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Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Identification of cDNA clones that code for protein-tyrosine kinases by screening expression libraries with
R A Lindberg1, D P Thompson, T Hunter
1Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92138.
Abstract:
A phage lambda gt11 human fibroblast cDNA expression library was screened with antibodies against phosphotyrosine to determine the feasibility of this approach as a method for the identification of clones that code for protein-tyrosine kinases. Several antibody positive clones were isolated. One clone also scored positive with degenerate oligonucleotides designed to identify sequences coding for protein-tyrosine kinases. This cDNA clone was partially sequenced and proved to be identical to part of lyn, a recently reported putative protein-tyrosine kinase. A portion of the cDNA was cloned into an inducible plasmid expression vector. Phosphorylation of many bacterial proteins on tyrosine was observed upon addition of inducing agent. The results demonstrate that this method of screening can identify cDNAs that encode active protein-tyrosine kinases and could prove useful for the identification of novel ones.
Insights
Researchers screened a human fibroblast cDNA library using phosphotyrosine antibodies to identify protein-tyrosine kinases. This method successfully identified a lyn kinase cDNA clone, demonstrating its utility for discovering novel kinases.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Protein-tyrosine kinases play crucial roles in cellular signaling pathways.
- Identifying novel protein-tyrosine kinases is essential for understanding various biological processes and diseases.
- Current methods for kinase identification can be labor-intensive and may miss certain kinase families.
Purpose of the Study:
- To evaluate the feasibility of using phosphotyrosine antibodies to screen expression libraries for protein-tyrosine kinases.
- To identify novel protein-tyrosine kinase-encoding cDNAs from a human fibroblast library.
- To demonstrate the functional activity of an identified kinase using an expression system.
Main Methods:
- Screening of a phage lambda gt11 human fibroblast cDNA expression library with anti-phosphotyrosine antibodies.
- Confirmation of positive clones using degenerate oligonucleotides specific for protein-tyrosine kinase sequences.
- Partial sequencing of a positive cDNA clone to identify the encoded kinase.
- Cloning of the cDNA into an inducible plasmid expression vector for functional analysis.
Main Results:
- Several antibody-positive clones were isolated from the cDNA library.
- One clone was confirmed to encode a protein-tyrosine kinase sequence, identified as part of the lyn kinase.
- Expression of the cloned lyn cDNA in bacteria resulted in the phosphorylation of bacterial proteins on tyrosine residues.
- The results validated the screening approach for identifying active protein-tyrosine kinases.
Conclusions:
- Screening cDNA expression libraries with phosphotyrosine antibodies is an effective method for identifying protein-tyrosine kinases.
- This approach can successfully identify known kinases, such as lyn, and has the potential to discover novel ones.
- The functional expression of the identified kinase confirms its activity and the validity of the screening strategy.
