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Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest
K Alevizopoulos1, B Sanchez, B Amati
1Swiss Institute for Experimental Cancer Research, Epalinges.
Abstract:
Ectopic expression of the CDK inhibitors (CKIs) p16INK4a and p27Kip1 in Rat1 fibroblasts induces dephosphorylation and activation of Retinoblastoma-family proteins (pRb, p107 and p130), their association with E2F proteins, and cell cycle arrest in G1. The growth-inhibitory action of p16, in particular, is believed to be mediated essentially via pRb activation. The 12S E1A protein of human Adenovirus 5 associates with pRb-family proteins via residues in its Conserved Regions (CR) 1 and 2, in particular through the motif LXCXE in CR2. These interactions are required for E1A to prevent G1 arrest upon co-expression of CKIs. We show here that mutating either of two conserved motifs adjacent to LXCXE in CR2, GFP and SDDEDEE, also impairs the ability of E1A to overcome G1 arrest by p16 or p27. Strikingly, however, these mutations affect neither the association of E1A with pRb, p07 and p130, nor its ability to derepress E2F-1 transcriptional activity in transient transfection assays. One of the EIA mutants, however, is defective in derepressing several endogenous E2F target genes in the presence of p16 or p27. Thus, CR2 possesses an essential function besides pRb-binding. We speculate that this function might be required for the full derepression of E2F-regulated genes in their natural chromatin context.
Insights
Cyclin-dependent kinase inhibitors (CKIs) like p16 and p27 cause cell cycle arrest by activating Retinoblastoma proteins. Adenovirus E1A protein
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Cyclin-dependent kinase inhibitors (CKIs) p16INK4a and p27Kip1 induce cell cycle arrest by activating Retinoblastoma-family proteins (pRb, p107, p130).
- The 12S E1A protein of human Adenovirus 5 interacts with pRb-family proteins via Conserved Regions (CR) 1 and 2, including the LXCXE motif in CR2.
- These E1A-pRb interactions are crucial for E1A to counteract CKI-induced G1 arrest.
Purpose of the Study:
- To investigate the role of conserved motifs adjacent to the LXCXE motif in Adenovirus E1A CR2.
- To determine if these motifs are essential for E1A's ability to overcome CKI-mediated G1 arrest.
- To elucidate functions of E1A CR2 beyond pRb-family protein binding.
Main Methods:
- Ectopic expression of CKIs (p16, p27) and wild-type/mutant Adenovirus E1A proteins in Rat1 fibroblasts.
- Analysis of pRb-family protein dephosphorylation and E2F association.
- Cell cycle progression assays (G1 arrest).
- Transient transfection assays to assess E2F-1 transcriptional activity.
- Evaluation of endogenous E2F target gene derepression.
Main Results:
- Mutations in conserved motifs (GFP, SDDEDEE) in E1A CR2 impaired its ability to overcome p16/p27-induced G1 arrest.
- These mutations did not affect E1A binding to pRb-family proteins or E2F-1 transcriptional activity.
- One E1A mutant showed defective derepression of endogenous E2F target genes in the presence of p16/p27.
- This suggests a pRb-binding independent function for E1A CR2.
Conclusions:
- Conserved motifs in E1A CR2, distinct from the pRb-binding LXCXE motif, are critical for overcoming CKI-induced G1 arrest.
- E1A CR2 has a function beyond pRb-family protein interaction, potentially involved in chromatin-context gene regulation.
- This novel function may be essential for the complete derepression of E2F-regulated genes.