v-Rel and c-Rel are differentially affected by mutations at a consensus protein kinase recognition sequence

Area of Science:

• Molecular Biology
• Oncology
• Virology

Background:

• v-Rel and c-Rel are site-specific DNA-binding proteins within the Rel family.
• A conserved protein kinase A (PK-A) recognition sequence (Arg-Arg-Pro-Ser) is present in the Rel homology domain of most Rel proteins.

Purpose of the Study:

• To investigate the functional importance of the PK-A recognition sequence in v-Rel and c-Rel.
• To determine how mutations within this sequence affect DNA binding, protein oligomerization, and transcriptional activity.

Main Methods:

• Site-directed mutagenesis to disrupt the PK-A sequence and phosphorylatable serine.
• In vitro DNA binding assays using kappa B sites.
• Glutaraldehyde cross-linking for protein oligomerization analysis.
• Domain-swapping experiments to identify structural differences.
• Reporter gene assays in yeast to assess transcriptional activation.

Main Results:

• Disruption of the PK-A sequence abolished DNA binding for both v-Rel and c-Rel.
• Mutation of the phosphorylatable serine (Ser-275 in v-Rel, Ser-266 in c-Rel) differentially affected DNA binding: v-Rel was less affected than c-Rel.
• Mutations reducing DNA binding also impaired protein oligomerization.
• Structural differences in the central region, specifically a v-Rel Ala-278/c-Rel Glu-269 substitution, explained the differential sensitivity of c-Rel to serine mutations.
• Mutations affecting DNA binding in c-Rel correlated with reduced transcriptional activation.

Conclusions:

• The PK-A site is essential for the DNA-binding activity of v-Rel and c-Rel.
• Differential functional consequences of mutations in the PK-A site between v-Rel and c-Rel are due to structural variations.
• These findings highlight the complex regulation of Rel protein function through post-translational modifications and structural determinants.