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MEKs/ERKs-mediated FBXO1/E2Fs interaction interference modulates G1/S cell cycle transition and cancer cell
Ga-Eun Lee1, Dohyun Jeung1, Weidong Chen1
1Basic Research Laboratory, BK21-4 th Research Team, College of Pharmacy, The Catholic University of Korea, 43, Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do, 14662, Republic of Korea.
Abstract:
E2F 1, 2, and 3a, (refer to as E2Fs) are a subfamily of E2F transcription factor family that play essential roles in cell-cycle progression, DNA replication, DNA repair, apoptosis, and differentiation. Although the transcriptional regulation of E2Fs has focused on pocket protein retinoblastoma protein complex, recent studies indicate that post-translational modification and stability regulation of E2Fs play key roles in diverse cellular processes. In this study, we found that FBXO1, a component of S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) complex, is an E2Fs binding partner. Furthermore, FBXO1 to E2Fs binding induced K48 ubiquitination and subsequent proteasomal degradation of E2Fs. Binding domain analysis indicated that the Arg (R)/Ile (I) and R/Val (V) motifs, which are located in the dimerization domain of E2Fs, of E2F 1 and 3a and E2F2, respectively, acted as degron motifs (DMs) for FBXO1. Notably, RI/AA or RV/AA mutation in the DMs reduced FBXO1-mediated ubiquitination and prolonged the half-lives of E2Fs. Importantly, the stabilities of E2Fs were affected by phosphorylation of threonine residues located near RI and RV residues of DMs. Phosphorylation prediction database analysis and specific inhibitor analysis revealed that MEK/ERK signaling molecules play key roles in FBXO1/E2Fs' interaction and modulate E2F protein turnover. Moreover, both elevated E2Fs protein levels by knockdown of FBXO1 and decreased E2Fs protein levels by sh-E2F3a delayed G1/S cell cycle transition, resulting in inhibition of cancer cell proliferation. These results demonstrated that FBXO1-E2Fs axis-mediated precise E2Fs stability regulation plays a key role in cell proliferation via G1/S cell cycle transition.
Insights
FBXO1 targets E2F transcription factors for degradation, controlling cell cycle and proliferation. Regulating E2F stability via FBXO1 is crucial for cancer cell growth.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- E2F transcription factors (E2Fs) are crucial regulators of cell cycle progression, DNA replication, and apoptosis.
- While transcriptional regulation of E2Fs is well-studied, their post-translational modification and stability control are increasingly recognized as vital for cellular processes.
- The role of E2F stability in cancer cell proliferation requires further elucidation.
Purpose of the Study:
- To investigate the role of FBXO1, a component of the SCF complex, in regulating E2F stability.
- To identify the mechanisms by which FBXO1 interacts with and degrades E2Fs.
- To determine the impact of FBXO1-mediated E2F regulation on cancer cell proliferation.
Main Methods:
- Co-immunoprecipitation to identify FBXO1 as an E2F binding partner.
- Ubiquitination assays to assess K48-linked ubiquitination of E2Fs mediated by FBXO1.
- Site-directed mutagenesis to analyze the function of degron motifs (DMs) in E2Fs.
- Western blotting and proteasomal degradation assays to measure E2F protein half-lives.
- MEK/ERK signaling pathway analysis using inhibitors and prediction databases.
- Gene knockdown (FBXO1) and knockdown (E2F3a) experiments to assess cell cycle progression and proliferation.
Main Results:
- FBXO1 directly binds to E2Fs, inducing K48 ubiquitination and proteasomal degradation.
- Specific Arg/Ile and Arg/Val motifs in E2Fs function as degron motifs for FBXO1.
- Mutating these degron motifs reduces ubiquitination and increases E2F half-lives.
- Phosphorylation of threonine residues near degron motifs modulates E2F stability.
- MEK/ERK signaling pathway influences FBXO1-E2F interaction and E2F protein turnover.
- Modulating FBXO1 or E2F3a levels impacts G1/S cell cycle transition and inhibits cancer cell proliferation.
Conclusions:
- FBXO1 acts as a key regulator of E2F stability through targeted ubiquitination and degradation.
- The FBXO1-E2F axis precisely controls E2F protein levels, impacting G1/S cell cycle transition.
- Targeting the FBXO1-E2F interaction offers a potential therapeutic strategy for inhibiting cancer cell proliferation.
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