not in extracts from clones cultured with Dox. 15–20 clones were analyzed for every construct. Mutant β-catenin levels in clones expressing ΔN90, ΔN131, or ΔN151 β-catenin were on average higher than that in clones expressing β-catenin* and ΔC β-catenin. However, the level of ΔN151 in clone ΔN151-D was similar to β-catenin* in clone β-catenin*–10 (Fig. c; KT3 blot). In the clones shown in Fig. , the ratios of ΔN90, ΔN131, ΔN151, or ΔC β-catenin to MDCK endogenous β-catenin were 5:1, 1:1, 0.5:1, and 1:1, respectively (Fig. a, lanes 7, 9, and 11; Fig. b, lane 5).
Figure 2 Dox-repressible expression of β-catenin mutant proteins in MDCK cells. MDCK clones were cultured for 4 d without or with Dox (−/+ Dox) and extracted with 1% SDS. 15-μg protein lysates were subjected to SDS-PAGE and immunoblotted (more ...)
Expression of E-cadherin leads to an increase in the cellular levels of catenins (Herrenknecht et al., 1991
; Weißig, 1993
). Therefore, we analyzed whether expression of β-catenin mutant proteins affected levels of endogenous β-catenin, E-cadherin, or α-catenin by comparing their amounts in lysates from clones cultured without or with Dox. Expression of ΔN90, ΔN131, or ΔN151 did not significantly affect the level of endogenous β-catenin (Fig. , a
, compare lanes 7
, and 11
, respectively). Expression of ΔC β-catenin resulted in a small but reproducible decrease in amount of endogenous β-catenin (Fig. , a
, lanes 5
). Although full-length exogenous β-catenin* and endogenous β-catenin were not always well separated by SDS-PAGE because of similarity in their electrophoretic mobilities, a decrease of endogenous β-catenin in response to the expression of β-catenin* was detectable in some of these clones (Fig. a
, lanes 3
The amount of E-cadherin was slightly higher in clones expressing ΔN90 β-catenin (ΔN90-A) and ΔN131 β-catenin (ΔN131-D) compared with levels in β-catenin–10* and ΔC clones (Fig. d). Differences in E-cadherin levels may be the result of clonal variation since the amount of E-cadherin in a particular clone was not significantly affected after repression of mutant β-catenin expression by addition of Dox (Fig. d; compare lanes 7 to 8, 9 to 10, and 11 to 12, respectively). Expression of mutant β-catenins did not have a significant effect on amounts of α-catenin in all clones except ΔN131-D in which the α-catenin level was slightly decreased when cells were cultured without Dox (Fig. e, lanes 9 and 10).
Mutant β-Catenin Proteins Compete with Endogenous β-Catenin for Binding to E-Cadherin and α-Catenin
Binding of β-catenin mutants to E-cadherin and α-catenin was analyzed by coimmunoprecipitation of protein complexes (Fig. ). The binding site for E-cadherin in β-catenin is located within the armadillo repeat domain of β-catenin (Aberle et al., 1994
; Hülsken et al., 1994
) and was retained in all mutant proteins. Accordingly, all β-catenin mutant proteins were coimmunoprecipitated by E-cadherin antibody (Fig. a
blot). Binding of ΔN90 and ΔN131 β-catenin to E-cadherin resulted in a reduction in amount of endogenous β-catenin complexed with E-cadherin compared with the amount of endogenous β-catenin in similar complexes isolated from the same clones cultured with Dox (Fig. b
blot; compare lanes 5
, and 7
, respectively). The binding site for α-catenin is located between amino acids 120 and 151 in β-catenin (Aberle et al., 1994
, 1996) and was incomplete or missing in ΔN131 and ΔN151 β-catenin. Accordingly, ΔN131 and ΔN151 β-catenin were not coimmunoprecipitated by α-catenin antibody (Fig. c
blot). Binding of ΔN90 and ΔC β-catenin to α-catenin resulted in a reduction in amount of endogenous β-catenin bound to α-catenin compared with endogenous β-catenin in complexes with α-catenin that were isolated from the same clones cultured in the presence of Dox (Fig. d
blot; compare lanes 5
, and 11
, respectively). These results confirm that ΔN90 and ΔC β-catenin retained both E-cadherin and α-catenin binding sites, and that ΔN131 and ΔN151 β-catenin retained the E-cadherin, but not α-catenin, binding site; all mutant β-catenin proteins competed with endogenous β-catenin for these binding partners.
