Substrate screening of protein kinases: Detection methods and combinatorial peptide libraries

Abstract

The study of protein kinases has become a matter of great importance in the development of new drugs for the treatment of diseases, including cancer and inflammation. Substrate screening is the first step in the fundamental investigation of protein kinases and the development of inhibitors for use in drug discovery. Towards this goal, various studies have been reported regarding the development of phospho-peptide detection methods and the screening of phosphorylated peptide sites by protein kinases. This review introduces the detection methods for phosphorylation events using the reagents with (γ(32)P)ATP, ligand-linked ATP, phospho-peptide-specific antibodies and metal chelating compounds. Chemical modification methods using β-elimination for the detection of phospho-Ser/Thr peptides are introduced as well. In addition, the implementations of combinatorial peptide libraries for screening peptide substrates of protein kinases are discussed. The phage display approach has been suggested as an alternative method of using synthetic peptides for screening the substrate specificities of protein kinase. However, a solid phase assay using a peptide library-bound polymer resin or a peptide-arrayed glass chip is preferred for high throughput screening (HTS).

Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains

“…Commercial phosphotyrosine antibodies typically recognize short mammalian phosphopeptides; in some cases even five amino acids were previously shown to be sufficient for recognition [35], [50], [55], [56]. To systematically analyze the recognition capabilities of phosphorylated CagA EPIYA-motifs by α-phosphotyrosine antibodies we first synthesized a series of peptides derived from the EPIYA-A motif exhibiting the phosphotyrosine residue in the middle +/− five, four, three or two flanking amino acids, including the STEPIYAKVNK (11-mer), TEPIYAKVN (9-mer), EPIYAKV (7-mer) and PIYAK (5-mer) sequences as indicated (Figure 1B, top).…”

“…It is well known that the α-phosphotyrosine antibodies were originally developed for mammalian proteins and typically recognize short amino acid stretches containing the phosphorylated tyrosine residue, including synthetic phospho-peptides [35], [50], [55], [56]. We therefore proposed that phospho-peptides derived from the CagA EPIYA-motifs would be useful for studying the recognition capabilities by seven commercial antibodies.…”

“…These 11-mer peptides were chosen to compare the three EPIYA-motifs because α-phosphotyrosine antibodies typically recognize short phosphopeptides [35], [50], [55], [56]. Commonly, 11-mer peptides are also used for immunizations to generate phospho-specific antibodies, which then recognize the corresponding phosphopeptides bound to affinity columns and in ELISA (Biogenes, Berlin, Germany).…”

Binding of small molecules to cavity forming mutants of a de novo designed protein

“…[6][7][8]15 (ii) Alternatively, high throughput methods, such as phage display or mRNA display, can be used to find rare binding proteins amidst large combinatorial libraries of nonbinding sequences. [16][17][18] The hydrophobic core of a protein is generally well packed, with few cavities. Previous studies showed that creation of new cavities in a protein core can produce binding sites where small molecules can bind.…”

Large-scale profiling of protein kinases for cellular signaling studies by mass spectrometry and other techniques

“…The randomized peptide library immobilized on beads was also coupled with chemical modification via beta-elimination [52] or incorporation of thiophosphoryl group [53] to detect phosphorylated substrate peptides and applied to profiling of other protein kinases.…”