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R.G. Morgan et al.
the observed translocation of β-catenin was sufficient to generate a transcriptional response. A caveat to these data is that these observations cannot be linked exclusively to the modulation of β-catenin nuclear-localization since LEF-1 expression is a co-variable in these experiments. Taken together, these data indicate LEF-1 can regulate the nuclear level of β-catenin and our observations are also consistent with a concomitant regulation of Wnt signaling activity.
LEF-1 protein is proteolytically cleaved in Wnt-unre- sponsive cells
In the experiments above, overexpression of full-length LEF-1 in Wnt-unresponsive cell lines resulted in the emer- gence of a 25-30kDa species of LEF-1 protein. To evaluate the contribution of this short LEF-1 form with the increased nuclear β-catenin level observed in these cell lines we examined the β-catenin binding capacity of this species. We performed β-catenin co-IP from both the cytosol and nucleus of CHIR99021 treated LEF1-over- expressing U937 cells and immunoblotted for LEF-1 pro- tein. Both the full-length and short-forms of LEF-1 protein co-immunoprecipitated with β-catenin from the cytosol implying both forms bind β-catenin in this cell line. In the nuclear fraction, β-catenin preferentially co-immunopre- cipitated with the short LEF-1 form, though the propor- tion of short-form was highly enriched in this fraction (Figure 8A; input lane). These data confirm that the short-
form LEF-1 has β-catenin binding ability and could mediate the increased nuclear β-catenin translocation observed in Wnt-unresponsive cell lines above upon LEF-1 overexpression with CHIR99021 treatment.
Finally, we examined the origin of the short LEF-1 polypeptide. This variant was unlikely to have derived from alternative splicing since the ectopic expression of LEF-1 was driven from cDNA. Furthermore, these short forms of LEF-1 were also observed endogenously in col- orectal cell lines (Online Supplementary Figure S9A) and in primary AML samples featured in Figure 5 (full blots in Online Supplementary Figure S9B). Therefore, the presence of short-form LEF-1 was most consistent with a proteolyt- ic cleavage mechanism. To investigate this, nuclear lysates from both K562 control and U937-LEF1 cells ± protease inhibitor cocktail (PIC) were incubated at 37°C and the relative proportions of full-length to short LEF-1 forms in each cell line observed over 0-60 min. In K562 control cells, full-length LEF-1 protein was stable with no detectable breakdown of full-length LEF-1 protein even in the absence of PIC (Figure 8B). In contrast, the full-length LEF-1 band present in U937-LEF-1 cells was reduced with concomitant enrichment of the short-form polypeptide after a 10 min incubation at 37°C (Figure 8C). The removal of the PIC reduced the half-life of full-length LEF-1 by approximately 50% with degradation occurring within 5 min. Together these data are consistent with the short- form of LEF-1 arising through proteolytic processing.
A
B
C
Figure 5. Correlation between nuclear LEF-1 and nuclear β-catenin in leukemia cell lines and primary acute myeloid leukemia (AML) samples. (A) Representative immunoblot screen of myeloid leukemia cell lines showing the relative cytoplasmic (C) and nuclear (N) abundance of candidate proteins influencing β-catenin nuclear localization detected by mass spectrometry. SW620 colorectal cells were used as a positive control. TCF-4 arrows indicate 58/79kDa transcriptional isoforms. (B) Representative immunoblots showing the cytosolic and nuclear expression of both LEF-1 and total β-catenin in primary AML patient samples. LEF-1 protein isoforms migrate as bands between 40-55kDa. Lamin A/C and GAPDH or α-tubulin were used to assess fractionation efficiency and equal protein loading. (C) Summary scatter plot showing correlation between relative percentage nuclear β-catenin localization and relative percentage nuclear LEF-1 localization in primary AML patient blasts.
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haematologica | 2019; 104(7)