Identification of ELK1 interacting peptide segments in the androgen receptor
Claire Soave1, Charles Ducker2, Seongho Kim1
1Department of Oncology, Wayne State University School of Medicine and Barbara Ann Karmanos Cancer Institute, Detroit, MI, U.S.A.
Abstract:
Prostate cancer (PCa) growth requires tethering of the androgen receptor (AR) to chromatin by the ETS domain transcription factor ELK1 to coactivate critical cell proliferation genes. Disruption of the ELK1-AR complex is a validated potential means of therapeutic intervention in PCa. AR associates with ELK1 by coopting its two ERK docking sites, through the amino-terminal domain (A/B domain) of AR. Using a mammalian two-hybrid assay, we have now functionally mapped amino acids within the peptide segments 358-457 and 514-557 in the A/B domain as required for association with ELK1. The mapping data were validated by GST (glutathione S-transferase)-pulldown and BRET (bioluminescence resonance energy transfer) assays. Comparison of the relative contributions of the interacting motifs/segments in ELK1 and AR to coactivation of ELK1 by AR suggested a parallel mode of binding of AR and ELK1 polypeptides. Growth of PCa cells was partially inhibited by deletion of the upstream segment in AR and nearly fully inhibited by deletion of the downstream segment. Our studies have identified two peptide segments in AR that mediate the functional association of AR with its two docking sites in ELK1. Identification of the ELK1 recognition sites in AR should enable further structural studies of the ELK1-AR interaction and rational design of small molecule drugs to disrupt this interaction.
Insights
Researchers identified two key segments in the androgen receptor (AR) that bind to the ELK1 protein, which is crucial for prostate cancer (PCa) cell growth. Disrupting this interaction may lead to new PCa therapies.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Prostate cancer (PCa) growth depends on the androgen receptor (AR) interacting with ELK1 to activate cell proliferation genes.
- Disrupting the ELK1-AR complex is a potential therapeutic strategy for PCa.
Purpose of the Study:
- To functionally map the specific amino acid segments within the AR's amino-terminal (A/B) domain responsible for binding to ELK1.
- To validate these findings and assess the impact of disrupting the interaction on PCa cell growth.
Main Methods:
- Mammalian two-hybrid assays were used to identify AR segments interacting with ELK1.
- GST-pulldown and BRET assays validated the identified interaction sites.
- Deletions in specific AR segments were created to assess their effect on PCa cell proliferation.
Main Results:
- Two distinct peptide segments (358-457 and 514-557) in the AR A/B domain were identified as crucial for ELK1 association.
- The downstream segment (514-557) deletion nearly fully inhibited PCa cell growth, while the upstream segment (358-457) deletion partially inhibited it.
- The binding mode of AR and ELK1 appears to be parallel.
Conclusions:
- Two specific peptide segments in the AR mediate its functional association with ELK1.
- These identified ELK1 recognition sites in AR can guide structural studies and the rational design of drugs to disrupt the ELK1-AR interaction for PCa treatment.