Figure 3 β-catenin mutant proteins compete with endogenous β-catenin for binding to E-cadherin and α-catenin. MDCK clones were cultured 4 d without or with Dox (−/+) and extracted with 1% Triton X-100 lysis buffer. Protein (more ...)
NH2-terminal–deleted Mutant Proteins of β-Catenin Are Enriched in APC Protein Complexes
Binding of mutant β-catenins to APC protein was also analyzed by coimmunoprecipitation. All mutant β-catenins were coimmunoprecipitated by APC protein antibody (Fig. ). However, ΔN90, ΔN131, and ΔN151 β-catenin were significantly enriched in APC protein immunoprecipitates compared with endogenous β-catenin, ΔC, or exogenous β-catenin*. Three times more ΔN90, ΔN131, or ΔN151 was coimmunoprecipitated with APC protein than endogenous β-catenin (Fig. c). Note that total amounts of mutant β-catenin in lysates used for immunoprecipitation were either similar to, or less than that of, endogenous β-catenin (Fig. a); the ratios of ΔN90, ΔN131, or ΔN151 β-catenin to endogenous β-catenin in Triton X-100 lysates were 1.6:1, 1.3:1, and 0.1:1, respectively. Very little ΔC β-catenin was detected in APC protein complexes, although as much ΔC as ΔN151 β-catenin was present in the Triton X-100 lysates of each clone (compare Fig. b and 4 d; KT3 blots).
Figure 4 ΔN90, ΔN131, and ΔN151 are enriched in APC protein complexes. Aliquots of the same Triton X-100 lysates as described in Fig. were either subjected to SDS-PAGE or used for immunoprecipitation with APC antiserum (more ...)
These data indicate that, compared with endogenous β-catenin, NH2-terminal–deleted β-catenins bind preferentially to APC protein. In addition, the amount of ΔN151 β-catenin that is complexed with APC protein is very similar to that of ΔN90 and ΔN131 β-catenin, even though there is much less ΔN151 β-catenin in clone ΔN151-D. Therefore, we suspect that binding of NH2-terminal–deleted β-catenin to APC protein approaches a maximum.
To further explore the degree of enrichment of mutant β-catenin in APC protein complexes, amounts of β-catenin* and ΔN131 β-catenin bound to APC protein were compared in two different clones (β-cat.*-7 and ΔN131-7; Fig. e). Portions of Triton X-100 lysates of these clones were used to immunoprecipitate both full-length and deleted β-catenin with β-cat.C antiserum, or to coimmunoprecipitate APC protein complexes. In β-cat.C immunoprecipitates, the ratios of endogenous β-catenin to β-catenin* in the β-catenin*-7 clone, and those of endogenous β-catenin to ΔN131 in the ΔN131-7 clone, were ~1:1. In APC protein immunoprecipitates, the ratio of endogenous β-catenin to β-catenin* was also ~1:1, but, in contrast, the ratio of endogenous β-catenin to ΔN131 β-catenin was ~1:3. ΔN131 β-catenin was considerably more abundant in APC protein immunoprecipitates than either endogenous β-catenin or exogenous full-length β-catenin* (Fig. e).
NH2-terminal Deletions Result in Increased β-Catenin Stability in APC Protein and E-Cadherin Complexes
Enrichment of ΔN90, ΔN131,and ΔN151 β-catenins bound to APC protein could be caused by increased stability of these β-catenins in the complex. To test this hypothesis, expression of β-catenin mutant proteins was repressed by addition of Dox to cultures for 0, 6, 12,or 18 h. Amounts of mutant β-catenin remaining in Triton X-100 lysates of these cells, and in E-cadherin and APC protein complexes isolated from those lysates, were analyzed by immunoprecipitation and/or Western blotting (Fig. ).
Figure 5 Increased stability of ΔN90, ΔN131, and ΔN151 in the E-cadherin– and APC protein–bound pools. MDCK clones were cultured 0, 6, 12, or 18 h with Dox and extracted with 1% Triton X-100 lysis buffer. Protein lysates (more ...)
