Abstract

Krüppel-like factor 5 (KLF5) is a zinc finger transcription factor that is highly expressed in the crypt epithelial cells of the intestine and plays a critical role in regulating proliferation of both normal intestinal epithelial cells and colorectal cancer cells. Stability of the KLF5 is mediated by proteasomal degradation via phosphorylation by glycogen synthase kinase 3β (GSK3β) and recognition by F-box and WD repeat domain-containing 7 (FBW7) of a phosphodegron sequence surrounding serine 303 in KLF5. A genomic analysis of colorectal cancer tissues identified a somatic mutation (P301S) in KLF5 within the phosphodegron sequence. We hypothesized that due to its close proximity to the phosphodegron sequence, the P301S mutation may affect signaling that is involved in proper KLF5 degradation. We demonstrated that the P301S KLF5 mutant has a longer half-life than wild type (WT) KLF5. Furthermore, P301S KLF5 has a higher transcriptional activity than WT KLF5 as demonstrated by luciferase assays using cyclin D1 and CDC2 promoter constructs. In contrast to WT KLF5, P301S KLF5 does not physically interact with FBW7α. Concomitantly, the P301S KLF5 mutant displays reduced levels of phosphorylation at serine 303 in comparison with WT KLF5. These results of our study indicate that amino acid residue 301 of KLF5 is critical for proper recognition of the phosphodegron sequence by FBW7α and that the P301S mutation inhibits this recognition, leading to a degradation-resistant protein with elevated levels and enhanced transcriptional activity. These findings raise a potentially oncogenic role for the P301S KLF5 mutant in colorectal cancer.

Highlights

  • Stability of Krüppel-like factor 5 (KLF5) is regulated by GSK3␤- and F-box and WD repeat domain-containing 7 (FBW7)␣-mediated proteasomal degradation

  • Ubiquitin Assay—human embryonic kidney (HEK) cells at 80 –90% confluence were transfected with wild type (WT), S303A, or P301S human KLF5 constructs in combination with FBW7␣ and ubiquitin-expressing plasmids

  • Because the identified mutation is localized close to or within the phosphodegron sequence in KLF5, we examined the impact of the P301S mutation on KLF5 protein stability

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Summary

Background

Stability of KLF5 is regulated by GSK3␤- and FBW7␣-mediated proteasomal degradation. Results: Colon cancer-derived P301S KLF5 mutant is degradation-resistant due to the inability to interact with FBW7␣. The P301S KLF5 mutant displays reduced levels of phosphorylation at serine 303 in comparison with WT KLF5 These results of our study indicate that amino acid residue 301 of KLF5 is critical for proper recognition of the phosphodegron sequence by FBW7␣ and that the P301S mutation inhibits this recognition, leading to a degradation-resistant protein with elevated levels and enhanced transcriptional activity. These findings raise a potentially oncogenic role for the P301S KLF5 mutant in colorectal cancer. Our results provide evidence that proline residue in amino acid position 301 in KLF5 is necessary for the recognition of human KLF5 phosphodegron sequence by GSK3␤ and FBW7␣

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