DETERMINATION OF CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN-KINASE SUBSTRATE-SPECIFICITY BY THE USE OF PEPTIDE LIBRARIES ON CELLULOSE PAPER

An iterative approach to the a priori determination of the substrate s pecificity of cAMP- and cGMP-dependent protein kinases (PKA and PKG) b y the use of peptide libraries on cellulose paper is described. The st arting point of the investigation was an octamer library with the gene ral structure Ac-XXX12XXX, where X represents mixtures of all 20 natur al amino acids and 1 and 2 represent individual amino acid residues, T he library thus contained all possible 2.56 x 10(10) octamers, divided into 400 sublibraries with defined amino acids 1 and 2 each consistin g of 6.4 x 10(7) sequences. After phosphorylation with the kinases in the presence of [gamma-P-32]ATP, the sublibrarys Ac-XXXRRXXX and Ac-XX XRKXXX were identified as the best substrates for PKA and PKG, respect ively. The second-generation libraries had the structures Ac-XXXRR12X and Ac-XXXRK12X for PKA and PKG and resulted in the most active sequen ce pools Ac-XXXRRASX and Ac-XXXRKKSX. After delineation of every posit ion in the octameric sequence and extension of the investigation to de cameric peptides, the best sequences, Ac-KRAERKASIY and Ac-TQKARKKSNA, were obtained for PKA and PKG, respectively. Promising octameric and decameric peptides were assembled 5 or 10 times each and assayed in or der to determine the experimental scatter inherent in the approach. Th e kinetic data of several octameric and decameric sequences were deter mined in solution and compared to data for known substrates. The recog nition motif of PKA was confirmed by this approach, and a novel substr ate sequence for PKG was identified. The approach can be expected to b e of generally applicable for the elucidation of protein kinase specif icity with linear peptide substrates.