INTERACTIONS BETWEEN THE AMINO-TERMINAL DOMAIN OF P56(LCK) AND CYTOPLASMIC DOMAINS OF CD4 AND CD8-ALPHA IN YEAST

The interactions between CD4 or CD8 and p56(lck) were tested using the two-hybrid protein interaction system in yeast. Plasmid constructs we re created which fuse the cytoplasmic domains of either CD I or CD8 al pha to the DNA-binding protein LexA, and the unique amino-terminal dom ain of p56(lck) fused to a transcriptional activation domain. These co nstructs were transfected into yeast bearing lacZ and LEU2 reporter ge nes controlled by upstream LexA operator sequences. Yeast transfectant s bearing either CD4 or CD8 alpha hybrid proteins in combination with the amino terminal p56(lck) hybrid protein exhibited increased beta-ga lactosidase activity and growth on leucine-deficient medium, indicatin g interactions between these protein domains. Quantitation of reporter activation indicated that the interaction of p56(lck) with CD8 alpha is at least 18-fold weaker than the interaction with CD4 in this assay . This reduced interactive capacity is apparently not due to competiti on by CD8 alpha interacting with itself, since homotypic or heterotypi c interactions between CD8 alpha and/or CD4 could not be detected. Tru ncation and point mutants demonstrated that the interactions of p56(lc k) with CD4 or CD8 alpha were dependent on the integrity of a pair of cysteines on each protein. The results indicate that these interaction s do not require any additional proteins. Additionally, expression of the entire p56(lck) molecule as a hybrid with LexA resulted in dramati c reduction in the growth of yeast. Though the two-hybrid system is a powerful tool for examining protein interactions, this result indicate s potential limitations in studying full-length src family tyrosine ki nases in yeast.