Intracellular Signalling, I Kinase Phosphates and SCR domains · Protein Phosphorylation-targeted program · Covalent Modification -by adding and removing phosphates. -good for regulation function · Via Kinase Enzymes (family of enzyme that catalyse a phosphate for serine, threonine, and tyrosine · To require control enzyme transfer ATP + protein kinase ADP + phosphorylated protein · ATP-provides source of phosphate. · 30% of all proteins are phosphorylated Kinases · Specific Amino Acids are phosphorylated: · 3 amino acids: -Serines-86%-most abundant -Thereonine-12%-middle abundant -Tyrosine-2%-least abundant · OH, group is phosphorylated -Giving a Polar Group -Which changes physical and chemical change. -Result in a physical shape. Protein phosphorylation changes: Shape of amino acid Charge of amino acid · Each protein kinase has a defined amino acid sequence that it will target. · Provides highly specific regulation Change in 3D structure. Result in a change in Function. Results in a change in binding site for signal. Signalling is for binding or inhibit binding Conserved amino acid sequence -> identify unknown kinases by sequence Conserved tertiary structure Each protein kinase has a specific target sequence.
-each protein kinase has different aa sequence that it will target. -high specific regulation Can find target phosphorylation sites by doing experiments to find actual sites. 2 major Types: 1. Serine / Threonine kinases 2. Tyrosine kinases Dynamic phosphorylation to regulate function Ligand Ligand- binding sites Bound ligand Exterior Transmembrane a helix ATP Activation lip P ADP Cytosol Poorly active protein tyrosine kinase 1 Receptor tyrosine kinases (RTKs) without bound ligand ATP P + ADP Active protein tyrosine kinase 2 Dimerization and phosphorylation of activation lip tyrosines P ATP P ADP + P P P ATP P + ADP P P 3 Phosphorylation of additional tyrosine residues Epidermal growth factor show tyrosine kinase activity Inhibition of tyrosine kinase activity provides a therapeutic action in non- small lung cancers localisation 1. Cytosolic -Target cytosolic or nuclear proteins -Controls Activity (structure) Binding Cellular localisation Degradation 2. Membrane Bound -Target membrane or cytosolic proteins -Controls -Activity (structure) -Cellular localisation
-Dimerization How to monitor phosphorylation? Antibodies to select specific phosphorylation protein. 1. Synthesis of oligopeptide with phosphorylated amino acids 2. Inject into animal to produce antibody 3. Booster to elevate titre 4. Purify 5. Radio-active isotope of phosphate, 32P can be used to label phosphorylated proteins of proteins 6. Using gamma 32P ATP in combination with a kinase 7. Blot an western blot with total antibody and phospho-antibody 8. Radioactive isotope phosphate 32P (used to label phosphorylated protein of protein. 32P is a beta-emitter Half life of 14 days Requires significant safety 9. Using Gamma 32p 10. Measured using photographic film Scintillation counter Phosphor imager (e.g. 'Typhoon') Old days- Used pre-incubated cells with radioactive phosphate. Simulates signalling cells Few minutes Extract protein SDS page electrophoresis Gel exposed to autoradiography Comparison between treated and untreated For some identify molecular weight Kinases · Ser/Thr kinases Function 1. Transcription of encoding genes. 2. Signal transduction regulation (phosphorylation etc). 3. Specific substrate recognition sites. 4. Localization with regulators