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A protein kinase-A recognition sequence is structurally linked to transformation by p59v-rel and cytoplasmic
G Mosialos1, P Hamer, A J Capobianco
1Department of Chemistry, Boston University, Massachusetts 02215.
Abstract:
The Rel family of proteins includes a number of proteins involved in transcriptional control, such as the retroviral oncoprotein v-Rel, c-Rel, the Drosophila melanogaster developmental protein Dorsal, and subunits of the transcription factor NF-kappa B. These proteins are related through a highly conserved domain of approximately 300 amino acids, called the Rel homology domain, that contains dimerization, DNA binding, and nuclear targeting functions. Also within the Rel homology domain, there is a conserved consensus sequence (Arg-Arg-Pro-Ser) for phosphorylation by cyclic AMP-dependent protein kinase (PKA). We used linker insertion mutagenesis and site-directed mutagenesis to determine the importance of this sequence for the transformation of avian spleen cells by v-Rel and the subcellular localization of c-Rel in chicken embryo fibroblasts (CEF). The insertion of 2 amino acids (Pro-Trp) within this sequence completely abolished transformation and transcriptional repression by v-Rel and resulted in a shift in the localization of c-Rel from cytoplasmic to nuclear in CEF. When the conserved Ser within the PKA recognition sequence was replaced by Ala, there was no significant effect on transformation and transcriptional repression by v-Rel or on cytoplasmic retention of c-Rel. However, when this Ser was changed to Asp or Glu, transformation and transcriptional repression by v-Rel were significantly inhibited and c-Rel showed a diffuse nuclear and cytoplasmic localization in CEF. Although a peptide containing the recognition sequence from v-Rel can be phosphorylated by PKA in vitro, this site is not constitutively phosphorylated to a high degree in vivo in transformed spleen cells incubated with okadaic acid. Our results indicate that the transforming and transcriptional repressing activities of v-Rel and the cytoplasmic retention of c-Rel are dependent on the structure of the conserved PKA recognition motif. In addition, they suggest that phosphorylation at the conserved PKA site could have a negative effect on transformation and transcriptional repression by v-Rel and induce the nuclear localization of c-Rel.
Insights
The Rel homology domain
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- The Rel family of proteins, including v-Rel and c-Rel, are crucial for transcriptional control.
- These proteins share a conserved Rel homology domain responsible for dimerization, DNA binding, and nuclear targeting.
- A conserved phosphorylation site for cyclic AMP-dependent protein kinase (PKA) exists within this domain.
Purpose of the Study:
- To investigate the role of a conserved PKA recognition sequence within the Rel homology domain.
- To determine the sequence's importance for v-Rel's transforming activity and c-Rel's subcellular localization.
Main Methods:
- Linker insertion mutagenesis and site-directed mutagenesis were employed.
- Mutations were introduced into the PKA recognition sequence (Arg-Arg-Pro-Ser).
- Effects on v-Rel transformation and c-Rel localization in chicken embryo fibroblasts (CEF) were assessed.
Main Results:
- Insertion of Pro-Trp abolished v-Rel transformation and repression, shifting c-Rel to the nucleus.
- Replacing Ser with Ala had no significant effect.
- Replacing Ser with Asp or Glu inhibited v-Rel transformation and caused diffuse c-Rel localization.
Conclusions:
- The structure of the PKA recognition motif is critical for v-Rel's transforming activity and c-Rel's cytoplasmic retention.
- Phosphorylation at this site may negatively impact v-Rel's functions and promote c-Rel nuclear localization.