Neuronal and Cellular Signalling Lecture 6 Notes: Membranes and Intracellular Signalling Part 2 Intracellular Signalling - Protein phosphorylation is the covalent modification of proteins via addition or removal of phosphates, often used to regulate function. This is the most common and important Active Target -0-P-0" protein 0=0-0 ADP. H2O post-translational modification of proteins; at any time, around 30% of proteins in the body are phosphorylated. Protein kinase Protein phosphatase ATP - A protein kinase uses energy from ATP to transfer a phosphate group (Pi) to the target protein (has a hydroxyl group). Often, this activates the target protein, but this process can also cause deactivation. Protein phosphatases will remove the phosphate from the target protein. Target protein -OH Inactive kinase ATP + protein ADP + phosphorylated protein - ATP is the source of phosphate/energy and is widely available in the cell. The phosphorylation requires enzymatic catalysis to control the energy transfer. Enzymes enable variable activity (fine-tuning control). Phospho- Phospho- threonine H H Phospho- tyrosine H serine H3N-C-COO- H3N-C-COO- H3N-C-COO- O - Specific amino acids (i.e. serine, threonine, tyrosine) are phosphorylated by protein kinases which transfer a phosphate group to the oxygen of the hydroxyl groups on the polar head. Serine and threonine represent 98% of kinase phosphorylation. CH2 HC-O-P-O- CH2 1 -O- CH3 O-P-O- O 0 -O-Q=O O-P-O O - This phosphorylation is a covalent modification (i.e. is stable) which adds a polar region (0) which changes the shape and charge of the amino acid on the target protein and creates a binding site for regulatory proteins. Protein kinases are a family of enzymes that catalyse the phosphorylation of Ser, Thr and Tyr. They have a conserved amino acid sequence (i.e. can identify unknown kinases by sequence) and tertiary structure. They are primarily classified by amino acids to be phosphorylated. There are two main types: serine/threonine kinases and tyrosine kinases (i.e. named after target enzyme). Each protein kinase has a defined amino acid sequence that it will target. This provides highly specific regulation. For example, glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase which can act on any polypeptide with the sequence S/TXXS/T (where X is any amino acid, can also be XXX for GSK3). A phosphate group will be added to the serine/threonine. - Kinase proteins can be membrane bound or cytosolic. Cytosolic kinases (e.g. GSK3) target cytosolic or nuclear proteins and controls activity (i.e. structure), binding, cellular localisation and degradation. On the other hand, membrane-bound kinase proteins (e.g. EGF receptors) target membrane or cytosolic proteins and control the activity, cellular localisation and/or dimerization of other proteins. - Tyrosine kinase receptors (single TM proteins) are an example of dynamic phosphorylation to regulate function. These activate after binding to ligands at the extracellular binding site. This triggers 2 tyrosine kinase receptors to dimerise, activating (i.e. phosphorylating) a C-terminal
internal tyrosine protein kinase. This enables the phosphorylation of additional tyrosine residues to signal a cascade of intracellular events. Epidermal growth factor (EGF) show tyrosine kinase activity. Inhibition of tyrosine kinase