Fluorescence-based cloning of a protein tyrosine kinase with a yeast tribrid system.
Post-translational modifications of proteins control myriad biological functions. However, relatively few methods exist for the identification of the enzymes that catalyze these modifications. To expand this repertoire, we report a yeast genetic approach that enables the identification of protein tyrosine kinases (PTKs) from cDNA libraries. Yeasts were transformed with four vectors encoding: 1) a potentially universal PTK substrate fused to the LexA DNA binding domain, 2) the Grb2-SH2 domain fused to the B42 activation domain, 3) a fluorescent reporter gene controlled by LexA DNA sites, and 4) a Jurkat cDNA library. Transient expression of PTKs, such as the lymphocyte-specific kinase Fyn, resulted in phosphorylation of the DNA-bound substrate, recruitment of the Grb2-SH2 domain, and activation of the fluorescent reporter gene. This brief induction of protein expression circumvented the potential toxicity of PTKs to the yeast. Fluorescence activated cell sorting (FACS) enabled isolation of PTKs, and these enzymes were further characterized by flow cytometry and immunoblotting. This approach provides a potentially general method for the identification and evaluation of enzymes involved in the post-translational modification of proteins.
Clark
Daniel D
DD
Department of Chemistry, The Pennsylvania State University, 104 Chemistry Building, University Park, PA 16802, USA.
Peterson
Blake R
BR
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D017930:Genes, Reporter; D006801:Humans; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases