Year: 1995
Type: article
Abstract: Association of the amino terminal domain of ~5 6 ' " ~ with the cytoplasmic domain of CD4 in T cells is one of the best characterized intracellular protein interactions involving immune cell receptors.p56Ick is an src family protein tyrosine kinase (PTK) and is activated upon CD4 ligation.Coimmunoprecipitation studies have shown that the noncovalent interaction is mediated by way of cysteine pairs in each protein.'**Interestingly, this interaction has not been successfully reproduced with fusion proteins in vitro, presumably due to the inability to mimic exact intracellular conditions or due to involvement of additional cofactors (e.g., a third coupling protein).To determine if the protein interaction was indeed binary, we made use of the yeast two-hybrid system.Plasmids and the Saccharomyces cerevisiae strain were from Dr. R. Brent and colleagues at H a r ~a r d .~. ~ Constructs of hybrid proteins were created by polymerase chain reaction (FIG.1A).The cytoplasmic domain of murine CD4 was fused to the LexA DNA binding protein.Full-length (Lck) and the amino-terminal domain (aLck; amino acids #1-67) of murine p56ICk were fused to the B42 acid patch domain, which exhibits moderate transcriptional activity.Transcription of the B42 hybrid protein is induced only in a galactose-based medium.Interacting cysteine pairs were eliminated by truncation of CD4 (tCD4; amino acids #420-442) and mutation of aLck (maLck; cysteines #20 and 23 changed to alanine).Constructs were introduced into yeastbearing chromosomal LEU2 and episomal lacZ reporter genes controlled by upstream LexA operator sequences.Transfected yeast were tested for growth on leucine-deficient medium (FIG.1B) and for P-galactosidase activity (TABLE 1).Yeast containing all combinations of plasmids grew well on glucose-based agarose plates containing leucine, whereas none grew on glucose-based medium lacking leucine.Full-length p56ICk significantly inhibited growth when induced on galactose-based medium containing leucine.This is presumably due to nonspecific inhibitory effects of protein tyrosine phosphorylation by ~5 6 ' " ~.Only yeast transfected with both aLck or Lck in combination with CD4 grew on galactose-based leucine-deficient medium, indicating that these proteins interact in a binary manner.Similarly, increased P-galactosidase activity above control 'The Basel Institute for Immunology was founded and is supported by F.
Institution: University of Basel
Language: English
Topic: T-cell and B-cell Immunology
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2044855538