A solid-phase screen for protein kinase substrate selectivity

Carmel, G., Kuret, J. (June 1992) A solid-phase screen for protein kinase substrate selectivity. Anal Biochem, 203 (2). pp. 274-80. ISSN 0003-2697 (Print)0003-2697 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/1416023
DOI: 10.1016/0003-2697(92)90313-V

Abstract

Cassette mutagenesis was used to synthesize an Escherichia coli expression library of unique phosphorylation sites. The cassette encodes a central serine residue surrounded by every combination of Ala, Arg, Gln, Glu, Gly, and Pro residues over a 7-residue segment (a total of 6(7) approximately 2.8 x 10(5) sequences). The cassette was inserted into the gene of a suitable carrier protein and expressed in E. coli with the T7 expression system, and the resultant library was subjected to solid-phase protein phosphorylation assays on nitrocellulose filters. When the library was screened with TPK1 delta, the modified catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase, individual colonies that expressed substrates for this kinase were identified. By DNA sequencing through the cassette region of positive clones, the consensus recognition sequence for TPK1 delta was deduced and found to conform with the well-established substrate selectivity of its mammalian homolog (Arg-Arg-Xaa-Ser). Because a large number of clones can be sequenced rapidly, and the positions of invariant residues composing a recognition site identified, this approach may be useful as a general screen of protein kinase substrate selectivity.

Item Type: Paper
Uncontrolled Keywords: Amino Acid Sequence Base Sequence DNA/chemistry/genetics Electrophoresis, Polyacrylamide Gel Escherichia coli/genetics Gene Expression Gene Library Molecular Sequence Data Mutagenesis, Insertional Phosphorylation Protein Kinases/*metabolism Proteins/chemistry/genetics/*metabolism Substrate Specificity
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene expression
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > kinase
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > mutations > mutagenesis
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression > phosphorylation
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date: June 1992
Date Deposited: 01 Oct 2015 20:54
Last Modified: 01 Oct 2015 20:54
Related URLs:
URI: http://repository.cshl.edu/id/eprint/31767

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