A significant increase in the relative stabilities of NH2terminal–deleted β-catenins compared with full-length β-catenin* or ΔC β-catenin was detected in E-cadherin and APC protein complexes from lysates. This difference in stability was most pronounced in the APC protein– bound pools. Very little or no full-length β-catenin* or ΔC β-catenin were detected in the APC protein complex after only 6 h of treatment with Dox. In contrast, there was little or no decrease in amounts of ΔN90, ΔN131, or ΔN151 β-catenin in the APC protein complex after 18 h of treatment with Dox. In the E-cadherin–bound pools of lysates, 10– 12% of the original amount of full-length β-catenin* and ΔC β-catenin was detected after 12 h of treatment with Dox. In contrast, amounts of ΔN90, ΔN131, or ΔN151 β-catenin bound to E-cadherin were not or little reduced after 18 h of treatment with Dox.
In the total Triton X-100 lysates, the amounts of all mutant β-catenin proteins were reduced after 12 to 18 h of treatment with Dox. The percentage of remaining mutant protein after 12 h of treatment with Dox is 17% and 18% of β-catenin* in clones β-catenin*–7 and β-catenin*–10, respectively; 18% of ΔC β-catenin; 44% of ΔN90 β-catenin; 32% of ΔN131 β-catenin; and 26% of ΔN151 β-catenin. The close similarity in the decrease in amounts of β-catenin* in two independent clones indicates that the rate and efficiency of Dox repression of gene expression were similar in different MDCK clones. Assuming that this is also the case in other clones, the half-life of ΔC is similar to that of full-length β-catenin*, whereas ΔN90, ΔN131, and ΔN151 β-catenin are slightly more stable than full-length β-catenin*. The mutant β-catenin proteins in the total TX100 lysates represent the sum of the E-cadherin– and APC-bound, and unbound pools. Although ΔN90, ΔN131, or ΔN151 β-catenin was very stable in E-cadherin and APC protein complexes, their overall stability in the lysates was not very different from that of full-length β-catenin* and ΔC β-catenin. This indicates that pools of ΔN90, ΔN131, and ΔN151 β-catenin that are not bound to either E-cadherin or APC protein turn over at a rate similar to that of full-length β-catenin* and ΔC β-catenin.
The high stability of the NH2
-terminal–deleted β-catenins in the APC protein complexes correlated with the enrichment of these mutant proteins in the APC protein complexes compared with endogenous β-catenin in the same cells (Fig. ). Surprisingly, we could not detect an enrichment of NH2
-terminal–deleted β-catenins in the E-cadherin pool when compared with endogenous β-catenin in the same cells (see Fig. c
), even though NH2
-terminal–deleted β-catenins were more stable than full-length exogenous β-catenin* in the E-cadherin complexes of different clones (Fig. ). We note that most of the E-cadherin in MDCK cells is normally bound to endogenous β-catenin in a 1:1 molar ratio (Hinck et al., 1994a
). In contrast, most of the APC protein appears to be free of endogenous β-catenin (Näthke et al., 1996
). Therefore, NH2
-terminal– deleted β-catenins must compete with endogenous β-catenin to bind to E-cadherin, but they may be enriched in the APC protein pool by binding unoccupied sites and remaining stabilized in these complexes. This may explain the difference in the ratios of the mutant proteins to endogenous β-catenin when comparing the E-cadherin– bound or APC-bound pools in the same cultures (Figs. and ).
NH2-terminal–deleted β-Catenin Prominently Localizes in Clusters with APC Protein at Tips of Plasma Membrane Protrusions
Our immunoprecipitation studies show that NH2-terminal–deleted β-catenin proteins form stable complexes with E-cadherin and APC protein. We examined the subcellular distribution of mutant β-catenins by immunofluorescence microscopy. Mutant β-catenins were epitope tagged and can be distinguished from endogenous β-catenin with the monoclonal anti-tag antibody KT3. We examined two types of cell cultures: low density cultures in which small groups of cells had initiated cell–cell contacts (Fig. ), and high density cultures in which mature cell–cell contacts had been established (Fig. ).
Figure 6 ΔN151, ΔN131, and ΔN90 localize to clusters near the plasma membrane in extending membranes of MDCK cells. MDCK clones were double stained with mAb KT3 against the epitope tag in β-catenin mutant proteins (a–e (more ...)
Figure 7 ΔN90, ΔN131, and ΔN151 colocalize with APC protein in MDCK cells. MDCK clones were double stained with mAb KT3 against the epitope tag in β-catenin mutant proteins and antiserum against APC protein. Full-length β-catenin* (more ...)
