Substrate specificity of protein kinases studied with synthetic peptides

Detta är en avhandling från Uppsala : Acta Universitatis Upsaliensis

Sammanfattning: This thesis concentrates on the substrate specificities of protein kinases A and C (PKA, PKC). Synthetic peptides are used for that purpose, both in soluble and cellulose membrane-bound form. The, stereospecificity of both protein kinases was studied with D-amino acid containing synthetic peptides.The reactions of protein kinases with peptides are often monitored by adsorbing the phosphorylatedpeptides onto phosphocellulose paper, an anionic adsorbent. The presence of at least two basicamino acid residues in the peptide has been considered to be sufficient for the adsorption. In thisthesis, it was found that none of the phosphopeptides studied were adsorbed completely and therecovery of the phosphopeptide could vary twofold even if the peptides contained an equal numberof basic residues.A PKC substrate peptide pEKRPSQRSKYL was gradually shortened from either end. Themodifications at the C-terminus had a weaker influence on the reaction parameters than those at theN-terminus. The less pronounced specificity of PKC compared to that of PKA did not permit us todefine a minimum length substrate for this enzyme.The stereospecifically sensitive regions he in different parts of the substrates for PKA (RRASVA)and PKC (KRPSQRAKY). In the case of PKA, major effects appeared at the N-terminal side of thephosphorylatable serine while the most sensitive part in PKC substrates lies at the C-terminal side.PKA has a strong requirement for L-configuration at the serine residue while PKC can recognizeboth D- and L-configurations at the phosphorylation center. PKA has a well-defined core regionsensitive to changes in the substrate (-RAS-). In the case of PKC the stereochemical requirementsaround the phosphorylatable residue are not as strong. In conclusion, the stereospecificity of PKC isnot as clearly pronounced as that of PKA.In this thesis, it has been shown that SPOTs-membrane-bound peptides can be used for evaluation ofprotein kinase substrates. Using that method combined with the analysis of soluble peptides wefound a substrate peptide for PKC which was both sensitive (Km = 0.5 μM) and selective, as itshowed low or negligible cross-reactivity with other kinases tested.

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