title: Delineation of selective cyclic GMP-dependent protein kinase Ialpha substrate and inhibitor peptides based on combinatorial peptide libraries on paper.
abstract: Peptide libraries on cellulose paper have proven to be valuable tools for the a priori determination of substrate specificities of cyclic AMP- and cyclic GMP-dependent protein kinases (cAMP-kinase and cGMP-kinase) on the basis of octa-peptide sequences. Here, we report the extension of our peptide library screens to 12-mer and 14-mer peptide sequences, resulting in highly cGMP-kinase Ialpha selective peptides. The sequences TQAKRKKSLAMA-amide and TQAKRKKSLAMFLR-amide, with Km values for cGMP-kinase Ialpha of 0.7 and 0.26 microM and Vmax values of 11.5 and 10.9 micromol/min/mg, respectively, display a high specificity for this enzyme. Furthermore, replacing the phosphate acceptor residue serine with alanine in TQAKRKKSLAMA-amide resulted in the highly cGMP-kinase Ialpha selective inhibitor peptide TQAKRKKALAMA-amide, with inhibitor constants for cGMP-kinase Ialpha and cAMP-kinase of 7.5 microM and 750 microM, respectively. Selective cGMP-kinase inhibitors have the potential to play an important role in the elucidation of the distinct cellular functions of cGMP-kinase separate from those activated by cAMP-kinases, and, therefore, may play an important role as pharmaceutical targets. Molecular docking experiments of the most cGMP-kinase selective sequences on a molecular model of the catalytic domain of cGMP-kinase Ialpha suggest that they adopt unique conformations, which differ significantly from those observed for the cAMP-kinase-specific inhibitor PKI(5-24). Our results suggest that despite their structural similarities, cAMP-kinase and cGMP-kinase use distinct peptide substrate and inhibitor conformations, which could account for their unique substrate specificities. These findings are further supported by cAMP- and cGMP-kinase-selective inhibitor analogs with (D)-Ala residues at the inhibitory positions.
authors.affiliation: Department of Pharmacology, University of Vermont, College of Medicine, Burlington 05405, USA.
authors.forename: W R
authors.initials: WR
authors.lastname: Dostmann
authors.forename: C
authors.initials: C
authors.lastname: Nickl
authors.forename: S
authors.initials: S
authors.lastname: Thiel
authors.forename: I
authors.initials: I
authors.lastname: Tsigelny
authors.forename: R
authors.initials: R
authors.lastname: Frank
authors.forename: W J
authors.initials: WJ
authors.lastname: Tegge
mesh_terms: D000818:Animals; D020134:Catalytic Domain / Q000502:physiology; D002482:Cellulose; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000502:physiology*; D006801:Humans; D015394:Molecular Structure; D010209:Paper; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000502:physiology; D010766:Phosphorylation; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid
chemical_list: D019151:Peptide Library; D010455:Peptides; D002482:Cellulose; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases