Abstract
Sal-like 4 (SALL4) is a transcription factor that enhances proliferation and migration in breast cancer cells. SALL4 expression therefore has the potential to promote cancer malignancy. However, the regulatory mechanisms involved in SALL4 protein expression have not been thoroughly elucidated. In this study, we observed that treating MCF-7 and SUM159 breast cancer cell lines with a proteasome inhibitor increases SALL4 protein levels, suggesting that SALL4 is degraded by the ubiquitin-proteasome system. Using immunoprecipitation to uncover SALL4-binding proteins, we identified an E3 ubiquitin-protein ligase, tripartite motif-containing 21 (TRIM21). Using an EGFP reporter probe of the major SALL4 isoform SALL4B, we observed that shRNA-mediated knockdown of TRIM21 increases cellular SALL4B levels. Immunostaining experiments revealed that TRIM21 localizes to the nucleus, and a K64R substitution in the nuclear localization motif in SALL4B increased SALL4B levels in the cytoplasm. These results suggested that TRIM21 is involved in nuclear SALL4 degradation. To identify the amino acid residue that is targeted by TRIM21, we fragmented the SALL4B sequence, fused it to EGFP, and identified Lys-190 in SALL4B as TRIM21's target residue. Amino acid sequence alignments of SALL family members indicated that the region around SALL4 Lys-190 is conserved in both SALL1 and SALL3. Because SALL1 and SALL4 have similar functions, we constructed a SALL1-EGFP probe and found that the TRIM21 knockdown increases SALL1 levels, indicating that TRIM21 degrades both SALL1 and SALL4. Our findings extend our understanding of SALL4 and SALL1 regulation and may contribute to the development of SALL4-targeting therapies.
Highlights
Sal-like 4 (SALL4) is a transcription factor that enhances proliferation and migration in breast cancer cells
tripartite motif– containing 21 (TRIM21) is involved in SALL4 degradation In breast cancer cell lines, a low SALL4 protein level suggests the possibility that SALL4 is regulated post-translationally (Fig. 1A), the protein level is regulated by several mechanisms
To identify the proteins binding to SALL4, we performed immunoprecipitation with anti-SALL4 antibody in SUM159 and MDA-MB231 cells followed by MS
Summary
Sal-like 4 (SALL4) is a transcription factor that enhances proliferation and migration in breast cancer cells. We observed that treating MCF-7 and SUM159 breast cancer cell lines with a proteasome inhibitor increases SALL4 protein levels, suggesting that SALL4 is degraded by the ubiquitin– proteasome system. Immunostaining experiments revealed that TRIM21 localizes to the nucleus, and a K64R substitution in the nuclear localization motif in SALL4B increased SALL4B levels in the cytoplasm. These results suggested that TRIM21 is involved in nuclear SALL4 degradation. Signal transducer and activator of transcription 3 up-regulates SALL4 expression by binding to and activating the SALL4 promoter in breast cancer cells [9]. The regulatory mechanism for SALL4 expression has not been fully understood, especially in post-translational regulation
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