A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP.
The Escherichia coli tet-repressor (TetR) operator system was used to develop a variation of the yeast two-hybrid assay in which disruptions of protein-protein interactions can be identified by a positive selection. This assay, designated the "split-hybrid system," contains a two-component reporter. The first component contains LexA binding sites upstream of the TetR gene and the second contains TetR operator binding sites upstream of HIS3. Interaction of one protein fused to the LexA DNA binding domain with a second protein fused to the VP16 activation domain results in TetR expression. TetR subsequently binds to the tet operators, blocking the expression of HIS3 and preventing yeast growth in media lacking histidine. The utility of the split-hybrid system was analyzed by examining the phosphorylation-dependent interaction of CREB and its coactivator CREB binding protein (CBP). CREB and CBP associate through an interaction that depends upon CREB phosphorylation at Ser-133. Mutation of this phosphorylation site prevents yeast growth in the standard two-hybrid assay but allows growth in the split-hybrid strains. The split-hybrid system was used to identify other CREB mutations that disrupt its association with CBP. These mutations localized around the site of CREB phosphorylation, indicating that only a small portion of the CREB activation domain is required for CBP interaction. The yeast split-hybrid system should be useful in identifying mutations, proteins, peptides, and drugs that disrupt protein-protein interactions.
Shih
H M
HM
Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.
Goldman
P S
PS
DeMaggio
A J
AJ
Hollenberg
S M
SM
Goodman
R H
RH
Hoekstra
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D050882:CREB-Binding Protein; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D012641:Selection, Genetic; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C034811:tetracycline resistance-encoding transposon repressor protein; D050882:CREB-Binding Protein; C494365:Crebbp protein, rat