Differential Specificity of Protein Kinases and Its Applications

This projects discusses about Protein Kinases and its applications. Protein kinases are enzymes which catalyse the phosphoryl transfer from the g-phosphate of ATP to acceptor amino acids in proteins. The uniqueness of chosen model protein kinases was analyzed at 3 distinctive levels making use of a) novel bi-substrate-analogue inhibitors, b) synthetic peptide substrates and c) mutated protein substrate analogues. A new type of protein kinase bi-substrate-analogue inhibitors was created based on adenosine-5’-carboxylic acid derivatives, in which a small arginine containing peptide was connected to the 5′-carbon atom of the adenosine sugar moiety via a linker chain. These compounds demonstrated high inhibitory potential against 2 basophilic protein kinases, the protein kinase A (PKA) and protein kinase C (PKC), with IC50 values in the nanomolar range, but no inhibitory activity for the acidophilic kinases CK1 and CK2. The inhibitors were successfully tried for affinity purification of PKA using MgATP in addition to L-arginine as eluting agents. Ca2+-dependent protein kinase (CDPK-1) was filtered from maize seedlings and its substrate specificity was analyzed utilizing a group of synthetic peptides. These were produced from the phosphorylatable sequence RVLSRLHS(15)VRER of maize sucrose synthase 2 (SuSy2), and a consensus sequence motif A/LXRXXSXRZR was described from a research making use of arrays of carefully varied peptides attached with cellulose membrane (SPOTsTM membranes). The SuSy2 derived peptides were also discovered to be efficient substrates for mammalian PKC, but demonstrated low reactivity when it comes to PKA. Based on this peptide motif, a positionally oriented peptide library technique according to ESI-MS detection of phosphopeptides in initial velocity circumstances was created for quantitative kinetic characterization of protein kinase specificity profiles. Based on the acquired data an ideal peptide substrate for PKC, FRRRRSFRRR, was created. The uniqueness of protein kinase A was analyzed using site-directed mutagenesis in the phosphorylation site of L-type pyruvate kinase (L-PK), and assessment of the obtained data eas done using past research on structurally altered peptide substrates revealed that amino acid variations in short peptide substrates cause robust effects on the phosphorylation rate than the related alterations in the protein substrate L-PK.

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1. The protein kinase superfamily
2. The catalytic domain of protein kinase A as a model for the protein kinase superfamily
3. Regulation of protein kinase activitie
4. Protein kinase C
5. Calcium-dependent protein kinase from plants
6. Studies on the substrate specificity of protein kinases
6. 1. Phosphorylation site specificity of protein kinases
6. 1. 1. Structurally varied peptides for study of substrate specificity of protein kinases
6. 1. 2. Use of peptide libraries for study of substrate specificity of protein kinases
6. 2. Overview of the substrate specificity of serine/threonine protein kinases
6. 2. 1. Basophilic protein kinases
6. 2. 2. Proline directed protein kinases
6. 2. 3. Acidophilic and phosphate-directed protein kinases
6. 3. Docking sites and anchoring proteins as secondary specificity
determining factors of protein kinases
7. Inhibitors of protein kinases
7. 1. Protein and peptide site directed inhibitors
7. 2. ATP site directed inhibitors
7. 3. Bi-substrate inhibitors
8. Affinity isolation of protein kinases using specific ligands targeted to substrate sites
Present investigation
Paper I
Paper II
Paper III
Paper IV………

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Source: Uppsala University Library

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