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Fluorescence‐Based Cloning of a Protein Tyrosine Kinase with a Yeast Tribrid System
Author(s) -
Clark Daniel D.,
Peterson Blake R.
Publication year - 2005
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500047
Subject(s) - biology , sh2 domain , biochemistry , complementary dna , grb2 , repressor lexa , microbiology and biotechnology , cdna library , protein tyrosine phosphatase , tyrosine kinase , gene , enzyme , gene expression , signal transduction , repressor
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.