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Screening for the Optimal Specificity Profile of Protein Kinase C Using Electrospray Mass-Spectrometry

Department of Medical Biochemistry and Microbiology, Uppsala University, 75 123 Uppsala, Sweden

Bo Ek

Swedish Agricultural University, 750 07 Uppsala, Sweden

Nikita Oskolkov

Institute of Organic and Bioorganic Chemistry, Tartu University, 51014 Tartu, Estonia

Ale Närvänen

Department of Organic Chemistry, Kuopio University, 70211 Kuopio, Finland

Jaak Järv

Institute of Organic and Bioorganic Chemistry, Tartu University, 51014 Tartu, Estonia

Pia Ek

Department of Medical Biochemistry and Microbiology, Uppsala University, 75 123 Uppsala, Sweden

A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phosphopeptides was designed for rapid and quantitative characterization of protein kinase specificity. The k_cat/K_m values for the protein kinase Cß (PKCß) were determined for a systematically varied set of individual substrate peptides in library mixtures by the ESI-MS method. The analysis revealed a complex structural specificity profile in positions around the phosphorylated serine with hydrophobic and/or basic residues being mostly preferred. On the basis of the kinetic parameters, a highly efficient peptide substrate for PKCß (K_mvalue below 100 nM) FRRRRSFRRR and its alanine substituted pseudosubstrate-analog inhibitor (K_i value of 76 nM) were designed. The quantitative specificity profiles obtained by the new approach contained more information about kinase specificity than the conventional substrate consensus motifs. The new method presents a promising basis for design of substrate-site directed peptide or peptidomimetic inhibitors of protein kinases. Second, highly specific substrates could be designed for novel applications such as high-throughput protein kinase activity screens on protein kinase chips.

Key Words: protein kinases • protein kinase inhibitors • protein kinase C • mass-spectrometry • peptide library

Journal of Biomolecular Screening, Vol. 10, No. 4, 320-328 (2005)
DOI: 10.1177/1087057104274353


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