Protein Structure and Function Lecture 8 Notes: Phosphorylation Part 2 - One way to investigate consensus sequences is looking at sequences around phosphorylated sites or to present a kinase with a mixture of peptides with a central Ser/Thr/Tyr and see which ones are phosphorylated. - Liver glycogen phosphorylase has species homology but no phosphorylation consensus at different Ser sites. However, the sequences are almost identical in mice and rats, showing this gene is highly conserved. Variation in, for example, Ser environment may enable kinase specificity and the consensus might apply to individual kinases rather than to targets. - The peptide specificity of PKC shows that target site selectivity is present but not absolute (i.e. cannot bind to some amino acids but binds to almost all others). - PhosphoSite plus is a PTM database which can show the phosphorylation modification sites of a protein and their flanking amino acid sequences to compare sites and organisms. - Alternative sequences can be compared for the substrates of two different enzymes. This shows that there are consensus sequences which are kinase specific. Predictions of sites of phosphorylation can be obtained by entering the protein ID or sequence into PhosphoNet. - The NetPhos is a database server which predicts phosphorylation sites in eukaryotic proteins - There are 518 kinases in the human genome which phosphorylate 1/3 of the proteome. Deregulation of kinase function has been demonstrated to play an important role in cancer as well as immunological, inflammatory, degenerative, metabolic, cardiovascular and infectious diseases. - Over 30 kinase-based drugs have been approved by the US FDA and many are undergoing clinical trials. The first was Imatinib (2001) which was used for various disorders, including CML, gastrointestinal stromal tumours (GISTs) and a number of other malignancies. Success in developing kinase inhibitor drugs has been limited, partly due to the fact that the majority of inhibitors bind to the evolutionarily conserved ATP binding pocket, common to all kinases, so achieving selectivity is difficult. - Imatinib (Glivec/Gleevec) is an inhibitor of an abnormal tyrosine kinase, used for treatment of chronic myelogenous leukaemia as it blocks the ATP binding site. Imatinib was the first clinical signal transduction inhibitor. - Protein phosphatases can be classified according to their: (a) Sequence (b) Structure (c) Substrate (i.e. phosphor- Tyr/Ser/Thr/His) (d) Requirement for a metal ion (e.g. Mg2+, Mn2+ or Ca2+) (e) Active site catalytic functional groups (Cys/Asp/Ser/Gln) - Relative to kinases, protein phosphatases are more structurally and catalytically diverse. Phosphatases co-regulate most biological processes with kinases, and like kinases, dys- regulation or mutation of phosphates leads to a wide variety of diseases. - The reaction catalysed by phosphatases is a simple hydrolysis wherein a substrate (i.e. a protein with a Thr/Ser/Tyr amino acid side chain) with a phosphate monoester is combined with H-O-H (water) to form a substrate with a free hydroxyl group and a monophosphate. O "O-P-O Ó H-O-H O-H 1 substrate + O O "0-P-O" H substrate
- Other monoester substrates and associated phosphatases exist (e.g. lipids, carbohydrates). The reaction