v-Rel and c-Rel are differentially affected by mutations at a consensus protein kinase recognition sequence.
The avian retroviral oncoprotein v-Rel and its cellular homolog c-Rel are members of a family of related site-specific DNA-binding proteins. Towards the carboxy-terminal end of the highly conserved Rel homology (RH) domain in the majority of Rel proteins, there is a consensus recognition sequence for protein kinase A (PK-A). We have investigated the importance of this sequence (Arg-Arg-Pro-Ser) for several functional properties of v-Rel and c-Rel. Disruption of the PK-A sequence by a two amino acid insertion between the arginine and the proline residues completely abolished the ability of v-Rel and c-Rel to bind a kappa B site in vitro. When the phosphorylatable serine in this sequence (Ser-275 in v-Rel, Ser-266 in c-Rel) was replaced by an alanine, DNA binding by v-Rel was not affected, whereas the ability of c-Rel to bind DNA was reduced approximately fourfold by this mutation. Similarly, a serine to tryptophan change greatly reduced the DNA-binding ability of c-Rel, whereas v-Rel was not appreciably affected by this change. When this serine was replaced by an acidic amino acid, DNA binding by v-Rel was reduced approximately twofold and the DNA-binding activity of c-Rel was nearly abolished. Glutaraldehyde cross-linking experiments indicated that mutations at the PK-A recognition site that reduced DNA binding also negatively affected protein oligomerization, which is likely to be responsible for the reduced ability of mutant v-Rel and c-Rel proteins to bind DNA. Domain-swapping experiments showed that structural differences between v-Rel and c-Rel in the central region of the proteins are primarily responsible for the higher sensitivity of c-Rel to a serine to alanine mutation in the PK-A site. One difference between v-Rel and c-Rel, a glutamine to alanine change in v-Rel located three amino acids carboxy-terminal to the PK-A phosphorylatable serine (Ala-278 in v-Rel; Glu-269 in c-Rel), is mainly responsible for the lack of an effect on DNA binding by v-Rel when Ser-275 is replaced by alanine. That is, a v-Rel double mutant (v-275A/278E) showed reduced DNA-binding and transforming abilities as compared with v-Rel and v-275A. Similarly, the mutations in c-Rel that affected DNA binding showed a corresponding effect on the ability of c-Rel proteins to activate transcription in yeast from a reporter gene containing upstream Rel binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)
Mosialos
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Department of Chemistry, Boston University, Massachusetts 02215.
Gilmore
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TD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002471:Cell Transformation, Neoplastic; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D020572:Oncogene Proteins v-rel; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020569:Proto-Oncogene Proteins c-rel; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation
D020572:Oncogene Proteins v-rel; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D015682:Retroviridae Proteins, Oncogenic; D004247:DNA; D011505:Protein-Tyrosine Kinases