ΔN90, ΔN131, and ΔN151 β-catenins prominently localized to clusters at the outer boundary of cell–cell contacts and at the tips of plasma membrane protrusions (Fig. , a–d
). Neither E-cadherin, endogenous β-catenin, nor α-catenin colocalize with the NH2
-terminal– deleted β-catenins in theses clusters (Fig. , a′–d′
). This distinctive subcellular distribution is very similar to that of APC protein in MDCK cells (Näthke et al., 1996
). Therefore, we examined colocalization of the mutant β-catenin proteins with APC protein in these clusters (Fig. ). ΔN90, ΔN131, and ΔN151 β-catenins colocalized precisely with APC protein in almost all clusters at the tips of plasma membrane protrusions; incidences of APC protein clusters without ΔN90, ΔN131, or ΔN151 β-catenins were very rare (Fig. , middle panels
). However, fulllength β-catenin* and ΔC β-catenin localized to intercellular contacts, and there was little overlap in distributions between these proteins and APC protein (Fig. , top and bottom panel
In low density cultures, ΔN90, ΔN131, and ΔN151 β-catenins were poorly localized at intercellular contacts, compared with strong staining for E-cadherin and α-catenin at the same sites (Fig. ). In these cell–cell contact areas, NH2-terminal–deleted β-catenin mutant proteins colocalized with E-cadherin, endogenous β-catenin, and α-catenin (Fig. , a–d, and a′–d′, arrows). Localization of ΔN90, ΔN151, and ΔN131 β-catenins to intercellular contacts was much more prominent in high density cultures (Fig. ; KT3 immunofluorescence). The distribution of β-catenin* and ΔC was identical to that of endogenous β-catenin (data not shown).
Note that some nuclear staining was detected with the KT3 antibody (see Fig. , a–d), but that the staining persisted in the absence of mutant protein expression (see Fig. e), indicating that nuclear staining was not specific to mutant β-catenin in these cells.
Distinct Differences in Morphologies of Cells Expressing NH2-terminal–deleted β-Catenin Compared With Cells Expressing β-Catenin* and ΔC β-Catenin
We examined the morphology and colony formation of cells expressing different mutant β-catenins. In low density cultures, the morphology of cells expressing ΔN90, ΔN131, or ΔN151 β-catenins was distinctly different from that of the same cells treated with Dox (i.e., without mutant β-catenin expression; Fig. ), parental MDCK cells, and cells expressing β-catenin* or ΔC β-catenin (data not shown). Cells treated with Dox, parental MDCK cells, and cells expressing β-catenin* or ΔC β-catenin produced compact cell colonies; note that cells at the edges of colonies rarely extended membrane protrusions onto the surrounding cell-free surface. In addition, we found very few individual cells that were not associated with colonies in these cultures. Neither addition of Dox to the cultures nor addition of the KT3 tag to full-length β-catenin affected the morphology of either parental MDCK cells (Fig. , top panel) or β-catenin*–expressing cells (data not shown), respectively. In contrast, the surface area of cells expressing ΔN90, ΔN131, or ΔN151 β-catenins (i.e., in the absence of Dox) was greater than that of control cells, indicating that the former were poorly compacted. Cells loosely associated in colonies also had many membrane extensions projecting onto the surrounding cell-free surface. In addition, many individual cells were not associated directly with colonies and were dispersed throughout the culture; in general, these cells have a more fibroblastic morphology than control cells (Fig. ). Repression of ΔN90, ΔN131, or ΔN151 β-catenin expression by treatment with Dox reverted the morphology of cells to compacted colonies typical of parental MDCK cells (Fig. , top panels; see also Fig. , e and e′).
Figure 8 Effect of ΔN90, ΔN131, and ΔN151 expression on colony formation in low density MDCK cultures. Cultures of MDCK clones were untreated or pretreated 3 d with Dox and plated at low density without (−) or with (+) (more ...)
At higher cell densities in which there was less free surface available for cell migration, differences in morphologies of cells expressing ΔN90, ΔN131, or ΔN151 β-catenins and control cells were less evident. Cells expressing ΔN90, ΔN131, or ΔN151 β-catenins established compact colonies and, eventually, formed complete monolayers similar to those of control cells (see Fig. ).